Nucleotide Facilitated Release Of Inorganic Phosphate And Hydrolysed Adenosine Triphosphate From Beef Heart Mitochondrial Adenos 6310 DK M B16550201
User Manual: 6310 DK-m
Open the PDF directly: View PDF 
.
Page Count: 204
| Download | |
| Open PDF In Browser | View PDF |
1%
National. ~ i b r a r ~
pf Canada
1
J
BibliothStque nationale
du Canada
Canadian Theses Service
Services des theses canadiennes
Ottawa, Canada
K I A ON4
CANADIAN THESES
THESES CANADIENNES
The qu6llty of this microfiche is heavily dependent upon the
quality of the original thesis sut~mittedfor microfilming. Every
effort has been made to ensure the highest quality of reproduco.
tion possible.
La qualit6 de cette microfiche depend grandement-de ki qualit6
de-lath2se soumise au microfilmage. Nous avons tout fait pour
assurer une qualit6 sup6rieure de reproduction.
If pages are meissing,contact the universit;'which'granted
degree.
the
S'il manque des pages, veuillez communiquer avec I'universit6 qui a confer6 le grade.
Some pages may have indistinct print especially if the original
pages were typed with a poor typewriter ribbon or ifathe university sent us an inferiq photocopy.
La qualit6 d'impression de certaines pages peut'laisser a
desirer, surtout si les pages originales ont 6t6 dactylographi&es
8 I'aide d'un ruban us6 ou s-i I'universit6 nous a fait parvenlr
une photqcopie de qualit6 inf6rieure.
Previously copyrighted materials (jourqal articles, published
tests, etc.) are not filmed.
Les documents qui font d6js I'objet d'un droit d'auteur (articles
de revue, examens publit%, etc.) ne sont pas microfllm~s.
'
-
\
Reproduction in full or in part of this film is governed by the
~anadianCopyright Act, R.S.C. 1970, c. C-30.
La reproduction, m6me partielle, de ce'microfilm est soumrse
8 la Loi canadienne sur le droit d'auteur, SRC 1970, c. C 3 0 .
\
3
.
THIS DISSERTATION
HAS BEEN MICROFILMED
EXACTLY AS RECEIVED
\
--
*
LA T H ~ S EA ETE
MOCROFILMEETELLE QUE
NOUS L'AVONS RECUE
\
.
4
I
b
AND ~ Y D R O L Y S E D ADENOSINE,*TRIP.HOSPHATE FROM BEEF HEART
&
x
MITOCHONDRIAL ADENOSINE T R I P H O S P H A T A S E
I
d
-
Seelochan B e h a r r y
IB.SC.,
u n i v e r s i t y of G u y a. n. a ,
. M.Sc.,
-
A THESIS
Acadia University,
SUBMITTED I N
1975
;
1979
FULFILLMENT
PARTIAL
O F THE REQUIREMENTS FOR THE DEGREE O F
-,
.-
DOCTOR OF PHILOSOPHY
.
i n the D e p a r t m e n t
of
Chemistry
.
.
I
1
@ . Seelochan
Beharry
L
b
SIMQN FRASER U N I V E R S I T Y
J u n e 1985
*2:
A l l r i g h t s reserved.
T h i s t h e s i s m a y n b t be
reproduced i n w h o l e or i n p a r t , by photocopy or
o t h e r m e a n s , w i t h o u t p e r m i s s i o n of t h e a u t h o r .
B
,
.
b
'8
Permission has been g r a n t e d
t o t h e N a t i o n a l ~ i b ~ a ro fy
Canada t o microfilm t h i s
t h e s i s and t o l e n d or s e l l
c o p i e s of t h e f i l m .
L' a u t o r i s a t i o n a 6 t G a c c o r d g e
Zi
l a BibLioFhSque n a t i o n a l e
du
Carrdda d e m & c r o f i l m e r
c e t t e t h s s e e t d e p r s t e r ou
de* v e n d r e d e s e x e m p l a i r e s du
film.
w
I
The a u t h o r ( c o p y r i g h t owner)
h a s
r e s e r v e d
o t h e r
p u b l i c a t i o n rig-hts, and
neither
the
thesis
e x t e n s i v e e x t r a c t s from i
may b e p r i n t e d o r o t h e r w k e
reproduced without his/her
w r i t t e n
p e r m i s s i o n .
,-"$
' a u t e u r ( t i t u l a i r e du d r o i t
se . r G s e r v e les
'auteur)
a u t r e s d r o i t s de publication;
ni l a thsse ni de longs
ne
e x t r a i t g de celle-ci
@ t r e i m p r i m e s ou
doivent
autremen r e p r o d u i t s s a n s son
a u t o r i s a t on E c r i t e .
h
-
r
1
APPROVAL
>
. Name':
.
0
~ e & i o c l w nB e h a r r y
-
Degree 5
Doctor pf P h i l o s p p h y
T i t l e of T h e s i s :
Nucleotide-Facilitated
<*
Release .of I n o r g a n i c
,'
P h o s p h a t e and
s e d ~denosi'ii'e-'-
T r i p h o s p h a t e from Beef H e a r t M i t o c h o n d r i a 1
Adenosine T r i p h o s p h a t a s e
Edamining Committee:
-
P r o f e s s o r F.W.B.
Chairman:
-
-
Dk. M . J .
Einstein
Gressef, S e K l o r S u p e r v i s o r
p
T
D
- -
m
--
-
-
-
. Slessor
--
D r . A.J,
Dav
I n t e r n a l Exam
Department o f K i n e s i o l o g y
P r o t e s s a r P a u l D." Boyer
E x t e r n a l Examiner
Molecular B i o l o g y ~ n s t i t u t e
U.C.L.A.
,Tule
fl U,. / 7 g~
Date ~ ~ ~ r o v e d :
+.
PARTIAL COPYRIGHT LICENSE
I he @by g r a n t t o Simon F r a s e r U n i v e r s i t y t h e . r l g h t t o l e n d
my t h e s i s , p r o j e c t o r extended essay ( t h e t i t l e o f w h i c h is'shown
t
below)
.
t o - u s e r s o f t h e Simon F r a s e r U n i v e r s i t y L i b r a r y , and t o make p a r t l a l ' o r
%
a
-
s i n g l e c o p-i e s o n l y f o r such u s e r s o r i n response t o a r e q u e s t from t h e
\
\a
l i b r a r y o f any o t h e r u n i v e r s i t y , o r o t h e r e d u c a t i o n a l I n s t i t u t i o p , on
A
d
i t s own b e h a l f o r f o r one o f i t s u s e r s .
.
I f u r t h e r agree t h a t permission
3
f o r m u t t i p l e c o p y i n g o f t h i s work f o r ' s c h o l a r l y - purposes may be g r a n t e d
by me o r t h e Dean o f Graduate S t u d i e s .
/
I t ' i s understood t h a t - c o p y i n g
o r pub1 l c a t i o n o f t h i s work f o r f I nanc l a 1
n sha l l n o t b@ a l lowed
w i t h d u t my w r i t t e n p e r m i s s i o n .
T i t l e o f Thesis-/Project/Extended Essay
i
" ~ u c l e i~d te - f a c i l i t a t e d
R e l e a s e o f ~ n o r g a n i cP h o s p h a t e and H y d r o l y s e d
0
i
A d e n o s i n e T r i p h o s p h a t e from Beef H e a r t ~ i t o c h o n d r i a lAdenosine
Author:
(signature)
seelochan Beharry
c5
(date)
b
#
\
-
-
iii
L,.
%
,
ABSTRACT
-
f
i
The Sephadex centkifuge column technique introduced by
Penefsky was modified and used t o perform preincubation and
pulse-chase type experiments with soluble beef h e a r t mitochon-
-
.
d r i a l adenosine t r i p h o s p h a t a s e , F1
expe-iments,
*
--
/
-.
In t h e preincubation-type
F1 was l a b e l l e d by incubation with [ 3 2 ~ ] ? i
before biin4 applied t o Sephadex G-50 columns whiche contained d
-/-.
1.0 cm nucleotide e q u i l i b r a t e d middle s e c t i o n .
.,
/
'
. Under
.
these
D
experimental 'condi,tions, ATP and AMPPNP bound equally well t o
+
F1 but ADP d i d ndt bind a s well.
.
.The order of e f f e c t i v e n e s s of
t h e nucleotides i n promoting t h e r e l e a s e of approximately 70%
( i . e . t h e s t e e p flhase of t h e biphasit. r e l e a s e ) of t h e -bound f
1
( 4 5mole
1
Pi
I
4
ADP.
pi/mole
F l ) from F1 was ATP > AMPPNP > >
r
H i g h oo&entrations o f ' nucleo'tides werb a b l e t o e f f e c t
t h e t o t a l r e l e a s e of P i from F1..
I t was concluded t h a t
4
bindi*
of -nucleotide, not hydrolysis, was necessary t o a f f e c t
a
P i release.
In t h e pulse-chase type experiment, F1 was l a b e l l e d when
it' passed through a 1.0 cm nucleotide e q u i l i b r a t e d g e l a t t h e
3
top ( p u l s e s e c t i o n ) of t h e column.
The l a b e l l e d F1 was exposed
t o unlabelled nucleotide present i n *the t h i r d . s e c t i o n (chase
s e c t i o n ) of t h e column.
(0.1 mole ~TP/moleF1 ) ,
pulse
P
when F1 was given a C ~ - ~ ~ P ] A T
the r e l e a s e of P i ( w i t h ADP present
.
.,
'
a t t h e same s i t e ) was e f f e c t e d equally well by chase ATP and
-/
AMPPNP.
B
*
Here a l s o t h e r e l e a s e of P i was b i p h a s i c with about
k
70% of t h e b o k d l a b e l being released i n t d e f i r s t phase.
using [ 3 ~ ] l~a b~e l~ i .n t h e pulse, it was shown t h a t ADP r e l e a s e
was s i m i l a r t o t h a t of P i b i n t h e presence o f . chase ATP.
a
[ 3 ~ ] purse
m ~ (0.1
~ ~
mole
~ A M P P N P / ~ o ~F~l ) ,
n d t e f f e c t i v e ' ( l e s s than
-30%) i n
With
c h a s e AMPPNP was
t h e r e l e a s e of bound
.
AMPPNP
A comparison of t h e r d u l t s from t h e two modes of inves-
t i g a t i o n reveal t h a t :
( i ) AMPPNP was more e f f e c t i v e i n
P i r e l e a s e when ADP is present
( m s e - c h a s e mode)
than when ADP i s absent (preincubation mode); and ( i i ) ATP was
more e f f e c t i v e i n promoting P i r e l e a s e when ADP is abs,en<
(preincubation mode) than when ABff i s present (pulse-chase
I"
mode )
>
.
\
The r e s u l t s a r e r a t i o n a l i z e d i n terms of t h e bindingchange mechanism f o r t h e F1 catalyzed hydrolysis of ATP involv-
i n g t h e ( i )nature of t h e nukleoti.de bound,
( i i ) number of
_*
occupied . s i t e s of F I before exposure t o incoming nucleotides,
and ( i i i ) t h e a b i l i t y of t h e nucleotides t o produce conformat i o n a l changes necessary t o e f f e c t t h e r e l e a s e of P i and/or
ADP
.
T o Martha and Matthew
ACKNOWLEDGEMENTS
.
I would l i k e t o expkess my appreciation t o Simon Fraser
.
.
University f o r providing finHncial support and adeq6a,,te ' f a c i l i -
t i e s t o 'enable my p a r t i c i p a t i o n i n t h i s study.
In p a r t i c u l a r ,
I would l i k e t o th9nk t h e Department of Chemistry for c o n t r i I
-
buting towards a productive and enjoyable s t a y .
*
I wouid l i k e t o e*presg my g r a t i t u d e and thankfuldebs t o
"
Dr.
6.
X
Michael J . ~ r e s s e r f' o r affording me t h e opportunity t o work
>
with him i n t h i s p r o j e c t .
More~ver, I appreciated t h e academic
i
s e t t i n g f o s t e r e d , t h e personal and p r o f e s s i o n a l growth encour+*
aged, t h e e x c e l l e n t d i r e c t i o n and invaluable a s s i s t a n c e given,
;
and t h e readiness and enthusiasm w i t h which hel$ was given.
.%
L a s t l y , I would l i k e a l s o l i k e t o thank him f o r t h e varipus
r
-
e f f o r t s made t o make my s t a y comfortable, enjoyable, educative,
and productive.
I would l i k e * t o
m R.
I
I
f
.
Keith N .
S l e s s o r and D r .
Richards f o r being onLmy superv'isory committee, and
f o r t h e i r h e l p , advice, and thoughts on t h e Comprehensive'
--
Examinations and t h e s i s .
I would a l s o l i k e t o express my
a p p r e c i a t i o n f o r t h e help given by D r . Kenneth E. Newman w i t h
t h e second Comprehensive Examination.
.
I w o u l d l i k e t o thank D r .
R i c h a r d L. c r o s s " ( D e p a r t m e n t o f
~ i o c h e m i s t r _~S, t a t e U n i v e r s i t y o f N e w York) f o r h i s h e l p f u l
-
d i s c u s s i o n s and s u g g e s t i o n s on t h i s project,,
-
1
G
'
I
I would l i k e t o e x p r e s s my d e ~ pa p p r e c i a t i o n and t h a n k s
,
t o t h e t e c h n i c i a n s , D e n i s e 1ul.k.
Moennich, and M i l d r e d E . L .
4
.
J o h n s o n , f o r $'heir i n v a f u a b l e h e l p i n t h i s p r o j e c t .
S u s a n Moore,
a l s o l i k e t o acknowledge t h e h e l p g i v e n by D r .
Amani Nour-Eldeen,
I would
T;?nia K a s t e l i c , Marcia C r a i g , E l i z a b e t h
b
B r a m h a l l , and Kevin D o h e r t y , e s p e c i a l l y i n t h e b e e f h e a r t m i t o chondrial preparatio-.
I n a d d i t i o n , t h e camaraderie, cheer-
f u l n e s s , good humour and t o l e r a n c e of t h e p e r s o n n e l m e n t i o n e d
i n t h i s paragraph w e r e appreciated.
My t h a n k s t o Marion J a c q u e s f o r t y p i n g t h i s t h e s i s . Thanks t o my f r i e n d s '
-
L a t i f Ayub and p h i l i p W h i t i n g ,
b r o t h e r s , and mother f o r t h e i r u n f a i l i n g e f f o r t s t o e n c o u r a g e
1
me.
I
S p e c i a l t h a n k s t o P a t r i c i a and J o e O r d f o r t h e i r k i n d n e s s ,
f r i e n d s h i p , and h o s p i t a l i t y t o w a r d s Martha and m y s e l f .
h'
0
L a s t l y , I would l i k e t o t ank Martha, my w i f e ,
.f o r
.
h
- -e r
u n d e r s t a n d i n g , h e l p , e n c o u r a g e m e n t , c o m p a n i o n s h i p , and l o v e
throughdut t h i s endeavour.
TABLE OF CONTENTS
1:
Paqe
................................................ iii
DEDICATION ...............................................
v
ACKNOWLEDGEMENTS ........................................ vi
TABLE OF CONTENTS ........................................ vi
LIST OF TABLES ...............*.......................... xii
LIST OF FIGUKES ......................................... x i J
LIST OF ABBREVIATIONS ...................................
xx
: INTRODUCTION .........................L............
.1
ABSTRACT
-.
- -
-
%+>
k ,;
:'4
- *L
-We-
v*.
n
$;&
-.,,R
.:
iu-,
t
.
h
$2
:
,-*++'-
,
(i)
Beef Heart Mitochondria1 Adenosine
. .,
,
I
a
Triphosphatase
G9
'+ .
.i
(ii)
............................
-.
1
Modified Sephadex Centrifuge Column
.................................
EXPERIMENTAL PROCEDURES ............................
(1)
Materials .................................
(ii)
Methods ...................................
Technique
14
C4
.
(a)
18
18
I
20
Preparation of Soluble Beef Heart
Mitochondria1 Adenosine
...........
*
for ATPase Activity[ ...........
,
Triphosphatase, F1-ATPase
20
(b)
Assay
21
(c)
Determination of Protein
.......................
Weight ......;. .............
the F1 Preparation ........
concentration
(d)
Molecular
21
23
**
(e)
Purity of
23
i
4
C
(f)
(9)
J
P u r i t y of t h e Radion c l e o t i d e s
........ 23
.
D
ATP and/or ADP
.i~onitor.i-ng
.......................
Measurement of Radioactivity .........
I
Preparation of Sephadex Centrifuge ...
Making a Longer Column Barrel ........
Concentrations
(h)
(i)
(j)
24
24
25
25
B
(k)
Modified Sephadex Centrifuge Column
Preincubation Mode
(b)
Pulse-Chase Mode
ReLease of P i frpm F1:
i
Method
............... 26
................. 28.
(a)
-
~reinaubation
......................................
i
.
Release of P i from F1:
30
Pulse-Chase
~ e l e a s ' eof AMPPNP from F l :
Pulse-Chase
.....................................
. /
Method
44
+
Binding
of Nucleotides t o F 1 i n t h e
-
.................
The Biphasic Release of P i from F1 ..... .I
DISCUSSION .......................................
~ e p h a d e xCentrifuge Column
IV
(i)
59
73
,/
The Release of P i from F1 :
Prsincubation Method
46
&
----I'
.................
.....
;
73
Page
A P P E N D I X 11
.......$.................................1 2 0
6
.1.
1
~ d a ~ t a t i oofn t h e Modified S e p h a d e x C e n t r i f u g e
'
I
Column.Technique f o r U s e i n Pulse-Chase
......................................... 1 2 0
1, R e s u l t s a n d discussion ...................... i 2 0
~xperigents
i
hi)
(a)
T h e ~ r r a n ~ e m e no ft t h e G e l ' s f o r
Pulse-Chase
(b)
Experiments
..I............ 1 2 0
D e t e r m i n a t i o n of. t h e Amount o f . ATP
7
t o U s e i n t h e Top o r P u l s e S e c t i o n
........................ 1 2 0
.................................;. 1 2 8
o f t h e Column
(ii)
Conclusion
,.
-
xii
-
LIST O F TABLES
React i o n s o f O x i d a t i v e ~ h o s p h o r y l a t i o nf o r
T a b l e 11,
The C o u p l e d Enzyme
.....................
A s s a y S y s t e m ..........
T a b l e 111
T h e B i n d i n g o f AX'P
i n t h e Column t o F1
w h i c h F1 i s R e q u i r e d
4
%
T a b l e I.V
.
...
The B i n d i n g b f ATP i n t h e Column t o F,
a n d t h e Release o f P i from F1
.........
The B i n d i n g o f C h a s e ATP i n t h e Column
to F1
...................................
+
.
Table V I
The B i n d i n g ok C h a s e AMPPNP i n t h e Column
Table I A
The q f f e c t o f
if f e r e n t L e n g t h s o f B u f f e r -
~ ~ u i l i b r a t eG de l s o n t h e Removal o f Label
from t h e R e a c t i o n Mixtures
Table I I A
..'............
The E f f e c t o f T o t a l L e n g t h o f t h e Column
o n t h e Removql o f L a b e l f r o m t h e R e a c t i o n
Mixtures
................................
.,
Table I I I A
'
.
T h d f f e c t o f ATP o n t h e Release o f S P i
f r o m FI when b i f f e r e n t Amounts o f P r o t e i n
w e r e u s e d i n $he R e a s t i o n M i x t u r e
-
,
/
i
-'
. , . .: . .
'117
-
Table .I'VA
xiii
-
The E f f e c t of Chase ATP on t h e Release
i
of Label Bound when F1 was Passed
-- Through
P u l s e Sections with D i f f e r e n t
Concentrations of ATP
...................
4
i
*
-
125
LIST OF F I G U R E S
F i g u r e I,
T h e e f f e c t s of ATP,
.
Figure 2
h
ADP,
t h e r e l e a s e of b o u n d P i f r o m F1
*
%
0
T h e e f f e c t s o f ADP, AMPPNP,
'
*
'
t,he r e l e a s e . o f P i f r o m F1
0
-
a n d AMPPNP o n
.
0
.......
31
a n d ATP an
.............
32
'
I
Figure 3
'
$
T h e e f f e c t of ,ATP o n t h e r e l e a s e , o f P i
I
<
from F1
......... ;......................*
4
.
T h e e f f e c t o f . A T P on' the release
Figure
J-3
f r o m ~1
34
%
'
.,
....... ..........................
4
*
35a.
T h e e f f e c t o f ADP o n t h e r e l e a s e o f P i
'Figure 5
E
f r o m F1
i
.................................
36
f
'dF i g u r e
-
6
i7-,
.,-'
C o m p a r i s o n of t h e e f f e c t s a f ATP,
in
Xr
p r e i n c u b a t i o n and pulse-chase
/
t h e r e l e a s e of l a b e l f r o m F1
modes;
I
on
............
39
TP on the- r e l e a s e of
,
Figure 8A
l a b e l bouhd when F1 was passed tfirousgh a
.................
10 pM ATP pulse s e c t i o n
Figure 9A
9's.
122
The e f f e c t of chase ATP on t h e r e l e a s e of
l a b e l bound when F1 was passed through a
t
10 pM ATP pulse s e c t i o n i . . . . . . . . . . . . . . . .
Figure 10A
hi'
124
e f f e c t o • ’chase ~ T Pon t h e r e l e a s e - o f
, label
bound when F1 was passed through
pulse s e c t i o n s with d i f f e r e n t
...................
I
concentrations of ATP
126
*
ADP
,-;.
LIST OF ABBREVIATIONS
Adenosine 5 ' -dip$sphate
.
ATP
~ d e n o s i n e5 '
AMPPNP
5'
GTP
Guanosi,ne 5 ' -tri~hosphate
ITP
Ionosine 5 ' -triph$sphate
I\. E
%U'
,,
-%-
,)I
,\
:
PK
PEP
P y r ~ v a t eKinase
.
Phosphoencd pyruvate
t
LDH
Lactate Dehydrogenase
p-NADH
@-Nicotinamide adenine dinucleotide
Pi
SDS
(-inorganic
phosphate
,
i
-&@kmr
dodebyl sulphate
;2',3'-0-(2,4,6-trihitrophenyl) adenosine
TNP-ADP
'
5 ' -diphosphate
2'". 3 ' -0-(2,4,6-trinitropheny-l) adenosine
TNP-ATg
%
5 ' -ifiphosphate
Introduction
In this research project the dissociation of bound molet
cules (ATP, ADY, AMPPNP, and Pi) from beef heart mitochondria1
adenosine triphosphatase was studied in the presence of other
4
'
molecules (ATP, ADP, AMPPNP, and Pi).
These studies were
performed with the modified Sephadex centrifige c~lumn~technique
described here.
(1) Beef Heart Mitochondria1 Adenosine Triphosphatase
a
Mitochondria1 adenosine triphosphatase (ATPase
-
E.C.
3.b.1.3) can be separated to give two fundamental units:
one, a
water-soluble unit, displays ATP hydrolytic activity; and the
other, a water-insoluble "~embr'anesector", is without ATP
hydrolytic activity, but is able to alter the
ties of the soluble ATPase (for appropriate
-
h
18).
The catalytically active soluble
referred to as F1-ATPase, or ,simply as F1; whereas the membrdne
sector of the complex is referred to as F o ,
plex (or membran
7-bound
,
intact com-
form of the enzyme) as ATPase (8) -
-
other
names include FIFo -ATPase, H + - A T P ~ S ~ATP
.
synthase. and
r ;
Complex V (19,20,21).
These two fractions were first recognized
by Racker and .co-workers (22,23) as playing essen,tial roles in
-
the coupling of oxidation to phosphorylation (see also Refs.
19,24).
Table I summarizes the reactions (or processes) in
which Fr is known to be involved (7).
,,
In the investigations
-.
Table I
f o r w h i c h Fl i s r e q u i r e d
Reactions of bOxidative phosihorylation
1.
ATP s y n t h e s i s
2.
ATP hydrolysis- _
(3.
Exchange r e a c t i o n s :
ATP
(c)
4.
~~~~0
ATP-dependent
ATP.
+
3
2
i
~2
ATP ( y - 3 2 ~ )
A T P + H 2 1 8 04-+
r e a c t i o n s of o x i d a t i v e
phosphorylation:
(a)
Reversal of t h e r e s p i r a t o r y .chains
Succinate
+
NAD+
...
(b)
Ion t r a n s p o r t .
(Taken from Ref.
7. )
Fumarate
+
NADH
+
fi+
+
P
i
b
r e p o r t e d h e r e , o n l y t h e ATP h y d r o l y t i c a c t i v i t y o f t h e @enzyme
was u t i l i z e d , and s o l u b l e b e e f h e a r t m i t o c h ~ n d r ~ i aa ld e n o 2 i n e
t r i p h o s p h a t a s e , Fl
, w a s used.
Q
A l t h o u g h t h i s eizyme
has b e e n i n t e n s i v e l y s t u d i e d ( t h e r e
~~
*
h a s b e e n a n i n c r e a s e i n A T P a s e p u b l i c a t i o n s from a b o u t 500 p e r *
y e a r i n t h e e a r l y s e v e n t i e s t o o v e r 1500 p e r y e a r i n t h e e a r l y
e i g h t i e s ( 1 3 ) ) by many ' w o r k e r s from d i f f e r e n t l a b o r a t o r i e s ,
7
t h e r e s t i l l r e m a i n s e v e r a l d e f i c i e n c i e s i n o u r k n o w l e d g e o f F1
( - 9 ) .
--
What r e m a i n s most e l u s i v e i s a n u n d e r s t a n d i n g o f t h e
r e s c t i o n bmechanism o f F1
.
r
No d o u b t some o f t h e ' c u r r e n t
uncer-
t a i n t i e s c o n c e r n i n g t h e r e a c t i o n mechanism r e s u l t from t h e
c o m p l e x i t y o r t h e p a t h w a y s o f ATP h y d r o l y s i s by F1 ('17.25).
Some o f t h e p r o p e r t i e s o f F1 t h a t must be c o n s i d e r e d when
s t u d y i n g t h i s c o l d - l a b i l e enzyirle, i n c l u d e i t s s p e c i f i c a c t i v i t y ,
m o l e c u l a r w e i g h t , and s t r u c t u r e .
I n addition,
i n these p a r t i c u -
-
l a r i n v e s t i g a t i o n s w i t h F1 r e p o r t e d here, some knowledge o f t h e
t i g h t l y bound n u c l e o t i d e s .and i n o r g a n i c p h o s p h a t e ( e g . t h e i r
number and p o s s i b l e r o l e ( s ) ) , w a s h e l p f u l ( 4 , 7 , 1 0 ) .
The s p e c i i f i c a c t i v i t y of F1 seems t o d e p e n d o n ' i t s h i s t o r y ,
which i s i n f l u e n c e d by t h e
of preparation,
chondria,
(1)s o u r c e
( i i ) method o f p r e p a r a t i o n o f F1 f r o m t h e m i t o -
( i i i ) method o f sto;age
t i o n s o f F1 ( 7 ) .
o f m i t o c h o n d r i a a n d method
o f F 1 , and ( i v ) a s s a y c o n d i -
I n t h e i n v e s t i g a t i o n s d e s c r i b e d here, b e e f
h e a r t m i t o c h o n d r i a .were p r e p a r e d by t h e m o d i f i e d method o f S m i t h
C
( 2 6 ) ; and t h e
P1
prepared a c c o r d i n g t o t h e method o u t l i n e d by
~ a
P e n e f s k y ( 2 7 , 2 8 ) . * T h e p u r i f i e d F1 w a s s t o r e d a t 4 " as
\
-
s u s p e n s i o n i n ammonium s u l p h a t e ( 2 , 2 9 ) .
T h e F1-ATPase
assay
p r o c e d u r e w a s t h e r e g e n e r a t i n g s y s t e m a s s a y ( 2 , 3 0 1 c o u p l e d -to
t h e o x i d a t i o n o f NADH ( 3 1 ) ( d e t a i l s are g i v e n i n t h e
. .+
a n d Methods s e c t i o n )
.
ater rials
d
'b
D i f f e r e n t m o l e c u l a r w e i g h t s ( r a n g i n g from 310,000 t o
400,000)s.
d e t e r m i n e d by d i f f e r e n t t e c h n i q u e s , h a v e b e e n r e p o r t e d
f o r F1 f r o m v a r i o u s s o u r c e s .
However,
f o r beef h e a r t
m i t o c h o n d r i a 1 F 1 , t h e a c c e p t e d m o l e c u l a r w e i g h t v a l u e s seem t o
be 347,000 ( d e t e r m i n e d by e q u i l i b r i u m s e d i m e n t a t i o n u l t r a c e n t r i -
8'
f u g q t i o n ( 3 2 ) ) a n d 3 6 0 , 0 0 0 ( d e t e r m i n e d by g e l ' f i l t r a t i o n ( 3 3 ) )
The . s t r u c t u r e o f t h i s m u l t i - s u b u n i t
(1,3,5,7,11,1).
(a,
p,
y, 6 ,
and
E)
enzyme, F 1 ,
.
is complex
T h e r e are f i v e d i f f e r e n t r e c o g n i z e d s u b u n i t s
o f F1 , w h i c h h a v e b e e n d e t e r m i n e d a n d c h a r a c -
t e r i z e d b y s o d i u m d o d e c y l s u l p h a t e , SDS, g e l e l e c t r o p h o r e s i s ,
gel filtration,
a m i n o a c i d a n a l y s i s , and e q u i l i b r i u m s e d i -
mentation s t u d i e s (32,34,
35).
A l l t h e approaches gave similar
ib
5
t h e molecular weight of each t y p e of subunit (54,000,
..
1 7 , 5 0 0 a n d 5 , 7 0 0 3 . ~ e x c e p t t h e SDS g e l e l e c t r o p h o r e s i s s t u d i e s w h i c h g a v e v a l a e S higher or lower t h a n t h o s e
o b t a i n e d by t h e b t h e r methods.
However, t h e e x a c t s t ~ i c h i o r n e t r ~
o f t h e s u b u n i t c o m p o s i t i o n o f F1 i s s t i l l u n c l e a r ,
i n v e s t i g a t i o n s i n v o l v i n g d i f f e r e n t methods,
.
I
e .g.,
despite
SDS g e l
t
/
using corrected molecular
electrophoresis (
g measurement ( 3 7 ) , a n d c h e m i c a l
weight ( 3 6 ) ,
/
cross-14inkjng s t u d i e s ( 3 8 ) .
The c h o i c e s f o r t h e s u b u n i t
@
n a t i o n s ) ; however, e v i d e n c e h a s accumulated i n f a v o u r of
3 $ 3 y 6 ~s u b u n i t c o m p o s i t i o n ( 1 , 3 , 4 , 7 , 8 , 1 0 , 1 7 , 2 1 ) .
\
$ h i s enzyme
on i s o l a t i o n c o n t a i n s " t i g h t l y bound" a d e n i n e
.
.
8
' n u c l e o t i d e s (AI'P a n d / o r A D P )
Fl
- t h e s e $re bound n o n - c o v a l e n t l y t o
, s i n c e t h e y . are l o s t
a r e c o n s i d e r e d t o be
-0
despite
ow ex
"tightly
~.
-%t i o n
lusiferase (41).
a t i o n of
5
t
w i t h EDTA i n a low i o n i c s t r e n 3 t h
I n a d d i t i o n , t i g h t l y boun4 ATP m o l e c u l e s a r e i n -
accesible t o A T P - u t i l i z i n g
enzymes s u c h as h e x o k i n a s e o r
T h e s e n u c l e o t i d e s a r e r e l e a s e d on t h e d e n ~ t u r -
by c o l d ( 0 " ~ or
)
acid treatment (e.9.
non-denaturing
4% H C 1 0 , )
..
A
way of removing t h e " t i g h t l y bound" n u c l e o t i d e s
from F1 i s t o p a s s t h e enzyme t h r o u g h a column o f S e p h a d e x G-25,
which h a s been p r e - e q u i l i b r a t e d
w i t h ' EDTA i n t h e a p p ~ p r i a t e
buffer of high ionia strength (42,43).
The e x a c t number of " t i g h t l y bound" a d e n i n e n u c l e o t i d e s p e r
m o l e of F
. .,
i s unknown, t h e numbers r e p o r t e d v a r y , e g
( 0 ATP and 2
mole
( 4 0 ) , and 2 ATP and 1
F1 ( 3 4 ) ) ,
mol mole
I
bound" i f t h e y a r e r e t a i n e d by F l
or c h a r c o a l t r e a t m e n t , ammonium s u l p h a t e p r e c i p i -
tation, or gel f i l t
buffer (40).
t u r a t i o n o f F1 (3w. N u c P e o t i d e s
t h r e e (1 ATP and 2
F1
( 3 3 ) ),
two
mole
F1
and f i v e ( 3 ATP and 2
--
-
6
-*
~ ~ ~ / m F1
o l (e3 2 ) ) n u c l e o t i d e s
-
(7,17,41,45,46).
?
G a r r e t t and
P e n e f s k y ('47) h a v e s u g g e s t e d t h a t f i v e n u c l e o t i d e b i n d i n g ' - s i t e s
.
are- f o u n d on E'l : @!hree o f t h e s e c o n t a i n " t i g h t l y bound" nucleo-r
t i d e s and t h e o t h e r t w o s i t e s ' p a r t i c i p a t e 'in r a p i d l y r e v e r s i b l e
b i n d i n g o f added a d e n i n e n u c l e o t i d e s .
C r o s s .and ~ d l i b(48)
n
have
found s i x n u c l e o t i d e b i n d i n g s i t e s on Fl
p a t e i n non-exchangeable
,
,'
t h r e e s i t e s -' p a r t i c i -
b i n d i n g and t h r e e s i t e s i n exchangeable
3
-
b i n d i n g . , T h i s v a r i a t i o n i n a m o u n t s / n u m b e r s < o f " t i g h t l y bound"
n u c l e o t i d e a s w e l l as t o t a l bound n u c l e o t i d e p e r mole o f F1 .'frb~n
d i f f e r e n t l a b o r a t o r i e s i s p r o b a b l y d e p e n d e n t o n t h e h i s t o r y of
t h e enzyme ( 7 )
.
The s i t e s o f t h e t i g h t b i n d i n g o f a d e n i n e n u c & e o t i d e s o n F,
a r e a l s o u n c e r t a i n ( 4 1 , 4 5 ) , t h o u g h most l i k e l y t h e a and p s u b u n i t s a r e involved.
8-azido-ATP
.
Studies w i t h photoaffinity labels, e.g.,
a n d 8-azido-ADP,
h a v e shown t h a t t h e s e s u b u n i t s a r e
6
involved i n n u c l e o t i d e binding (49-52).
The
- . r o l e of t h e sub-
u n i t s h a s .been s t u d i e d w i t h a n t i b o d i e s , c h e m i c a l m o d i f i c a t i o n ,
partial proteolysis,
r e c o n s t i t u t i o r ~ , binding,
l a b e l l i n g experi%ents.
and p Q o t o a f f i n i t y
'
a
The n o n c a t a l y t i c nuc l e o t i d e b i n d i n g
s i t e s a r e t h o u g h t t o be o n t h e
subunits (48,53-55),
' c a t a l y t i c s i t e s o n t h e $ s u b u n i t s (45,56-59):
and t h e
---
k'or example,
K o z l o v a n d Milgrom p r o v i d e d s t r o n g e v i d e n c e when t h e y showed
t h a t t h e c o v a l e n t b i n d i n g o f t h e d i a l d e h y d e d e r i v a t i v e o f ADP
(OXADP,
f o r m e d as a r e s u l t o f t r e a t m e n t o f ADP w i t h p e r i o d a t e )
4
P
'
,
t o t h e a s u b u n i t o f Fl o c ciu r r e d w i t h o u t l o s s i n t h e h y d r o l y t i c
/
\
4
a c t i v i t y o f F1 ( 5 4 ) .
I n a d d i t i o n , Dunn a n d F u t a i ,
$
in their
s t u d i e s with i s o l a t e d subunits-of
ATPase,
f o u n d by e q u i l i b r i u m
a subunit (not isolated p,
[4,
1
-
3
~
p
]
~
8 - ~ H ] A T or
P
y,
(approximately'
~
~
"I'heir r e s u l t s s u g g e s t e d t h a t e a c h a s u b u n i t c o n t a i n s a s i n g l e
t i g h t nucleotide binding site.
I t m u s t be n o t e d t h a t Kagawa a n d
4
co-workers
-
found i n t h e i r c i r c u l a r d i c h r o i s m s t u d i e s t h a t b o t h
i s o l a t e d a and 6 s u b u n i t s o f t h e t h e r m o p h i l i c b a c t e r i u m P S 3
bound A D Y a n d ATP ( 6 0 )
.
However,
it w a s f o u n d by H a r r i s et al.
i n t h e i r s t u d i e s w i t h b e e f , h e a r t m i t o c h o n d r i a 1 ATPase t h a t ' o n l y
15-30% o f t h e bound n u c l e o t i d e h a d e x c h a n g e d w i t h l a b e l l e d
medium n u c l e o t i d e ( L 3 ~ ] ~ l ' opr L 3 ~ ] ~ ~ epv )e n, a f t e r 24 h o u r s i n
t h e p r e s e n c e o f klg2+ ( 3 9 , 4 1 ' ) ; t h u s t h e p o s s i b i l i t y o f t h e a c t i v e
i n v o l v e m e n t o f t h e t i g h t n u c l e o t i d e b i n d i n g s i t e s i n t h e catal y t i c mechanism o f F 1 i s r e s t r i c t e d ( 4 , 1 0 , 2 4 , 3 9 , 4 1 ) .
The
n u c l e o t i d e s bound a t t h e n o n c a t a l y t i c s i t e s o f t h e a s u b u n i t s '
'
may b e s e r v i n g a s t r u c t u r a l ( t h e n u c l e o t i d e AT? i s r e q u i r e d f o r
C
t h e r e c o n s t i t u t i o n o f F1 f r o m t h e t h r e e major s u b u n i t s a ,
( 5 3 ) ) o r . r e g u l a t o r y role ( 4 1 , 4 8 , 5 5 ) .
t h a t t.he b i n d i n g o f ADP t o t h e
p , and
Ohta et. al. thought
s u b u n i t s o f TF1 ( t h e r m o s t a b l e
F 1 - s o l u b l e ATPase f r o m t h e r m o p h i l i c b a c t e r i u m P S ~ )w a s r e g u -
--r
l a t e d b y t h e b i a d i n g o f AUP t o t h e a s u b u n i t s o f TF1, p o s s i b l y
through a l l o s t e r i c i n t e r a c t i o n s ( 6 0 ) .
,?
The n u c l e o t i d e s bound a t t h e p s u b u n i t s a r e t h o u g h t t o exc h a n g e r a p i d l y w i t h t h e medium n u c l e c t i d e s , t h e r e b y r e f l e c t i w
*
t h e process of t h e s u b s t r a t e binding a t t h e c a t a l y t i c sites (48, ,
60).
C r o s s and N a l i n i n t h e i r s t u d i e s w i t h 5 ' - a d e n y l y l - @ , y -
i m i d o d i p h o s p h a t e (AMPPNP, a n o n - h y d r o l y s a b l e
a n a l o g o f ATP i n
i
which t h e oxygen b r i d g e between t h e
@
and y-phosphorus atoms h a s
b e e n r e p l a c e d by a NH g r o u p ) were a b l e t o d e m o n s t r a t e # t h a t t h r e e
s
exchangeable n u c l e o t i d e binding sites (probably @-subunits) are
t+
\
and t h a t t h e s e sites are
mitochondria1
from t h r e e
.
/
noncatalytic
s-i . I ' s
(probably a - ~ ~ b "
Grubmeyer a n d p & n e f s k y u s e d t h e r i b o s e - m o d i f i e d
nucleotides 2',3'-0-(2,4,6-tr
P.
itrophenyl) adenosine 5'-triphos-
p h a t e (TNP-ATP) a n d TNP-ADP i n t h e i r s t u d i e s and showed t h e
-.
p r e s e n c g o f t w o h i g h a f f i n i t y n u c l e o t i d e b i n d i n g s i t e s on
-
-
-
'
-
The r o l e o f t h e n u c l e o t i d e b i n d i n g s i t e s i n t h e mechanism
I
I
of m P t o c h o n d r i a 1 ATPase i s au a r e a o f a c t i t r e r e s e a r c h ( s e e a l s o
I
J
Refs'.
63-68).
he m u l t i p l i c i t y o f ' , b i n d i n g s i t e s r a i s e s some
<-
/
,,
-
---.
very i n t e r e s t i n g questions,
-
1
binding'.si&*
1
e.g.
-
,.
")ow
m&y
are involved'in the &talytic
of t h e nicleoticle
a c t i v i t y of t h e
\
P e r h a p s F,&wt
.---
enzyme?"/(7).
f
,
w h e t h e r or n o t t . 4 , ' 7 $ # 1 t "
i n v o l v e d in!'the
-3
,
'
d i r e c t method o f d e t e r m i n i n g
n u c l e o t i d e b i n d i n g s i t e s on F 1 a r e
ATp l n y & o l y t i c
\
r e a c t i o n is t o determine t h e
*
1.
-\
,/
t u r n o v e r k a t e o f n u c l e o t i d e s bound, t o t h e s e s i t e s ( 6 9 , 7 O )
However,
",
4s
1
p o i @ t e d o u t by Boyer a n d c o - w o r k e r s ,
.
their alterna-
.
t i v e non-destructive
a p p r o a c h e s ( f i l t e r b i n d i n g , EDTA q u e n c h i n g
a n d hexokinase-accessibiiity p r o c e -
i n r a p i d mixing experiments,
d u r e ) h a v e t h e i r own l i m i t a t i o n s ( 6 9 , 7 0 ) .
I t is undetermined
w h e t h e r . o r n o t m o s t o f t h e bound n u c l e o t i d e s a r e c a t a l y t i c
intermediates.
The r o l e o f t h e t i g h t l y bound n u c l e o t i d e s i n t h e
mechaniirn o f t h e F 19
-catalysed
hydrolysis
et. al.
a s p o i n t e d o u t by T i e g e -
af ATP
i s a l s o uncklear.
s using fluores-
in their
il
cence. t e c h n i q u e s a n d i s o t o p e
a n a l o g u e s (65),=--Penefsky
binding
ADP a n d ADP
a
( 7 ) and o t h e r s (S4,S9,63-68,7l172)
',
b e l i e v e t h a f t h e s e s h e s , i n s o m e way, d o p a r t i c i p a t e i n t h e
/
a
f u n c t i o n i y g o f t h e enzyme.
Inorganic phosphate, P i ,
- /
h y d r o l y s e s ATP.
i s o n e o f t h e p r o d u c t s when F1
F1 a l s o h a s a t l e a s t o n e b i n d i n g s i t e f o r P i
IS
(73).
~ a s a h a r aa n d P e n e f s k y ( 7 4 , 7 5 ) h a v e d e m o n s t r a t e d , u n d e r
c e r t a i n c o n d i t i o n s ( ~ n a~n d+ a u r o v e r t i n [ a n a n t i b i o t i c u s e d as a
f l u o r e s c e n t p r o b e of c o n f o r m a t i o n a l c h a n g e ] ) ,
t h e presence of
4
t w o t y p e s of P i b i n d i n g
- a high a f f i n i t y saturable binding,
and a second l o w a f f i n i t y n o n s a t u r a b l e b i n d i n g .
Their studies
on t h e b i n d i n g of P i by F1 showed a pH d e p e n d e n c e , a n d c o m p e t i t i v e i n h i b i t i o n b y t h e p i a n a l o g , t h i ~ h o s p h o r i ca c i d ; t h l s b
f i n d i n g s l e d t h e m t o s u g g e s t t h a t ' t h e monoanion i s t h e c h a r g e d
f o r m o f P i w h i c h i s bound by F1.
was shown t o be i ; f l u e n c e d
The b i n d i n g of P i by F1
by t h e s t i m u l a t o r s ( e . g .
divalent
"&
metal i o n s [ M ~ ~ + co2+,
,
ate],
and i n h i b i t o r s
ca2+] a n d
oxyanions [chromate,
,
bicarbon-
( e .g . i n h i b i t o r p r o t e i n , e f r a p e p t i n ) o f
.J
ATPase a c t i v i t y a n d of o x i d a t i v e p h o s p h o r y l a t i o n
(73,75)'.
They
o b s e r v e d t h a t t h e ATP a n a l o g AMPPNP i n h i b i t e d P i b i n d i n g ever1
--
i n t h e presence ~f aurovertin,
add s u g g e s t e d it was l l k e l y t h a t
t h e P i w a s b i n d i n g a t a s i t e o c c u p i e d by t h e y - p h o s p h a t e
o f ATP ( 7 4 ) .
group
Thus t h e r e is a s t r o n g p o s s i b i l i t y t h a t . t h e P i
b i n d i n g s i t e c a n p l a y a major role I n o x i d a t i v e p h o s p h o r y l a t i o n .
et. a l . i n t h e i r s t u d i e s u s e d t h e new p h o t o a f f i ~ l i t y
Lauquia d e r i v a t i v e of i n o r g a n i c phosphate, f4-azido-2-nitrophenyl
phate
(ANPP),
h
phos-
t o d e t i r m i n e t h e P i b i n d i n g s i t e ( s , ) on i s o l a t e d
F1 a n d i n s i d e - o u t
p a r t i c l e s . f r o m beef h e a r t mitochondria
(76).
They f o u n d t h a t P i was m o s t p r o b a b l y b i n d i n g t o t h e @ - s u b u n i t
i
o f F1-ATPase,
and t h e P i c a r r i e r p r o t e i n .
T h i s work ( 7 6 )
t h e r e f o r e l e n d s s u p p o r t t o t h e p r o p o s a l s made by K a s a h a r a and
-*
I
Penefsky (73-75).
,
b
A s s e e n from t h e s e v e r a l r e v i e w s ,
t h e mechanism o f t h e
~ ~ ~ a s e - c a t a l y s reeda c t i o n i s s t i l l n o t w e l l u n d e r s t o o d (1-18,
77-83).
The rna:.n e n e r g y - r e q u i r i n g
s t e p s i n o x i d a t i v e phosphory-
-e
l a t i o n and p h o t o p h o s p h o r y l a t i o n ( 1 0 , 2 4 ) a r e :
A'PP F r o m ATPase ( 8 4 - 8 7 ) ,
ATPase ( 8 8 - 9 0 )
;
( i )t h e r e l e a s e o f
a n d ( i i ) t h e b i n d i n g o f ADP and P i t o
v
evidence f o r these conclusions w e r e f i r s t
o b t a i n e d from i s o t o p e e x c h a n d s t u d i e s .
*,-,
ddition, there
s e e m s t o b e c o o p e r a t i v e i n t e r a c t i o n b e t w e e n 'the d u b u n i t s of
ATPase.
i
v
F o r e x a m p l e , A d o l f s e n and M o u d r i a n a k i s showed t h a t t h e
r a t e o f d i s s o c i a t i o n o f bound ADP from 1 3 s c o u p l i n g f a c t o r
'e'
A l c a l i q e n e s f a e c a i i s w a s i n c r e a s e d on therp a d d i t i o n o f n u c l e o t d e
\
N
--
t o ~&,&dium
(92).
They p r o p o s e d t h a t t h e b i n d i n g o f n u c l e o J
t i d e t o one s i t e o f t h e 1 3 s c o u p l i n g f a c t o r c a u s e d a conformat i o n a l change which f a c i l i t a t e d t h e d i s s o c i a t i o n o f bound
n u c l e o t i d e a t t h e o t h e r s i t e . ' T h e s e w o r k e r s , however, d i d n o t
show w h e t h e r o r n o t c a t a l y t i c s i t e s were i n v o l v e d ( 1 0 , 9 2 , 9 3 ) .
The a l t e r n a t i n g c a t a l y t i c s i t e model o f Boyer a d co-
/"
w o r k e r s ( 2 4 , 9 3 - 9 6 ) was p r o p o s e d on t h e b a s i s o f i s o t o p e exchange
experiments.
h
T h i s model hag rlow e v o l v e d i n t o w h a t
" b i n d i n g change mechanism" (10.13.97-99)
is
called the
which i n c o r p o r a t e s t h e ,
c o o p e r a t i v e i n t e r a c t i o n s between t h e s u b u n i t s and t h e e n e r g y d e p e n d e n t b i n d i n g c h a n g e s ( b i n d i n g of AQP and P i , and release
;4
d f ATP i n n e t s y n t h e s i s , , a n d v i c e v e r s a i n n e t h y d r o l y s i s ) .
.,
In
t h i s model, d u r i n g n e t o x i d a t i v e p h o s p h o r y l a t i o n , ATP i s p r o d u c e d a t one s i t e on t h e ATP s y n t h a s e where it i s t r a n s i t q r i l y
t ' g h t l y bound, and i s o n l y r e l e a s e d when ADP and P i b i n d a t a
4
s e c o n d s i t e , and t h e membrane ATPase complex i s e n e r g i s e d .
S i m i l a r l y , ' u n d e r c o n d i t i o n s o f n e t h y d r o l y s i s , ATP b i n d i n g
o n e s i t e i s accompanied by t h e r e l e a s e o f t h e t r a n s i t o r i l y
t i g h t l y bound ADP and P i ( h y d r o l y s e d A T P ) a t a s e c o n d s i t e .
T h i s b i n d i n g c h a n g e mechanism i s a t t r a c t i v e , e s p e c i a l l y sin'ce it
c a n accommodate a wide r a n g e o f e x p e r i m e n t a l o b s e r v a t i o n s ( 2 4 ) .
E v i d e n c e i n s u p p o r t of t h i s mechanism h a s b e e n f o r t h c o m i n g from
d i f f e r e n t s o u r c e s (10,61,62,69,70,97-104). F o r example, s t r ' o n g
I
B
e v i d e n c e ' f o r c o o p e r a t i v i t y b e t w e e n t h e c a t a l y t i c s i t e s o f Fl was
p r e s e n t e d by Grubmeyer aod P e n e f s k y " ( 6 1 , 6 2 ) i n t h e i r i n v e s t i g a t i o n with t h e ribose-modified
n u c l e o t i d k s 2 ' , 3 ' -'o- ( 2 , 4 , 6 - t q i n i -
t r o p h e n y l ) a d e n o s i n e 5' - t r i p h o s p h a t e ,
-e
and TNP-ADP.
( w l t h two b i n d i n g s i t e s f o r TNP-adenine
L
They f o u n d t h a t . F l
i
'
TWP-ATP,
'.
m
n u c l e o t i d e s ) bound b o t h TNP-ATP and TNP-ADP,
?
2
~
]
.
b e~i n g~ h y~ a r o. l y s e d l?y F1
' P . .
.
w i t h t h e TNP;[~
T ~ & Yo b s e r v e d e t h a t on t h e
h
a d d i t i o n o f e x c e s s n o n - z - a d i d a c t i v e TNP-ATP
( s i ff i c i e n t to f ill
t h e s e c o n d c a t a l y t i c s i t e ) t o t h e Fl'reac
fuge column consists of a 1.0 mL tuberculin syringe fitted with
_ a porous polyethylene disc.
equil&rated
with the
is then &nt.,rifuged
The columm packing is Sephadex G-50
appropriate
(1050
x
P
puffer, and the filled column
The enzyme sarfiple .
g for 2 minutes).
is then agplied to the column before the second centrifugation
(1050
x
g for 2 minutes).
The centrifugate is analysed for the
hound ligand, activity and protein concentration of the enzyme.
T h e ~ e ~ h a d ecentrifuge*
x
column
in essentially its
-
original form has been used iri several studies (48,73-75,96,104,
-
110).
~ r o k sand Nalin (.48) int.roduced the use o•’an extension
tube at the topeof the column, which allowed (i) a number of
samples to be indestigated simultaneously, and ( i i ) other addi-
*
I
t i o n s t o be made t o t h e samples j u s t p r i o r t o c e n t r i f u g a t i o n .
Their second modification involved t h e a d d i t i o n of 'bovine serum
'
albumin (BSA) to.samples with very small amounts of F1 '(10 t o 30
p g / r n ~ ) . This f a c i l i t a t e d the g r e a t e r recovery of F1 from t h e
c e n t r i f u g e columns when t h e concentration of F1 was lower than
.
0 . 3 rng/rn~.'
Cardon wrote an evalbation of t h e Sephadex c e n t r i -
fuge column technique i n t h e Appendix of h i s Ph.D.
t h e s i s (ill),
i n which he pointed out the b a s i c assumptions t h a t he made i n
' t h e construction of h i s model t o determine t h e necessary r a t e
.-
constants.
SO
f a r no major evaluation, c r i t i q u e , o r t h e o r e t i c a l
assesment of t h i s teahnique has been published.
P
.
.The Sephadex c e n t r i f u g e colurtin technique was modified t o
--
demonstrate the r e l e a $ e of enzyme-bound s p e c i e s , e.g.
ADP,
P i and
i n the presence of nucleotides ( A T P , ADP, and AMPPNP).
It
*
i s known t h a t FI-ATPase contains a t l e a s t , one binding s i t e f o r
$
Pi
(73-75,109), however, it i s undetermined whether o r not t h e
bound P i i s a c a t a l y t i c intermediate, or whether t h e P i i s
1
.
'
bound, a t a c a t a l y t i c s i t e .
I n t h e absence of nucleotide i n t h e
a-
.
mediuk, the bound P i d i s s o c i a t e s slowly from F1; whereas, 'on.,.
t h e a d d i t i o n af AT,P or ADP t o t h e medium, t h e d i s s o c i a t i o n of
*pi from F1 5 s a c c e i e r a t e d t o t h e point where t h e r e l e a s e i s
too rapid t o observe using conventional techniques ( 9 6 ) .
The
approach t o t h i s problem u s i n g t h e Sephadex c e n t r i f u g e column
technique,
is o u t l i n e d below:
&
.
.( i ) A column w i t h t h r e e section& i s
layer contains nucleotide,
e.g.
ATP.
t h e middle
( i i )T h e s a m p l e o f ' F ~
becomes bound t o F1 as i n d i c a t e d i n t h e f o l l o w i n g e q u a t i o n : I
f i i i ) T h e [ 3 2 ~ ] ~ li a b e l l e d F1 i s a p p l i e d - t o t h e column. which
i n t u r n i s p l a c e d i n t h e c e n t r i f u g e t u b e , and c e n t r i f u g e d a t
1050
x
g f o r 2 minutes;
( i v ) The column c e n t r i f u g a t e i s
analysed as' b e f o r e .
*/
I n t h e f i r s t ( u p p e r m o s t ) l a y e r of t h e column, t h e l o o s e l y
U
h e l d species (-ATPIADP, and P i ) a r e removed.
I n the second
l a y e r ( m i d d l e ) , t h e s u b s t r a t e , e. g . ATP becomes *bound t o t h e F1
A
w i t h o r w i t h ' o u t r a d i o l a b e l l e d P i , and h y d r o l y s i s o f ATP
+
occurs.
L a s t l y , ' i n the third (bottom) l a y e r
&
8
t h e column, t h e
l o o s e l y - b o u n d n u c l e o t i d e and P i r e l e a s e d f r o m F1 a r e removed.
Any t i g h t l y b o u n d ' m o l e c u l e ( n u c l e o t i . d e a n d / o r P i ) c a n be found
i n the centrifugate.
I f L a b e l l e d P i is f o u n d i n t h e ' F1 - c e n t r i f u g a t e ,
it means
t h a t t h e P i r e m a i n s on t h e F1 as a t i g h t l y bodhd mplec,~le; and
-
t h e r e f o r e most l i k e l y d o e s n o t p a r t i c i p a t e as a c a t a l y t i c i n t e r mediate, nor occupies a c a t a l y t i c s i t e .
l a b e l l e d P i i s n o t found
the
On t h e o t h e r ' h a n d , i f
'
-
that on the binding of nucleotide, the Pi is released from F 1 ,
and thus most probably participates as a catalytic intermediate
\
If the dissociation of the bound
or occupies a catalytic site.
Pi can be shown to occu-r w i t\h ~ " o n eenzyme turnover of ATP
'
,
B
P
hydrolysis, then the dissociatXon of the bound Pi can be
4f the catalytic mechanism of ATP
?
judged capable of being part
hydrolysis.
I
In addition to the preincubation experiments outlined
above, the Sephadex centrifuge column technique was us'ed for
performing pulse-chase experiments.
The approach was very
similar to that described above except that the first section
contained the pulse molecules, and the third section the chase
molecules.
Thus two approaches, pr
were used to investigate the effe&
ion and pulse-chase modes,
of the nucleotides ATP, ADP,
and AMPPNP on the release of Pi, ADP, ATF, and AMPPNP from
soluble beef heart mitochondria1 adenosine triphosphatase.
,
'
Experimental Procedures
Materials
All reagents used in these investigations were reagent
grade, ACS, or enzyme grade quality
-
whichever was applicable.
The common laboratory reagents were obtained from the usual
sources, except if otherwise mentioned.
The following chemicals
and enzymes were obtained from Sigma Chemical Co.: 'adenosine
5 ' -triphosphate (disodium salt, Grade IX); adenosine 5 ' -
,
*
diphosphate (disodium salt, Grade IX); adenylyl imidodiphosphate
(tetralithium salt); ammonium sulphate; 1,4-bis-2-(5phenyloxazoy1)-benzene (POPOP); bovine serum albumin; 7-chloro7
2,5-diphenyloxazole (PPO); p-nicotinamide adenine dinucleoti.de,
reduced form (Grade 111, from yeast j; phosphoenol pyruvate;
Sephadex G-50-80 (fine); sucrose; tris (hydroxymethyl) aminomethane; Triton X-114;
hexo$inase (Type C-300, from yeast*\;
L-lactate dehydrogenase (Type XI, from rabbit muscle) ; and
pyruvate kinase (Type 111, from rabbit muscle).
D E A E - S ~ P ~ ~ ~ ~ X
G-50-120 (fine) and Sephadex G-50-80 (fine) were also obtained
I
from Pharmacia.
The 1.0 mL tuberculin syringes were from Mandel
L
Scientific Co.
The polyethyleneimine thin layer chromatography
I
plates were from E. Merck Co.
0
The radiochemicals:
[ 2 , 8 - 3 ~ ].adenosine 5'-triphosphate
(ammonium salt); and adenosine 5 ' - [ y - 3 2 ~ ]
triphosphate (triethyl
ammonium s a l t ) w e r e f r o m Amersham C o r p .
[ 2 , 8 - 3 ~ ] adenosine 5 ' -
diphosphate ( t r i s o d i u m s a l t ) w a s f r o m New E n g l a n d N u c l e a r C o .
[2,
141 a d e n y l y l 5 ' - i m i d o d i p h o s p h a t e
C h e m i c a l and R a d - i o i s o t o p e D i v i s i o n .
w a s obtained f r o m ICN,
Phosphorus-32
( a s ortho-
phosphate i n d i l u t e H C 1 s o l u t i o ~ ,p H 2 . 3 ) w a s p u r c h a s e d f r o m
both A m e r s h a m C o r p .
and New E n g l a n d N u c l e a r C o .
j
Methods
~reparakionof S o l u b l e B e e f Heart Mitochondrial4Adenosine
Triphosphatase, Fl rATPase
F'resh b e e f h e a r t s w e r e o b t a i n e d f r o m , a l o c a l s l a u g h t e ; I
h o u s e i n S u r r e y , B r i t i s h Columbia, Canada.
The b e e f h e a r t
m i t o c h o n d r i a w e r e i s o l a t e d by t h e method o u t l i n e d b y S m i t h
(26).
Bcxth l i g h t a n d h e a v y f r a c t i o n s o f b e e f h e a r t m i t o c h o n d r i a
were u s e d i n t h e p r e p a r a t i o n of F1-ATFase
.
as d e s c r i b e d by
From t h e ammonium s u l p h a t e s u s p e n s i o n o f F 1 , t h e enzyme
w a s prepared f o r s t u d i e s i n a procedure s i m i l a r t o ' t h a t described by P e n e f s k y
.*
I
(73).
An a l i q u o t o f t h e ammonium s u l p h a t e
s u s p e n s i o n o f F1 wa's p l a c e d i n a c a p p e d 2-mL p o l y e t h y l e n e microc e n t r i f u g e t u b e , which i n t u r n w a s p l a c e d i n a l a r g e r p o l y e t h y l \
ene centrifuge tube.
$
\
The l a r g e c e n t r i f u g e t u b e ( i n i t s a d a p t o r
t u b e ) w a s c e n t r i f u g e d i n a SS-34 r o t o r f o r 1 0 m i n u t e s a t 1 2 , 0 0 0
r p m a t 4•‹C.
o f t h e 2-nL
T h e , s u p e r n a t a n t w a s d e c a n t e d , and t h e i n n e r w a l l s
polyethylene rnicroceiitrifuge tube c a r e f u l l y d r i e d
w i t h f = l t e r paper.
The
p r e c i p - i t a t e w a s d i s s o l v e d by
a d d i n g a volume o f 50 mM T r i s - a c e t a t e ,
pH 7 . 5 ,
b u f f e r a t 30•‹C.
T h e F1 s o l u t i o n w a s d e s a l t e d by c e n t r i f u g a t i o h o f 100-125 ;E
a l i q u o t s t h r o u g h S e p h a d e x G-50-80
a c e t a t e , p H 7.5,'
e q u i l i b r a t e d w i t h 50 mM T r i s -
b u f f e r a t 30•‹C, u s i n g t h e S e p h a d e x c e n t r i f u g e
, column
technique of Penefsky ( 7 3 ) .
The degalted F1 c e n t r i f u g a t e
*
was then ready f o r use with t h e a p p r o p r i a t e r e a c t i o n m i x t u r e ( s ) .
The r a t e s of ATP h y d r o l y s i s by F1-ATPase were monitored
usiny t h e coupled assay ( 3 1 ) .
The r e a g e n t s ' and t h e i r r e s p e c t i v e
c o n c e n t r a t i o n s and volumes used a r e shown i n Table 11.
The
f i r s t s i x reagents were added i n t h e q u a n t i t i e s i n d i c a t e d t o a
1-mL
cuvette.
The r e a c t i o n mixture i n t h e c u v e t t e was allowed
t o stand i n the c u v e t t e holder of a spectrophotometer (Vari-an
*
Technotron Model 6 3 5 ) a t 340 n m f o r about 10 minutes a t 30•‹C.
l'he spectrophotometer reading was a d j u s t e d t o read zero absorbance a t 340 nm.
The required amount of @-NADH was added t o t h e
cuGette, and a s t a b l e base l i n e a t 340 nm e s t a b l i s h e d .
A
measured volume ( X P L ) of t h e F1 s o l u t i o n was then added t o
a lk
t h e c u v e t t e . and t h e dec-
photometrically via a ch r t
e i n absorbance monitored s p e c t r o ecorder.
'\
The r a t e of h y d r o l y s i s of
A T P was c a l c u l a t e d u s i n g an e d t i n c t i o n c o e f f i c i e n t of
l i t ' r e r n ~ l e . - ~ . c r n - f~o r NADH ( 0 . 1
6.22
x
lo3
mole of NADH i n 1.0 mL of
**
\
s o l u t i o n gives
qf 0 . 6 2 2 ) .
ATPase a c ' t i v i t y was !*ally
In t h e s e F l p r e p a r a t i o n s . t h e
between 80-100 pmole.min.-l.mg-l
protein.
,Determination of P r o t e i n Concentration
t
A l l p r o t e i n c o n c e n t r a t i o n determinations were according
et. a l . procedure ( 1 1 2 ) .
t o t h e Lowry -
The p r o t e i n bovine serum
.
,-I
/-'
Table I 1
\
I
/
i
I---
b
The Coupled Enzyme Assay System
'
i
---
v
Vo 1 urnel
Cl L
----
2.
0.1 M ATP
3.
0.1 M PEP
4.
( 5 mg/rn~) LDH
7.
8.75
8.
FI-ATPase solution ( m g / m ~ )
.
mnM NADH
X
-
NADH, E3,," = 6 . 2 2
i.e.
x
103
litre mole.-1 .cm-1 or (M-1.crn-l )
0.1 pmole.min-I
of NADH in 1.0 mL of s o l u t i o r r g i v e ' s
4
a l b u m i n ( B S A ) was u s e d as t h e s t a n d a r d i n a l l assays/. The
v
I
p r o t e i n c o n c e n t r a t i o n o f t h e BSA s t a n d a r d s w a s d e t e t m i n e d b y
\
m e a s u r i n g t h e a b s o r b a n c e s a t 280 nrn; t h e e a b s o r b a n c e v a l u e s
I
6
( A 2 8 0 ) t o g e t h e r w i t h an e x t i n c t ~ c o e f f i c i e n t ( E 2 8 0 ) o f 0 . 6 7 7
rl.mg-l
.cm-l
tions.
[ C o n c e n t r a t i o h o f P r o t e i n = A b s o r b a n c e a t 280 nm/
w e r e t h e n u s e d i n t h e Beer-Lambert
J
law zalcula-
i'
( E x t i n c t i o n C o e f f i c i e q a t 280 nm
x
path length)].
Molecular ~ e i $ h t
i
A m o l e c u l a r w e i g h t o f 3 4 7 , 0 0 0 f o r F1 w a s u s e d i n a l l
c a l c u l a t i o n s ( 73 )
.
P u r i t y o f "the F1 P r e p a r a t i o n s .
-%
,
The p u r i t y o f t h e i s o l a t e % n a t i , V e F, p r e p a r a t i o n s e was
'
t e s t e d w i t h t h e u s e o f p o l y i ~ c r y l a m i d eg e l d i s c e l e c t r o p h o r e s i s
t e c h n i q u e s a s d e s c r i b e d by D a v i s ( 1 1 3 ) . m o d i f i e d as m e n t i o n e d by
8
Knowles and P e n e s k y ( 2 j ) .
--
-
'
e
I
One m a j o r enzyme band w a s s e e n w h i c h
had a r e l a t i v e m o b i l i t y of 0 . 3 w i t h r e s p e c t t o t h e dye f r o n t ;
w i t h 25 pg o f p r o t e i n a p p l i e d p e r g e l . a m i n o r band w i t h a r e l a ,
ti;e
These b b s e r v i t i o n s w e r e s i m i l a r
m o b i l i t y o f '0.7 was s e e n .
t o t h o s e of Knowles and P e n e f s k y ( 2 7 ) .
enzyme on a DEAE-Sephadex A-50
R e p u r i f i c a t i o n of t h e
'
'
.
I
-
column ( 2 8 ) d i d n o t e . r e s u l t i n t h e
d i s a p p e a.r a n. k e o f t h e t r a c e b a n d .
P u r i t y o f t h e ~ a d i o n u c l e o t i @ e s. ,
'
The p u r i t y o f t h e r a d i o n u c l e o t i d e s
ATP,
( [ y - 3 2 ~ ] ~ ~[ Z~. B, - ~ H ] -
and L2, 8-IH]ADP) w a s t e s t e d on p d l y e t h y l e n e i m i n e ( P E I )
I ,
p l a t e s u s i n g the-ocedure
of Randerath and Randerath ( 1 1 4 ) . - A
s a m p l e of t h e l a b e l l e d n u c l e o t i d e w a s a d d e d t o a m i x t u r e o f t h e
n u c l e o t i d e s (AMP, ADP,
a n d ATP), w h i c h w a s s p o t t e d o n t h e P E I
p l a t e b e s i d e separated spots o f AMP, ADP,
mM each, pH 7 . 0 ) .
a n d ATP (1 VL o f 2 . 5
The.plate w a s placed i n a tank with 1.0 M
L i C l a n d d e v e l o p e d f o r 20-25 m i n u t e s .
.
The d r i e d p l a t e w a s
p l a c e d u n d e r UV l i g h t a n d t h e n u c l e o t i d e s p o t s e n c i r c l e d .
The
spots w e r e c u t o u t and p l a c e d i n s c i n t i l l a t i o n c o c k t a i l f o r
T h e p e r b e n t a g e p u r i t y i n e a c h c a s e w a s f o u n d t o be
counting.
a b o u t 9 0 % ( % P u r i t y = [ c p m of l a b e l r e c o v e r e d / c p m of l a b e l
applied]
x
/
100).
t
M o n i t o r i n g ATP a n d / o r ADP C o n c e n t r a t i o n s
M ) w e r e monitored
T h e c o n c e n t r a t i o n s o f ATP a n d ADP
u s i n g t h e Hexokinase Assay ( 3 1 ) .
I
.
-
yeasurement of R a d i o a c t i v i t y
The r a d i o a c t i v i t y o f t h e s a m p l e s was d e t e r m i n e d by a d d i n g
a n ba l i q u o t o f t h e s a m p l e t o 1 0 mL o f w a t e r ( C e r e n k o v c o u n t i n g ) ,
or t o 1 0 mL o f ~ r i t o n / ~ o l u e nsec i n t i l l a n t .
-.
The s c i n t i l l a n t w a s t)
p r e p a r e d b y a d d i n g 1 5 g PPO a n d 1 c j o f POPOP t o 1 . 8 7 5 L xylene,,J
/
//
t o t h a t m i x t u r e w a s t h e n a d d e d 1 . 2 5 L T r i t o n X-114 a n d 1.87S,/L
x y l e n e t o b r i n g t h e E i n a l volume t o 5 L.
Each r a d i o a c t i v e
w
p r e p a r a t i o n ( C e r e n k o v c o u n t i n g or l i q u i d s c i ? t i l l a t i o n c o u n t i n g
s a m p l e ) w a s c o u n t e d f o r 5 m i n u t e s u s i n g a LKB i j a l i a c 17 l i q u i d
s c i n t i l l a t i o n counter.
k
'\*
P r e p a r a t i o n o f Sephadex C e n t r i f u g e Columns
The 1-mL Sephadex c e n t r i f u g e columns u s e d f o r ( i )
d e s a l t i n g P1. and ( i i ) p r e p a r a t i o n of t h e g e l s - f o r t h e l d n g e r
i
a n d / o r r e g s s e m b l e d columns, were p r e p a r e d as o u t l i n e d by
4
Penefsky ( 2 8 , 7 3 ) .
The columns used f o r d e s a l t i n g F1 were packed
'.
L
w i t h Sephadex G-50-80
7.5.
e q u i l i b r a t e d w i t h 50 mM \ ~ r i s - a ' c e t a t e , pH'
'
The columns .used i n t h e i n v e s t i g a t i o n s w i t h F1 were' p a c k e q
.
w i t h Sephadex G-50-80
equilibrated w i t h a buffer containing 90'
i
mM T r i s - a c e t a t e
Nucleotide-eq
(pH 7 . 5 ) ,
1 . b mM MgSb4, and 47 pM P i .
l i b r a k e d g e l s had t h e p a r t i c u l a r n u c l 6 o t i d e added
t o the buffer t o give the h d i c a t e d concentration.
fugations
' ,
using t h e
A l l centriII
s e p h a d e x c e n t r i f u g e column t e c h n i q u e were
performed with a t a b l e t o p clink-
centrifuge (with a
I.E.C.
\
s w i n g i n g b u c k e t r o t o r , 'model 5 2 1 ) a t 1050
g ( S e t t i n g - No. 5 )
x
f o r 2 minutes.
+
,,
- --
--
/-
1
.
Making a Longer Column B a r r e l
i
\
T o make a l o n g e r column barr"e1, two 1-mL
tubercupin
s y r i n g e s were c-u.t , , ( o n e a t t h e 1 . 0 rnL mark and t h e o t h e r a t t h e
0 . 5 5 mL m a r k ) , and j ~ i n e dby means o f a p i e c e o f t y g o n t u b i n g .
The l e n g t h of t h e column b a r r e l w a s 10 cm, w i t h t h e l e n g t h o f
L
t h e whole a s s e m b l y ( f r o m h e a d t o o u t l e t t i p
a
,
b e i n g a b o u t 11 c m .
M o d i f i e d S e p h a d e x C e n t r i f u q e Column T e c h n i q u e
(a)
reinc cub at ion Mode
B a s i c a l . 1 ~t h e t e c h n i q u e w a s t h e * s a m e a s d e s c r i b e d i n .
A p p e n d i x I ( P i g . l ~ ) , w i t h t h e optimum c o n d i t i o n s m e n t i o n e d
-4
3
Sephadex c e n t r i f u g e columns ' w e r e
a
u s i n g t h e c o n v e n t i o n a l S e p h a d e x ce t r i f u g e column t e c h n i q u e
(28,73).
Two o f t h e c o l u m n s w e r e p a c k e d y i t h S e p h a d e x G-50-80
equilibrated w i t h a buffer containing:
7.5,
90 rnM T r i s - a c e t a t e ,
'
equilibrated with a buffer consisting of:
pH 7 . 5 ,
1 . 6 mM MgS04, 47 p M P i ,
i n g c o n o e n t r a t i o n s ' o f nucleotide'.
a
S e p h a d e x G-50-80
pH
The t h i r d column w a s p a c k e d
1 . 6 rrdvl MgS04, a n d 47 p M P i .
w i t h S e p h a d e x G-50-80
9 0 mM T r i s - a c e t a t e ,
I n both cases,
and v a r y -
5 g of
w e r e a d d e d t d 1 0 0 mL b u f f e r ( o r r e d u c e d a m o u n t s
i n t h e s a m e p r o p o r t i o n s ) and a l l o w e d t o s t a n d o v e r n i g h t a t 4 • ‹ C .
C a r e w a s t a k e n i n p o u r i n g t h e g e i s ( e q u i l i b r a t e d t o room
temperature), since tyapped'air bubbles/usually
resulted in
I n a d d i t i o n , t h e columns{ were n o t allowed t o r u n
' I
d r y u n t i l t h e c o l u m n b a r r e l s were f i l l e d w i t h g e l .
broken g e l s .
,'
9
The colurnns w e r e c e n t r i f u g e d a t 1 50
I.E.C.,
'
Rotor 2 2 1 ) f o r 2 m i n u t e s .
x
g (Setting No.
5,
/'
The g e l s w e r e removed f r o m '
-
t h e c o l u m n b a r r e l s b y g e n t l y t i l t i n g t h e c o l u m n s 'so t h a t t h e
gels slipped out.
o f 2.5 and 4 c m ,
T h e f i r s t t w o g e l s were c u t t o g i v e l e n g t h s
respective-ly.
'
The t h i r d g e l ( i . e . w i t h n u c l e o -
t i d e ) was ,cut t o give a 1-cm p o r t i o n .
,
The measured lengths of
gels were reassembled i n t h e lengthened column b a r r e l (prepared
a s described above).
The 4-cm ge1:was
placed i n t h e co.lumn
4
'
b a r r e l (on top of. a pdrous polyethylene f r i t ) ; t h e 1-cm
nucleotide-containing gel was .placed on top o f . t h e 4-cm g e l ; and
t
l a s t l y t h e 2.5-cm g e l ' was placed on the nucleotide-containing
gel.
The bottom p o r t i o n of a cut 1-mL p i p e t t e t i p (with about
C
200 pL c a p a c i t y ) was i n s e r t e d i n N t h e column.
The t i p of t h e
I
p i p e t t e t i p was not allowed t o touch t h e top g e l nor t h e i n s i d e
f
?
of t h e column b a r r e l .
The e n t i r e assembly was i n s e r t e d i n a
15-mL conical c e n t r i f u g e tube, which i n t u r n was placed
,
in' t h e
swinging bucket r o t o r ( ~ o d e l2 2 1 ) of t h e t a b l e top c l i n i c a l
c e n t r i f u g e ( I. E . C . )
.
A t
150-pL a l i q u o t of t h e r e a c t i o n mixture'
which was preincubated f o r a t l e a s t 30 minutes a t 2 2 - 2 5 • ‹ C was
placed i n the cut p i p e t t e t i p .
of:
90 mM T r i s - a c e t a t e ,
c3 *P]E'~.
and F 1 .
The r e a c t i o n mixture consisted
pH 7 . 5 ,
1.33 mM MgS04, 47 pM P i with
The c o n t r o l experiment was performgd without
E l i n the r e a c t i o n mixture.
Whenever F l was used, t h e concen-
t r a t i o n was approximately 2 . 8 pM,
or 0.96 mg protein,/mL.
However, i n each experiment the s p e c i f i c a c t i v i t y of t h e
[ 3 2 ~ ~and
~ it h e
,concentration of F1 used a r e i n d i c a t e d .
loaded reassembled column was centrifuged a t 1050
..
minutes.
x
The
g for 3
A sample ( 2 0 ar 2 5 F L ~ of
)
the c e n t r i f u g a t e was used t o
I F ' passed
~
through t h e
determine t h e amount of c ~ ~ ~ which
.
column.
Y
',
O t h e r s a m p l e s o f t h e c e n t r i f u g a t e were u s e d t o Meter=\
- m i n e t h e p p o t e i n c o n c e n t r a t i o n a n d / o r a c t i v i t y o f t h e F1 i n t h e
-
centrifugate.
;
Each. e x p e r i m e n t was p e r f o r m e d i n t r i p l i c a t e f o r
each n u c l e o t i d e c o n c e n t r a t i o n .
,The amount of P i . i n a f i x e ~ o l u m go f c e n t r i f u g a t e w a s
c a l c u l a t e d u s i n g t h e amount o f r a d i o a c t i v i t y f o u n d i n t h a t
volume o f t h e c e n t r i f u g a t e a n d t h e s p e c i f i c a c t i v i t y o f the'
'
[ 3 2 p ] p i label i n t h e r e a c t i o n m i x t u r e a p p l i e d t o t h e column.
T h e amount o f F1 i n a n ' e q u i v a l e n t volume o f c e n t r i f u g a t e was
?
*
c a l c u l z t e d u s i n g t h e p r o t e i n c o n c e n t r a t i o n o f F1 i n t h e c e n t r i f u g a t e f o u n d b y t h e Lowry a s s a y s .
P i divided
The v a l u e o f t h e amount o f
by t h e v a l u e o f t h e amount o f F1 g i v e s t h e r a t i o o f
mole p i / m o l e F 1 .
.
On t h e x - a x i s
are' shown'the n u c l e o t i d e
c o n c e n t r a t i o n s used i n the e q u i l i b r a t i o n buffers-- of t h e middle
Each v a l u e p l o t t e d is t h e a v e r a g e o b t a i n e d f r o m t h e
gels.
triplicate experiments,
and t h e l o w e s t an@ h i g h e s t e x p e r i m e n t a l
r e s u l t s g i v e v a l u e s which f e l l w i t h i n the e n c l o s e d c i r c l e ,
square, o r triangle.
(b)
P u l s e - C h a s e Mode
.
The e x p e r i m e n t a l c o n d i t i o n s a n d p r o c e d u r e were b a s i c a l l y
IS/.
t h e s a m e as described f o r t h e p r e i n c u b a t i o n mode o f t h e m o d i f i e d
S e p h a d s x c e n t r i f u g e column t e c h n i q u e .
T w n g t h s o f t h e four
s e c t i o n s ( f r o m t o p t o b o t t o m ) w e r e 1 , 3 , l a n d 2.5' c m ,
' I
&
w
I
.-
respective-
1
ly.
These were arranged as shown i n Appendix 11, Fig. 7A.
In
some cases, t h e t h i r d and fourth s e c t i o n s were combined t o g.ive
one longer chase s e c t i o n ( 3 . 5 cm)
.
The r a d i o l a b e l l e d nucleotide
( e . g . [ y - 3 2 ~ ] ~ was
~ ~ ) added t o t h e nucleotide-containing b u f f e r
used t o prepare t h e pulse gel (N.B. NO [ 3 2 ~ ] ~ was
i
added t o
t h e \action
m i x t u r e applied t o t h e column).
e q u i l i b r a t i o n b u f f e r contained:
rnM MgSO,,
The pulse gel
90 mM T r i s - a c e t a t e ,
pH 7.5.
1.6.
4 7 pM P i , 'and 1 p M nucleotide: whereas t h e chase gel
e q u i l i b r a t i o n b u f f e r s had t h e various concentrations of nucleot
t i d e indicated.
h he experiments were performed i n t r i p l i c a t e
1
f o r each d i f f e r e n t chase nucleotide concentration.
Results
Release of Pi,from F1 : Preincubation Methakl
.J
T h e e f f e c t s o f ATP, ADP, a n d AMPPNP on t h e r e l e a s e o f P i
bound t o F1 w e r e s t u d i e d u s i n g t h e p r e i n c u b a t i o n mode o f t h e
m o d i f i e d S e p h a d e x c e n t r y f u ~e column t e c h n i q u e described i n d e r
Methods and Materials a n d i n Appendix I .
I n these s t u d i e s , t h e
.f
c o n c e n t r a t i o n s o f each a d e n i n e h u c l e o t i d e i n t h e e q u i l i b r a t & n
b u f f e r s o f t h e m i d d l e s e c t i o n s of t & ' c d l u m n s
w e r e -as i n d i c a t e d
d
and, u n l e s s o t h e r w i s e s t a t e d , the i e n g t h of the n u c l e o t i d e
@-
c o n t a i n i n g middle g e l w a s always 1.0 cm,
F i g u r e 1 shows the
e f f e c t s o f t h e t h r e e a d e n i n e n u c l e o t i d e s ATP, ADP, a n d AMPPNP o n
I t is e v i d e n t f r o m F i g u r e 1 t h a t
t h e release of P i f r o m F 1 .
t h e s e n s i t i v i t y of t h e P i r e l e a s e r e a c t i o n "to t h e c o n c e n t b r a I
2
L i o n o f n u c l e o t i d e i n t h e m i d d l 6 s e c t i o n o f t h e ~ o l u m ni s d i f c
'+
f g r e h t for each o f t h e t h r e e n u c l e o t i d e s ; a n d t h a t P i r e l e a s e
r e s p o n s e t o A ~ ~ . * a nAMPPNP
d
i n t h e middle
shows a biphas;c
section.
I n t h e s e biphasic r e s p b n s e s , a p p r o x i m a t e l y 7.QF o f t h e
I
1
,
t o t a l P i bound i s r e l e a s e d i n a r e l a t i v e l y h i g h l y * s e n s i t i v e
*
p h a s e ( s t e e p s l o p e ) , and t h e r e m a i n i n g P i i s released i n a
\
*
.
0
r
less s e n s i t i v e p h a s e (shallow s l o p e ) .
F i g u r e , 2 shows t h a t a t r e l a t i v e l y l o w c o n c e n t r a t i o n s
'
t i . e.
0
lees t h a n 1.0
pM n u c l e o t i d e ) , the: e f f e c t s o f WP a n d AMPPNP on
t h e r e l e a s e o f bound P i f r o m F l a r e e s s e n t i a l l y . t h e same.
<
e his
-
Figure 1
e
i
anu AMPPNP on t h e r e l e a s e 6f bound
I
The e f f e c t s of ATP,
4.
ADP,
s
The modified. Sephade'x c e n t r i f u g e column technique
P i from F 1 .
was used as described i n Methods.
For the ADP p l o t
(
0) :
the
--.
concentration oE F1 i n t h e r e a c t i o n mixture was 0.64 mg
protein.&-I
o r 1.85 + M I
t h e s p e c i f i c a c t i v i t y of t h e [32p,]pi
>.
%as 1.9
x
lo5
P
cpm/nmole, and t h g 100% P i bound corresponded t o
3
e S a t i o of 0 . 2 2 mole ~ i / m o l eof F1
.
Fot t h e AMPPNP p l o t
t h e concentration of F1 was 0.75 mg p r 0 t e i n . a - 1
~
1:5
s p e c i f i c a c t i v i t y of t h e L ~ * P ] Pwas
x
( A ):
or 2.2 p M I
the
yo6 cpm/nmole, and
t h e 100%P i ohound corresponded t o a r a t i o of 0.26 mole
*
~ i / m ~ Fl le
.
For t h e ATP
(0):
the
concentration of F1 was 0.8
b
mg protein=mL-l o r 2 . 3 2
'
r ~
~
~ was
p 1I. 9 px
l~o 6
pM, t h e s p e c i f i c a c t i v i t y of t h e
cpm/nmolei and t h e 100% P i bound
corresponded t o a r a t i o of 0 . 2 8 mole' ~ i / m o l eFl
A AMPPNP,
.
and ~ A T ) P
. ( 0 ADP,
,
Figure 2 .
The e f f e c t s o f ADP, AMPPNP, and ATP on t h e r e l e a s e o f P i
The m o d i f i e d Sephadex c e n t r i f u q e column t e c h n i q u e w a s
from Y 1 .
,
( 0) :
u s e d as d e s c r i b e d i n ~ e t h o d s . F o r . ' t h e ADP p l o t
c e n t r a t i o n o f Fl w a s 1.1 mg p r 0 t e i n . d - 1 2 0 r
a c t i v i t y of t h e
w a s 1.8
x
t h e con-
3..2 pM, t h e s p e c i f i c
lo6 cpm/nmole,
and t h e 1 0 0 % '
,
P i bound c o r r e s p o n d e d t o a r a t i o o f 0,27
F o r t h e AMPPNP p l o t
(
A
):
r n o l e j ~ i / m o l e F1.
t h e concen,tration
,
~
o f F1 was 0 . 7 6 mg
p r o t b i n * m L - l o r 2.23 pM, t h e s p e c i f i c a c t i v i t y . o f t h e [ 3 2 ~ ] ~ i
was 1 . 6
lo5
x
cpm/nmolel
and t h e 1 0 0 % P i bound c o r r e s p o n d e d t o
a r a t i o o f 0.29 mole ~ i / m b l eFl,.
c o n c e n t r a t i o n o f F1
bas
plot
For t h e ~ T P
0 . 8 mg protein.mL-I
i a s 1.9
s p e c i f i c a c t i v i t y of t h e [ 3 2 ~ ] ~ w
x
(
0) :
o r 2 . 3 pMI t h e
lo6
cprn/nmole,
t h e 1 0 0 % P i bound c o r r e s p o n d e d t o a * r a t i o o f 0 . 2 8 m o l e p i /
m o l e Fl
I
.
(
A
A ~ P ,
AMPPNP, and
0ATP )
the
,
.
and
!'
-.
"
p o i n t e d o u t the n e c e s s i t y o f e x p l o r i n g a r a n g e o f e x p e r i m e n t a l
.
c o n d i t i o n s , L b e f o r e a t t e m p t i n g t o draw g e n e r a l c o n c l u s i o n s a b o u t
t h e r e l a t i v e effectivness of t h e d i f f e r e n t adenine nucleotides
.. .
.4
i n f a c i l i t a t i n g r e l e a s e o f P i f r o m F 1 . F i g u r e s 3-5 s h o w t h e
-
e f f e c t s o f h i g h c o n c e n t r a t i o n s o f ATP and ADP o n P i r e l e a s e
from F 1 .
From t h e s e r e s u l t s ,
it i s e v i d e n t t h a t h i g h
c o n c e n t r a t i o n s o f ATP a n d ADP a p p e a r t o h a v e s i m i l a r e f f e c t s ,
a n d t h a t i n both cases $hey a r e a b l e t o e f f e c t t h e r e l e a s e o f
e s s e n t i a l l y a l l t h e b o u n d P i f r o m F1
.
Figure 3
,
The e f f e c t of ATP on t h e r e l e a s e of P i from F 1 .
.
*
The
modified Sephadex ~ e n t r i f " ~column
d
technique was used a s
I
, d e s c r i b e d i n Methods.
The c o n c e n t r a t i o n of F1 i n t h e r e a c t i o n
mixture was 0.96 mg protein.mL'l
a c t i v i t y of t h e [ 3 2 P ] P i
was 1 . 3
o r 2 . 7 8 pM,
x
The s p e c i f i c ,
lo6 cpm/nrnole.
P i bound corresponded t o a 5 r & t i oof 0.,2
The 100%
m o l e ~ i / m o l eF 1 .
F i g u r e ,4
The e f f e c t of ATP on t h e r e l e a s e of Pi-from F 1 .
The
modified Sephadex cenfrifuge column tech'nique was used as
*
described i n Methods.
The concentration of- F1 i n t h e r e a c t i o n
mixture was 0.96 mg proteineml-I
a c t i v i t y of t h e
Pi
c32
~ ] ~was
i
1.5
or 2 . 7 8 pM.
x
lo5
The s p e c i f i c
'
b
I
cpm/nmole.
The 100%
bound corresponded t o a r a t i o of 0.18 mole ~ i / m o l eF I .
I
'
Figure 5
The e f f e c t of ADP on t h e r e l e a s e of P i from
PI.
The
modified Sephadex c e n t r i f u g e column technique was used as
described i n Methods.
The concentration of F 1 i n the r e a c t i o n
mixture was. 0.64 mg p r 0 t e i n . d - l
a c t i v i t y of t h e
PIP^
was 1 . 9
or 1 . 8 5 pM.
x
lo5
The s p e c i f i c
cpm/nmole.
P i bound corresponded t o a r a t i o of 0 . 2 2
T h e 100%
mole ~ i / m o l eF 1 .
Release of Pi from F1:
Pulse-Chase
Method
The pulse-chase mo'de of t h e modified Sephadex c e n t r i f u g e
r
column technique, as described under Methods and i n Appendix II',
+
u
was used t o study t h e e f f e c t s of ADP, ATP, and AMPPNP on t h e
r e l e a s e of bound l a b e l from F 1 .
'
The l a b e l i n t h i s approach, was
given t o F I i n i t s passage through the 1 cm "pulse s e c t i o n " a t
t h e top of t h e column.
This pulse s e c t i o n was prepared from a
gel which had t h e d e s i r e d l a b e l l e d nucledtide i n t h e e q u i l i b r a -
*
t i o n buffer of t h e Sephadex.
Below the pulse s e c t i o n , t h e
column contained t h r e e s e c t i o n s ( 3 , 1, and 2 . 5 cm) se;ving
s i m i l a r functions as those used i n the columns f o r t h e s t u d i e s
employing t h e preincubation mode of "he
,
Sephadex c e n t r i f u g e t
\
column technique.
The t h i r d s e c t i o n $ ? o mt h e top i s r e f e r r e d t o
as t h e "chase s e c t i o n " of t h e
column".^
The s t u d i e s of P i r e l e a s e using the pulse-chase
node a r e
valuable : f o r comparison w i t h those using t h e preincubation
mode.
In t h e preincubation s t u d i e s :
when P i (from the reac-
t i o n mixture) i s bound t o F 1 , t h e P i presumably occupies an
empty s i t e which contains no other ligands.
I n t h e pulse-chase
-
b
s t u d i e s [ y - 3 2 ~ ] i~s ~present
~
i n t h e pulse g e l , t h e r e f o r e any
bound C~ 2
~
(found
~
~ i n i t h e c e n t r i f u g a t e ) o r i g i n a t e d from the
C ~ - ~ ~ P I Awhich
T P
bound and hydrolysed, and it a l s o shares i t s
binding s i t e w i t h ADP ( t h e other product of h y d r o l y s i s ) .
Eublished s t u d i e s have shown t h a t when ATP binds t o F1 under
s i n g l e s i t e occupancy
i t i o n s (such as those used i n t h e
s t u d i e s reported h e r e ) , it hydrolyzesbr a p i d l y and ADP ahd P i
a r e released slowly "(103).
T h u s by comparing t h e r e s u l t s from
the preincubation and pulse-chase methods, it i s p o s s i b l e t o
0
assess t h e e f f e c t of ADP being bound a t t h e same s i t e with P i ,
on t h e - s e n s i t i v i t y of t h e P i r e l e a s e r e a c t i o n t o nucleotides
,
i n the chase s e c t i o n .
Figures 6 and 7 show t h e e f f e c t s of ATP
and AMPPNP ( i n t h e chase s e c t i o n ) , r e s p e c t i v e l y , on t h e r e l e a s e
-
of l a b e l bound as [ y - 3 2 ~ ] from
~ ~ ~t h e pulse s e c t i o n .
For ease
,
of comparison, t h e r e s u - l t s from t h e corresponding s t u d i e s w i t h
I
t h e preincubatio6 mode a r e included i n t h e f i g u r e s .
The sensi-
b
t i v i t y of P i r e l e a s e t o ATP is decreased i n t h e pulse-chase
experiment r e l a t i v e t o t h e r e l e a s e observed i n t h e preincubation
experiment ( F i g . 6 ) , whereas t h e opposite e f f e c t i s observed
w i t h AMPPNP i n t h e pulse-chase
(Fig. 7 ) .
and preincubation experiments
The s e n s i t i v i t y of P i r e l e a s e t o ATP and AMPPNP i s
e s s e n t i a l l y t h e same i n t h e pulse-cnase
experiments ( c f . Figs. 6
and 7 ) ; whereas t h e s e n s i t i v i t y of P i r e l e a s e t o ATP i s much
?p
g r e a t e r than t o AMPPNP. i n t h e preincubation experiments.
The
4
biphasic response of P i r e l g a s e on exposure t o ATP and AMPPNP
1s observed with both experimental methods.
~
-
E
&
L
J L
Q
Figure 6 , ,
Comparison of the effecrs of ATP. in preincubationtand
v
q'
pulse-chase niodes, on the release of label-from F1.
preincubation experimentaS)plot,
Figure 2.
0.
Fpr the
the data were taken from
The pulse-chase technique was as described in
Methods, with four qections (1. 3, 1 and 2.5 cm) being used.
Note that in both modes, a 1 cm nucleotide-containing middle
t
section (preincubation mode) or chase section (pulse-chase mode)
was used.
The concentration of F 1 in the reaction mixture 0.96
mg protein*ml-l or 2.78 pM.
The p u l ~ e - ~ eequilibration
l
buffer
contained 1.0 pM ATP with '
2 ~ ] ~
the~ specific
~ ,
activity of
C
which was 3.07
x
lo5 cpm/nmole.
to a ratio of 0.,1 mole
e
c 3 *p]pi
label bound as.
mol mole
The 100% ATP bound corresponded
/9
F1 (
0 ATP
(preincubation mod:);
rel'ease of label bound C y
-3 2
~
,
on the release of
. and
ATP on
(pulse-chase
]
~
~ mode)
~ 1.
L
Fiqure 7
Comparison of t h e e f f e c t s of ANPPNP, i n preincubation and
4A
,
-
.
pulse-chase
~ o d e s ,on t h e r e l e a s e of l a b e l from F1.
preincubation experimental p l o t .
3
Figure 2 .
The pulse-chase
0,
For t h e
t h e data were taken from
technique was a s described i n
U
%
Methods, with four s e c t i o n s (1, 3 , 1 and 2 . 5 cm) being used.
Note t h a t i n both modes, a 1.0 cm nucleotide-containing m i d d l e
s e c t i o n (preincubati.on mode) or chase s e c t i o n (pulse-chase mode)
was used.
The concentration of F1 i n t h e r e a c t i o n mixture was
1.07 mg proteinmi&-I
/.
o r 3.09 VM.
The pulse-gel
equilibration
4
b u f f e r contained 1.0 p M ATP with [ y - 3 2 ~ ATP ( 2 . 6 8 K,
nrnolg).
lo5
cpm/
b
The 100% ATP bound corresponded t o a r a t i o o'f 0.1 mole
.
A T P ~ O
F l ~ ~(
0 AMPPNP
(Preincubation mode), and
on r e l e a s e of l a b e l bound as C 3 * p ] p i
AMPPNP on r e l e a s e of
~ ~ ~
mode)).
a s [ y - 3 2 ~ ] (Pulse-chase
l a b e l bound
6
I n t h e pulse-cha,se experiments just described (Figs. 6 and
7 ) , 1 cm chase s e c t i o n s were used i n order t o have t h e
nucleot-ide-containing s e c t i o n s i d e n t i c a l t o those used (1 cm
kiddle s e c t i o n ) i n t h e preincubation e x p e r i ~ e n t s . T h u s t h e
experimental conditions aid
p o s s i b l e i n both methods.
procedures were made as c l o s e as
.
Figures 8 and 9 show t h e r e s u l t s of
pulse-chase experiments i n which e n t i r e lower 3.5 cm (1 cm chase
gel p l u & 2.5'cm bottom g e l ) of t h e column contained chase
nucleotide.
A comparison of Figure 8 w i t h Figures 6 and 7 shows
t h e r e s u l t s w i t h [ y - 3 2 ~ ] pulse
~ ~ ~ and an ATP or AMPPNP chase
were e s s e n t i a l l y i d e n t i c a l i n both cases, i . e .
w i t h the column
containing e i t h e r a 3 . 5 cm chase s e c t i o n ( i . e . t h e e n t i r e
bottom) or a 1 cm chase 'section followed by a 2 . 5 cm spacer
section.
Figure 8 shows t h e r e l a t i v e s e n s i t i v i t y of t h e r e l e a s e
ADP,
of l a b e l , bound a s [ y - 3 2 ~ ] t ~
o ~
~ , ATP, and AMPPNP i n the
3.5 cm chase s e c t i o n .
The nucleotides ATP and AMPPNP were
equally e f f e c t i v e i n f a c i l i t a t i n g t h e r e l e a s e of label, whereas
ADP was considerably l e s s e f f e c t i v e i n promoting the r e l e a s e of
label.
Figure 9 shows t h a t whether l a b e l was bound a s C 3 ~ ] - A ~ p
or as [ y - 3 2 ~ ] from
~ ~ ~ t h e pulse s e c t i o n , approximately t h e same
s e n s i t i v i t y of l a b e l r h e a s e t o chase ATP was seen i n both
cases
%
/
Figure 8
The e f f e c t s o f ADP, ATP, and AMPPNP on t h e r e l e a s e of l a b s 1
bound a s [ Y - ~ ~ P ] A T Pfrom F 1 .
The p u l s e - c h a s e
t e c h n i q u e w a s as
d e s c r i b e d i n Methods, w i t h t h r e e s e c t i o n s (1, 3 , and 3.5 cm)
b e i n g used.
Note t h a t t h e e n t i r e bottom s e c t i o n ( 3 . 5 &I)
F o r t h e ADP p i o t
tained chase nucleotide.
(
0) :
con-
t h e concenP
t r a t i o n of F1 i n t h e r e a c t i o n m i x t u r e w a s 1 . 0 9 mg p r o t e i n . & - I
or 3.16 ,,M;
i
t'Me s p e c i f i c a c t i v i t y o f t h e [ y - 3 2 ~ ] i ~n ~+..he
~
1.0
pM ATP-containing
x
lo5
cpm/nmole;
e q u i l i b r a t i o n b u f f e r d f t h e p u l s e g e l was 2 . 0 4
and t h e 100% ATP bound c o r r e s p o n d e d t o a r a t i o
o 1l . e F o r t h e ATP p l o t
o f 0 . 1 mole ~ ~ ~ / r n F
t r a t i o n of F l was 1 . 1 6 mg p r o t e i n m a - l
~
a c t i v i t y of t h e [ y - 3 2 ~ ] w~a s~ 2.15
x
(
0) :
o r 3.36
lo5
.
F o r t h e AMPPNP p l o t
mg p r o t e i n - a - 1
was 2.35
x
lo5
0
):
and t h e
mol mole
t h e c o n c e n t r a t i o n o f F1 was 0.95
o r 2.75 pM; t h e s p e c i f i c a c t i v i t y o f t h e [ y - 3 2 ~ ]
cpm/nmole;
a r a t i o of 0 . 1 m o l e
A AMPPNP )
(
the specific
cpm/nmole;
100% ATP bound c o r r e s p o n d e d t o a r a t i o o f 0 . 1 2 mole
FI
t h e concen-
and t h e 100% ATP bound c o r r e s p o n d e d t o
mole
F1.
( 0 ADPI
0 ATP,
and
.
,
-
Figure 'i
The e f f e c t s of. ATP on t h e releaqe of l a b e l bound a s
[ v - 3 2 ~ ] and
~ ~ ~(2,8
- 3 ~ from
] ~ E~l . ~ The pulse-chase .technicjue
was as described i n Methods, with t h r e e s e c t i o n s (1, 3 , and 3 . 5
cm) being used, and t h e e n t i r e bottom 3.5 cm Section containing
chase anucleotide.
plot (
of
For t h e r e l e a s e of l a b e l bound a s [ y - 3 ? ~ ] ~ ~ ~
t
03,t h e . d a t a
were taken from Figure 8.
l a b e l bound as [ ~ H ] A T P p l o t
(0) :
e;uilibration
3
~
For t h e & l e a s e
t h e concentration of F1 i n -
t h e reaction mixture was' 0.94 mg protein.mL;l
s p e c i f i c a c t i v i t y of the [
o r 2 . 7 2 pM: t h e
i~
] t~
h e ~1.0~ pM ATP-containing
@
buffer of t h e pulse g e l was 2 . 2 8
-.
x
lo5
cpm/nmole:
and t h e 100% ATP bound correspondedpto- a r a t i o o f ' 0 A 5 mole
. (0ATP
~ ~ ~ / m oF.l l e
on t h e r e l e a s e cf l a b e l bound as
Cy
-32~]-
I
AI'P,
end
t
ATP on the r e l e a s e o f ' l a b e l
bound as C3i3]~W).
I
~ e i e a sof
~
-
W P N P f r o m PI:
Pulse-Chase Method
F i g u r e 1 0 shows t h e r e s u l t s of a p u l s e - c h a s e
experiment i n
u s e~d ~i n~' t )h e p u l s d s e c t i o n
w h i c h l a b e l l e d AMPPNP . ( ' [ 3 ~ ] ~w a~s ~
a n d u n l a b e l l e d AMPPNP i n t h e 1 . 0 c m chase s e c t i o n .
A compari-
s o n o f F i g u r e 1 0 w i t h F i g u r e 7 shows t h a t chase AMPPNP i s more
e f f e c t i v e i n f a c i l i t a t i n g t h e d i s s o c i a t i o n o f l a b e l b o u n d as
?
L ~ - ~ ~ P ] A tTh Pa n t h e d i s s o c i a t i o n o f l a b e l b o u n d ' as
C3 H~AMPPNP.
These f i n d i n g s s u p p o r t ( b u t do n o t p r o v e ) t h e h y p o t h e s i s t h a t
A
l a b e l b o u n d as [ y - 3 , 2 ~ ]d ~i s ~s o~c i a t e s f r o m F 1 a s C 3 * P ] p i
'
\
rather t h a n a s t h e u n h y d r o l y s e d [ y - 3 2 ~ ] ~ o~ n~ t, h e b i n d i n g of
chase AMPPNP'.
" I t w a s shown t h a t u n d e r s i n g l e s i t e o c c u p a n c y
c o n d i t i o n s , a n e q u i l i b r i u m i s a t t a i n e d i n which t h e r a t i o of
b o u n d ATP t o bound h y d r o l y s i s p r o d u c t q , FiDP a n d P i (1.e. t h e
e q u i l i b r i u m c o n s t a n t , K ) i s 2.
The c a l c u l a t e d f o r w a r d and
-
r e v e r s e r a t e c o n s t a n t s f o r t h i s s t e p of t h e E l - c a t a l y s e d
t i o n were 1 0 s'l
a n d 20 s - l , r e s p e c t i v e l y ( 1 0 3 ) .
o f medium n u c l e o t i d e s ,
,
I n the absence
t h e d i s s o c i a t i o n o f ADP a n d P i f r o m F1
is s l o w w i t h the'rate c o n s t a n t s of 4
s
reac-
respectively (lO3j.
x
s - I sand 3 x
I f t h e b e h a v i o u r of
AMPPNPi s s i m i l a r
t o t h a t o f ATP, t h e n t h e r e s u l t s d i s p l s y e d i n F i g u r e 1 0 i n d i c a t e
?
t h a t i n a n 'AMPPNY chase, t h e d i s s o c i a t i o n of b o u n d ATP i s f a r
.
.less f a v o u r a b l e t h a n t h e d i s s o c i a t i o n b f b o u n d ADP a n d P i ,
,
Figure 10
W
The e f f e c t of AMPPNP on t h e r e l e a s e of l a b e l bound a s
C ~ H ] A M P ~ N Pfrom F 1 .
The pulse-chase technique was a s described
i n Methods, with f o u r s e c t i o n s (1, 3 , 1 and 2 . 5 cm) being used,
i.e.
t h e 1.0 cm chase g e l was ~ o l l o w e dby 2 . 5 cm spacer g e l .
The c o n c e n t r a t i o q of F I i n t h e r e a c t i o n mixture "a$
p_rotein.mL-1 o r 2.49 pM.
0.83 mg
he pulse-gel e q u i l i b r a t i o n b u f f e r
d
contained 1.0 pM AMPPNP with [
B
3
~
(1.63
]
x~
l o 5~
cpm/nmole).
~
~
~
The 100%AMPPNP bouilil corresponded t o a r a t i o of 0.1 mole
~
Binding of Nucleotides t o F1 in the Sephadex Centrifuge Column
--
F i g u r e 11 shows t h e r e s u l t s o f a n e x p e r i m e n t i n w h i c h P I
I
*
w a s - p a s s e d * t h r o u g h a column of t h e t y p e u s e d i n t h e p r e i n s u b a -
t i o n mode.
I n t h i s c a s e t h o u g h , t h e E l was n o t p r e i n c u b a t e d
w i t h l a b e l l e d P i f i.e.
[ 3 2 ~ ] ~ i ,b
c o n t a i n i n g 1.0 c m middle s e c t i o n
t t h e nucleotide-
1
\*
-,
a d i a b e l l e d ATP ( [ y - 3 2 P ] A T Y ) .
Assuming t h a t t h e volume o f t h e c e n t r i f u g a c e w a s t h e s a m e as t h e
volume a p p l i e d t o t h e column, Table 111 shows t h e r e s u l t s of
>calculations
[ y - 3 *P]ATP
o f t h e p e r c e n t a g e o f t h e t o t a l amount o f t h e
c o n t a i n e d i n t h e column which w a s bound.
of c a l c u l a t i o n i s described i n t h e t a b l e l e g e n d .
Th4
+ T a b l e 111
shows t h a t u n d e r t h e c o n d i t i o n s o f t h e e x p e r i m e n t , w h e r e ATP was
p r e s e n t i n s u b s t o i c h i o m e t r i c amount w i t h r e s p e c t t o F 1 , a p p r o x i 1
m a t e l y a l l o f t h e ATP bound t o E l .
T h i s o b s e r v a t i o n i s cons's-
t e n t w i t h t h e v e r y r a p i d b i n d i n g o f ATP t o F1 ( s e c o n d o r d e r r a t e
" c o n s t a n t of 6
f r o m Fl
x
lo6
a n d t h e veFy s l o w r e l e a s e o f ATP
M-~.S-~)
( r a t e c o n s t a n t of 7
x
s u b s t o i c h i o m e t r i c a m o u n t s (103).
- 1 ,
when ATP i s p r e s e n t a t
I t is a l s o c o n s i s t e n t w i t h the
r e s u l t s shown i n F i g u r e 1 f o r t h e e f f e c t o f ATP ( i n t h e m i d d l e
s e c t i o n ) o n t h e release o f
t i o n e x p 95%.
I
"
This r e s u l t supports the Hypoth~'
e s i s t h a t t h e ATP b i n d s a t c a t a l y t i c s i t e s .
-?-
3
F i y u r e 20 s h o w s t h a t p r e i n c u b a t i o n o f F1 w i t h ATP d i d n o t
a l t e r t h e a m o u n t o f ATP b o u n d t o F 1 ; a n d n e i t h e r w a s t h e ,amount
b
of
I
P i released f r o m F1 a l t e r e d i n t h e p u l s e - c h a s e
experiment using
b
a 3.5 c m 0 . 2 5 p M A T P - c o n t a i n i n g
'
chase section.
show t h a t ' p r e i n c u b a t i o n o f F 1 w i t h e i t h e r
Av
F i g u r e s 2 1 a n d 22
por
EDTA d i d n o t
0
a f f e c t t h e amount o f l a b e l
C S f pIpi
bound Tfuga&
acetat-el p H 7 . 5 ,
1 . 6 mM MgS04, a n d 47 p M P i w i t h [ 3 2 ~ ~
a n d~ i ) l
t h i s a l l o w e d t o s t a n d d o r 8 m i n u t e s b e f o r e a p p l i c a t i o n t o thg,
column.
The m o d i f i e d Sephadex c e n t r i f u g e column t e c h n i q u e
( p r e i n c u b a t i o n mode) w a s u s e d ,as d e s c r i b e d i n M e t h o d s .
The.
c o n c e n t r a t i o n o f F1 i n t h e u s u a l r e a c t i o n m i x t u r e w a s 0 . 7 5 rng
or 2 . 1 7 pM.
pr0tein.a-l
The s p e c i f i c a c t i v i t y o f t h e [ " P ] P ~
u s e d i n t h e u s u a l r e a c t i o n m i x t u r e w a s 1.18
x
1 0 5 cpm/nmole.
The
F1 r e a c t i o n m i x t u r e s were t r e a t e d t h e s a m e e x c e p t f o r t h e a d d i t i o n
or e x c l u s i o n o f ADP.
F o r t h e r e a c t i o n m i x t u r e w i t h o u t ADP-treated
.t h e 100% P i bound c o r r e s p o n d e d t o a r a t i o o f 0 . 2 5 m o l e
f
Fl
1
Pi/mol.e F l
(
0 F1
c u b a t e d ~ 9 t hADP)
.
n o t p r e i n c u b a t e d w i t h ADP.
and
FI p r e i n -
,
-- /
F i g u r e 22
T h e - e f f e c t o f p r e i n c q b a t i o n o f F1 w i t h EDTA b e f o r e i t s a p p l i /
c a t i o n t o t h e column.
The Fl
( i n 50 $@
T rI
is-acetate.
a d d e d t o a s o l u t i o n of EDTA ( a l s o i n
t o g i v e a f i n a l c o n c e n t r a t i o n of 1 7
I
pH 7 . 5 ) was
0 mM ~ r i s ~ a c e t a t epH
. 7.5)
EDTA.
The m i x t u r e o f F1
a n d EDTA w a s allowed t o s t a n d f o r 1 m i n u t e s b e f o r e i t s a p p l i c a t i o n
I
r
t o t h e u s u a l S e p h a d e x c e n t r i f u g e column ( 2 8 , 7 3 ) .
The centrifu-
g a t e w a s added t o the r e a c t i o n m i x t u r e t o g i v e f i n a l concentrat i o n s o f 90 mM T r i s - a c e t a t e ,
plp 7 . 5 .
1 . 6 rnM MgSds, a n d 47 p M P i
w i t h C32pIpi, and t h i s a l l o w e d t o s t a n d f o r 8 m i n u t e s b e f o r e
A-
-
a p p l i c a t i o n t o t h e column.
The m o d i f i e d S e p h a d e x c e n t r i f u g e
column t e c h n i q u e ( p r e i n c u b a t i o n mode) w a s u s e d as d e s c r i b e d i n
~ e t h o d s ; ;The c o n c e n ' t r a t i o n o f FI i n t h e r e a c t i o n m i x t u r e was 0 . 6 8
mg p r ~ t e i q b m l ' ~or 1 . 9 7 vM.
The s p e c l f i c a c t i v i t y of t h e
C ~ ~ . P iI nP t~h e r e a c t i o n m i x t , g r e w a s 1 . 2 2
x - l o 5 cpm/nmole.
,
The
F1 r e a c t i o n m i x t u r e s w e r e t r e a t e d t h e same e x c e p t f o r t h e a d d i t i o n
or e x c l u s i o n o f EDTA.
F o r t h e r e a c t i o n m i x t u r e w i t h o u t EDTA-
treated F1
t h e 1 0 0 % P i bound c o r r e s p o n d e d t o a r a t i o o f 0 . 2 4
- ,
t
m o l e ~ i / m o l eF I
.
40
Fl not preincubated
p r e i n c u b a t e d w i t h EDTA).
kith
EDTR. a n d
FI
'
F i g u r e 23
+
F1.Pi
The e f f e c t o f ATP on t h e r e l e a s e o f P i from F 1 , when t h e
complex was g i v e n
The p u l s e - c h a s e
p u l s e o f ADP.
tech-
n i q u e w a s u s e d as d e s c r i b e d i n Methods, w i t h f o u r s e c t i o n s (1, 3 ,
Note t h a t i n t h i s case t h e r e a c t i o n
1 and 2.5- c m ) b e i n g used.
m i x t u r e c o n t a i n e d lab ell;^ P i as c 3 * p ] p i
mole).
(1.24
x
lo5
cpm/
The c o n c e n t r a t i o n o f F1 i n t h e r e a c t i o n m i x t u r e w a s 1 . 0
mg pkotein.mL-l
The'pulse-gel
or 2 . 8 9 pM.
c o n t a i n e d 50 p M ADP w i t h [
3
~
F l o p = complex n o t g i v e n p u l s e
qf
used.insteadj,
mole Pi/mole
(]3 . 5~
ADP
x
equilibration buffer
~l o 4
~ cpm/nmole).
(i.e,
For t h e
a 1.0 cm spacer gel
1 0 0 % P i bound c y r e s p o n d e d t o a r a t i o o f 0 . 2 1
F1;, a n d f o r F l o p = complex g i v e n a p u l s e o f A D P ,
%
1 0 0 % P i bound c o r r e s p o n d e d t o a r a t i o o f 0 . 1 8 mole ~ i / m o l eF 1
(
0 Fl
n o t g i v e n p u l s e o f ADP, and.
0
FI g i v e n p u l s e of ADP).
[ N o t e c h a t t h e t o t a l amount o f ADP a v a i l a b l e i n t h e 1 crn o f AUPp u l s e - g e l was 800 p m o l e ,
and t h e t o t a l ADP bound by F1 in t h e
c e n t r i f u g a t e was 1 0 . 4 p m o l e . ]
0.02.
The r a t i o o f mole
mole
F l w$s
The m e t h o d s o f d e t e r m i n a t i o n and c a l c u l a t i o n w e r e as
d e s c r i b e d i n t h e legend o f T a b l e 111 . I
U
brief pulse of ADP
F given brief pulse of ADP
0 F, not &en
:
e x p e r i m e n t , w i t h a 1 . 0 cm 5 . 0 M
,
ADP ( i 3 ~ ] ~ ~ ~ ) - c o n t a i nt oi pn g
s e c t i o n , t h a t b o t h t h e amount of P i bound t o F I a n d t h e amount
o f P i . r e l e a s e d w e r e n o t i n f l u e n c e d by t h e W P - p u l s e
batment.
I n a d d i t i o n , t h e e x p e r i m e n t showed t h a t a r a t i o o f 0 . 0 2 m o l e XDP/
m o l e F1 was o b t a i n e d u n d e r t h e e x p e r i m e n t a l c o n d i t i o n s ( b r a b o u t
0 . 0 1 o f t h e a v a i l a b l e ADP f r o m t h e 1 . 0 c m t o p s e c t i o n was b o u n d ) .
T h i s l a s t s e r i e s o f e x p e r i m , o n t s ( F i g s . 20-23) was p e r f o r m e d i n
o r d e r t o t e s t t h e p o s s i b i l i t y t h a t P i b i n d i n g or A T P - f a c i l i t a t
4d
P i r e l e a s e m i g h t be a f f e c t e d by t h e p r e s e n c e o f Mg*,x&l>P
abound'to
L.
F1.
I
4
$
Bound ADP, w h i c h c o u l d be removed f r o m F 1 by t r e a t m e n t w i t h
EDTA or i n c u b a t i o n w i t h p y r u v a t e k i n a s e and P E P ,
*
h a s been proposed
a s t h e c a u s e o f l a g s i n ATP h y d r o l y s i s c a t a l y s e d by F1 ( 1 1 5 - 1 1 9 ) .
The p u r i t y g f t h e F 1 p r e p a r a t i o n s u s e d i n t h e s e e x p e r i m e n t s
w a s s i m i l a r t o t h a t o f Knowles a n d P e n e f s k y ( 2 7 ) a s d e s c r i b e d
under methods.
-
The NBD-chloride
i n a c t i v a t i o n e ' x p e r i m e n t shown i n
F i g u r e 1 9 i n d i c a t e s t h a t e v e n i f some p r o t e i n i m p u r i t i e s w e r e p r e < 5 8 ) o f ATP, and
s e n t , t h e y d i d n o t b i n d s i g n i f i c a n t amounts ( -
t h e r e f o r e c a n n o t a c c o u q t f o r t h e o b s e r v e d b i p h a s i c r e l e a s e of
label.
A t present,
t h e r e i s no s a t i s f a c t o r y e x p l a n a t i o n a v a i l a b l e
for t h e b i p h a s i c P i r e l e a s e obsekved.
I t is i n t e r e s t i n g ' t h a t a t
s u f f i c i e n t l y h i g h ATP or ADP c o n c e n t r a t i o n s ,
w a s r e l e a s e d ( F i g s . 3 and 4 ) .
a t c a t a l y t i c s i t e s , bi;t
a l l o f t h e bound P i
P o s s i b l y a l l - o f t h e P i was bound
t h e F1 e x i s t s i n d i f f e r e n t s t a t e s a n d some
,
of these cannot be a c t i v a t e d by exposure t o low ATP concentrations
- .
on the time s c a l e of t h e Sephadex c e n t r i f u g e column experiment.
$
I t was found by o t h e r workers t h a t F1, l a b e l l e d by exposure t o
subsioichiornetric amounts of ATP, did not r e l e a s e a l l of i t s l a b e l
upon subsequent exposure t o high concentrations of medium ATP
Other published s t u d i e s a r e a l s o cons
with t h e biphasic
'
P i r e l e a s e observed i n t h e i n v e s t i g a t i o n s
_here.
I n the
o r i g i n a l study of P i E n d i n g t o and i t s r e l e a s e from F1 , about
.
\
108 of the bound P i did not d i s s o c i a t e even a t 48 minuteg a f t e r
-
0,
t h e addition of unlabelled P i t o t h e r e a c t i o n mixture ( 7 3 ) .
semilog p l c t of t h e published d a t a (Fig. 3 of Ref.
A
7 3 ) , using t h e
48 minute point a s t h e i n f i n i t y value, i s shown i n Figure 2 4 .
Phis f i g u r e shows t h a t ' t h e P i d i s s o c i a t i o n which occurred did
"
not follow c l e a r f i r s t order k i n e t i c s .
This r e s u l t i s c o n s i s t e n t
with t h e r e being more than one kind of bound P i .
Similar
r e s u l t s were obtained i n another published study ( 9 6 ) , and Zn t h e
..
preliminary work which led t o t h e development af t h e methods'used
.
i n t h e s t u d i e s reported here.
I
s t u d i e s a r e shown i n Figure 25.
The r e s u l t s from t h e present
A semilog p l o t of t h e P i
r e l e a s e data from t h i s experiment a l s o does not show clean f i r s t
order k i n e t i c s .
The biphasic nature of t h e
r e l e a s e i s evident from t h e experiment shown i n
which U P was added t o unlabelled P i which was then i n t u r n
Pi
a
Figure 24
.)
-
A s e m i l o g p l o t showing t h e r e l e a s e of P i from F 1 w i t h
time.
T h e d a t a w e r e tak-en from F i g u r e 3 o f Ref.
m i n u t e p o i n t b e i n g u s e d as t h e i n f i n i t y v a l u e *
y-axis
r e p r e s e n t s { m o l e P i / m o l e bound a t t min
mole bound a t 48 min)
,
.
7 3 , w i t h t h e 48
~ [ P i l b o u n don
-
mole P i /
0-
*
P
0
*
0
10
TIME (min)
I
20
"
-I:
-
*
F i g u r e 25
F1.
The e f f e c t o f n u c l e o t i d e s on t h e release o f P i bound t o
,
0
( R e p e a t o f H u t t o n and B o y e r ' s e x p e r i m e n t shown i n ~ i ~ 2 uo f 3
Ref.
9e.)
A r e a c t i o n m i x t u r e c o n t a i d i n g 90 rnM T r i s - A c e t a t e ,
* .
7 . 5 , -1.6 mM MgSOq, 47 $4
P i w i t h C3*p]pi (1.4
x
lo3
pH
cpm/
1.
p m o l e ) . , and 3 . 7 pM F1-ATPase w a s i n c u b a t e d f o r 20 m i n u t e s a t
23•‹C.
80 pL a l i q u o t s o f t h e r e a c t i o n m i x t u r e w e r e a p p l i e d i n t h e
Sephadex c e n t r i f u g e column t e c h n i q u e a s d e s c r i b e d by P e n e f s k y ( 2 8 ,
73).
The s e p h a d & c G-50-80
g e l w a s e q u i l i b r a t e d i n a b u f f e r ,con-
O t h e r 80 pL a l i q u o t s w e r e mixed w i t h 20 pL o f P i ,
P i and ATP,
o r P i and ADP t o g i v e f i n a l c o n c e n t r a t i o n ' s o f 1 2 mM P i and 2 0 rnM ATP o r ADP,
respectively.
80 pL a l i q u o t s o f t h e s e m i x t u r e s
:
1
w e r e a d d e d t o t h e S e p h a d e x columns a t ' t h e t i m e s i n d i c a t e d , and
5
-
c e n t r i f u g a t i o n performed immediately.
The 100% P i bound c o r r e s -
ponded t o a r a t i o o f 0 . 1 6 mole ~ i i m o l eF 1 .
12
Pi
+
20 p M ADPI and
0
1 2 mM P i
+
(
A
1 2 m~ P i ,
20, p M ATP).
0
added t o t h e F 1 r e a c t i o n mixture t o i n i t i a t e [ 3 2 ~ ] ~ ri e l e a s e
+' •’rom F 1 .
This experiment (Fig. 25) was performed u s i n g e x a c t l y
t h e same c o n d i t i o n s and procedure used i n a s i m i l a r study (96),
and .the r e s u l t s obtained i n t h e two s t u d i e s were e s s e n t i a l l y
identical
.
t
4
Although it has t h u s f a r proved impossible t o e i t h e r eliminI
a t e o r adequately r a t i o n a l i z e th'e b i p h a s i c P i r e l e a s e ,
'a
c o n s i s t e n t with published r e s h l t s ( 7 3 , 9 6 ,
16;).
it i s
I n the analysis
of t h e r e s u l t s i n t h e d i s c u s s i o n s e c t i o n , a t t e n t i o n w i l l be
focused on t h e s e n s i t i v e o r s t e e p phase of n u c l e o t i d e - f a c i l i t a t e d
P i r e l e a s e from P1
.
A case w i l l be made f o r t h e hypothesis t h a t
t h e P i r e l e a s e d from F1 i n t h i s phase is r e l e a s e d from a c a t a l y t i c s i g e , and t h a t t h i s , r e a c t i o n i s a s t e p i n t h e c a t a l y t i c
mechanism of F1-catalysed ATP h y d r o l y s i s .
The r e s u l t s w i l l be
r a t i o n a l i z e d i n terms; of t h e c a t a l y t i c mechanism.
3
I
-
73
--
DISCUSSION
The Release of P i from F,:
Preincubation Method
F i g u r e 2 shows t h a t a t r e l a t i v e l y low c o n c e n t r a t i o n s o f t h e
n u c l e o t i d e s , ATP w a s a b l e t o e f f e c t t h e release o f P i from F1,
..
whereas ADP and AMPPNP had no o b s e r v a b l e e f f e c t .
The e f f e c t o f
ATP c a n be e x p l a i n e d by t h e " b i n d i n g c h a n g e mechanism" ( 1 0 , 1 3 ,
\
97-99,122
? illustrated
as shown i n F i g u r e 26.
F1 b i n d s P i
d u r i n g t h e p r e i n c u b a t i o n p e r i o d t o g i v e t h e F l * P i complex
2 m i n u t e s ) from t h e F1 * P i complex ( 7 3 , 9 6 ) .
However, when t h e
F 1 * P i . complex p a s s e s t h r o u g h t h e ATP-containing
s e c t i o n of t h e
column, ATP becomes bound a t o n e o f t h e a v a i l a b l e ATP-binding
sites.
The b i n d i n g o f ATP a t a n a l t e r n a t e s i t e i s a b l e t o
e f f e c t t h e conformational change n e c e s s a r y t o promote t h e f a s t e r
r e l e a s e o f P i from F1.
Grubmeyer and P e n e f s k y ( 6 2 ) h a v e
d e m o n s t r a t e d t h a t a t l e a s t t w o b i n d i n g s i t e s are i n v o l v e d i n
s i t e - s i t e c o o p e r a t i v i t y i n t h e mechanism o f a c t i o n o f F1.
The
r e s u l t s o b t a i n e d w i t h ADP i n t h e m i d d l e s e c t i o n o f t h e column
a r e e x p l a i n e d by t h e f a i l u r e o f Fl t o b i n d ADP u n d e r these
e x p e r i m e n -t a l c o n d i t i o n s .
T h iL
2x p l q n a t i o n
i s s u b s t a n t i a t e d by
t h e o b s e r v a t i o n i n F i g u r e 23 i n t h a t t h e r a t i o o f mole
F1 w a s 0 . 0 2 ,
used.
when a 1 . 0 cm p u l s e - s e c t i o n
mol mole
( w i t h SPM ADP) w a s
Thus w i t h l o w e r 'ADP c o n c e n t r a t i o n s i n t h e m i d d l e s e c t i o n s
F i s u r e 26
P i release from F1 o n b i n d i n g o f ATP ( f o r s i m p l i c i t y o n l y
J
two b i n d i n g s i t e s are s h o w n ) .
(1)
E represents FI,
The d e s i g n a t i o n s a r e as f o l l o w s :
( i i ) > or ~< r e p r e s e n t s a t r a n s i t o r i l y
r.
t i g h t bound s u b s t r . a t e o r g x o d u ! i mnolecule, and ( i i i ) a center
t-\
dot r e p r e s e n t s a more l o o s e l y bound s u b s t r a t e o r p r o d u c t
<
molecule.
(See Refs.
7O,93, 94. )
tight binding
of Pi
loose binding
of ATP
Conformational
change
P
pi
Conversion of
(I)tightly bound Pi
to loosely bound Pi
(11) loosely bound ATP
to tightly bound ATP
.
pi
HOH
Pi*
F
~
j
A pi>
D p >
conversion of
tightly bound ADP and Pi
to loosely bound ADP
and Pi
*
k
F
Release
of Pi
~
<3>
Reversible
Hydrolysis
t h e amount o f ADP bound was n o t s i g n i f i c a r l t .
Grubmeyer et. al.
h a d f o u n d t h a t t h e r a t e o f ADP b i n d i n g t o F L ,
(lo3
M - ~.s-l
t h r e e orders o f m a g n i t u d e lower t h a n t h a t o f ATP ( 1 0 4 ) .
%
1, i s
Since
- -.
l o w c o n c e n ' t r a t i o n s of AMPPNP b i n d as w e l l a s ATP t o F l u n d e r
t h e s e e x p e r i m e n t a l c o n d i t i o n s ( T a b l e s I11 and I V ,
F i g u r e 1 5 and
R e f . 1 2 4 ) , it c a n be c o n c l u d e d t h a t t h e bound AMPPNP (Law
c o n c e n t r a t i o n s ) wa,s, u n l i k e ATP, u n a b l e to e f f e c t t h e c o n f o r m a t i o n a l c h a n g e which r e s u l t s i n t h e r e l e a s e o f P i ( F i g , 2 7 9 .
-
H i g h c o n c e n t r a t i o n s o f a l l t h r e e n u c l e o t i d e s w e r e , able t o
e f f e c t t h e t o t a l r e l e a s e o f P i from F1 ( ~ i g s ' . 3-5).
A t these
h i g h ATP c o n c e n t r a t i o n s ( F i g s . 3 , 4 ) , s i n c e ATP i s p r e s e n t i n
g r e a t e r t h a n s t o i c h i o h e t r i c amounts, t h e p r o b a b i l i t y of each
complex b i n d i n g a n ATP m o l e c u l e i s g r e a t l y e n h a n c e d ,
t h u s r e s u l t i n g i n t h e g r e a t e r release of P i f r o m t h e F1 * P i
complex ( F i g . 2 6 ) .
I t is also p o s s i b l e t h a t the h i g h n u c l e o t i d e
c o n c e n t r a t i o n s w e r e a b l e t o make more F1 a c t i v e u n d e r t h e s e experimental conditions, there&ccounting
r e l e a s e of P i f r o m t h e
complex.
for the greater
I n a d d i t i o n , it i s
p o s s i b l e i n t h e p r e s e n c e o f e x c e s s ATP for e a c h F i o P i complex
t o b i n d a s e c o n d ATP m o l e c u l e , t h u s a c c e l e r a t i n g t h e r e l e a s e o f
P i from1 F1 a t l e a s t t w o - f o l d
(104).
-
.-. -
>.:
1
*
F i g u r e 27
..-
P i r e l e a s e f r o m F 1 o n e x p o s u r e t-o low AMPPNP
concentrations (Figure 2 ) .
symbols. )
( S e e F i g u r e 26 f o r e x p l a n a t i o n o f
i
.-
Ci
t-
Pi
AMPPNP
AMPPNP
t
Effective
con formational
change
not achieved
E
Pi
;k-
Conversion of (I)tightly bound
Pi to loosely bound Pi
not achieved, (Il) loosely b o m d
AMPPNP to tightly bound
AMPPNP not achieved
/
i
-
\
i
77
-
Grubmeyer a n d P e n e f s k y f o u n d t h a t a t h i r d b i n d i n g s i t e was
o c c u b i e d B f t e t p r o l o n g e d ( 3 0 t o 60 m i n u t e ) i n c u b a t i o n w i t h h i g h
concentration6
( 5 t o 1 0 0 pM) o f TNP-adenine
2
present i n buffer,).
.
nucleotides ( ~ g * +
The presellce o f a t h i r d s i t e f o r t h e
n u c l e o t i d e s AMPPNP a n d ADP o n F1 w a s a l s o o b s e r v e d u n d e r s i m i l a r
u
c o n d j t i o n s o f i n c u b a t i o n ( 6 O I 6 l)
.
However, w h e t h e r t h i s t h i r d
'h.
n u c l e o t i d e b i n d i n g s i t e is a c a t a l y t i c s i t e o r a l o w a f f i n i t y
....
C
i
*
exchang-ble
s i t e which seryes-a
\
--.
-
r e g u l a t o r y role w a s n o t c l e a r l y
-/
1-
d i ~ t i n ~ u i s h e ~ - ~ . (O~p e4 )p .o s s i b i l i t y i n v o l v i n g t h r e e s i t e s a n d
-
3
' t h e b i n d i n g o f a s e c o n d ATP m o l e c u l e i s shown i n F i g u r e 28.
E v i d e n c e h a s b e e n p r e s e n t e d f o r t h r e e separate i n t e r a c t i n g s u b u n i t s p e r m o l e c u l e o f F1 ( 4 8 , 9 8 , 9 9 ) .
F i g u r e 11 a n d T a b l e I11
show t h a t t h e amount o f ATP bound by F1 i n c r e a s e s as t h e ATP
c o n c e n t r a t i o n i n c r e a s e s f o r l o w c o n c e n t r a t i o n s o f ATP.
Figure
1 5 a n d T a b l e V show t h a t t h e amount of ATP b o u n d i n c r e a s e s u n t i l
a maximum v a l u e i s r e a c h e d ,
a n d t h a t it i s p o s s i b l e f o r F 1 t o
b i n d more t h a n o n e ATP m o l e c u l e a t h i g h n u c l e o t i d e c o n c e n t r a t i o n
(100 p M ) .
Note t h a t i n t h e e x p e r i m e n t s d e s c r i b e d i n F i g u r e s 3
a n d 4,' 10 t o 1 0 0 a n d 0 . 1 t o 25 mM ATP c o n c e n t r a t i o n s ,
i v e l y , w e r e used,
respect-
thus t h e p o s s i b i l i t y of F1 binding m o r e than
o n e ATP m o l e c u l e i s l i k e l y .
v
However,
as p o i n t e d o u t b y
Grubmeyer a n d P e n e f s k y ( 6 1 , 6 2 ) ' , .it i s n o t n e c e s s a r y t o i n v o k e a
t h i r d s i t e t o e x p l a i n p r o m o t i o n o f t h e release o f h y d r o l y z e d
nucleotides;
r
'-.,
F i g u r e . 28
P i r e l e a s e . f r o m F1 o n e x p o s u r e t o h i g h ATP
concentrations.
( F o r e x p l a n a t i o n of s y m b o l s , see F i g u r e 2 b . )
Q
Conformational
Change
I
The e f f e c t o b s e r v e d w i t h h i g h c o n c e n t r a t i o n s o f ADP ( F i g u r e
5 ) c a n be a c c o u n t e d f o r i f a s m a l l amount o f
p r e s e n t i n t h e s e ADP p r e p a r a t i o n s
(103.123).
(-1.4%)
o f ATP is.
T h i s very small
I
p e r c e n t a g e (w 1 . 4 % ) o f ATP i n t h e 0 . 1 t o 10 mM ADP w%uld y i e l d a
B
,
r a n g e o f a b o u t 1 . 4 t o 1 4 0 pM AllP c o ~ l c e n t r a t i o n s , w h i c h would be
s u f f i c i e n t t o e f f e c t , t h e r e l e a s e o f P i i r o m F1 ( F i g u r e 5 ) .
This explanation,
i n a d d i t i o n t o the l o n g e r t i m e o f i n c u b a t i o n
of F1 * P i w i t h n u c l e o t i d e ,
may a c c o u n t f o r t h e e f f e c t o f ADP on
c
t h e a c c e l e r a t e d r e l e a s e of P i from F1 o b s e r v e d b
1n t h e i r s t u d i e s a b o u t 0 . 2 8
Boyer ( 9 6 ) .
-.
Hutton and
pM ATP - m a y h a v e b e e,
n
,
-
p r e s e n t i n t h e i r 2 0 pM ADP p r e p a r a t i o n i n a d d i t i o n t o t h e i r
t
l o n g e r i n c u b a t i o n t i m e of a b o u t a m i n u t e .
i n c u b a t i o n would a l l o w ADP t o b i n d t o F 1 .
,This
longer ' t i m e of
Grubmeyer et.; al.
\
.
q..
f o u n d t h a t t h e r a t e o f ADP b i n d i n g t o F1 was 103 M - ~ s-1 b 0 3 )
P e n e f s k y ( 7 3 ) h a s a l s o o b s e r v e d t h a t h i g h c o n % e n t r a r i o n s (30-1pM) o f ATP a n d ADP
( F i g u r e 9 o f Ref.
of P i . t o a b o u t the same e x t e n t .
7 3 ) i n h i b i t e d the binding ,
$ P e n e f s k y 8 sADP p r e p a r a t i o n s
.
i n t h e r a n g e s t u d i e d may h a v e c o n t a i n e d a b o u t 0.42 t o 1.68 pM
ATP, a n d i n a d d i t i o d he used. a l o n g i n c u b a t i o n p e r i o d o f 30
minutes.
he
r e s u l t s r e p o r t e d here ( F i g u r e s 1 - 5 ) show t h a t t h e
r e l e a s e of P i i s more s e n s i t i v e t o ATP t h a n t o A D P ,
and t h a t
t h e P i r e l e a s e o b s e r v e d w i t h h i g h ADP c o n c e n t r a t i o n s c a n be
I
.
.
a c c o u n t e d f o r b y t h e p r e s e n c e o f t r a c e a m o u n t s (-1.4%)
_-
i n t h e ADP p r e p a r a t i o n s . /
High n u c l e o t i d e c o n c e n t r a t i o n s
gf ATP
'
-
( F i g u r ~ s3-5)
e n a b l e d tkie release' aof t h e P i (N
r e m a i n e d bound a t l o w ATP c o n c e n t r a t i o n s .
3 0 % ) which
One p o s s i b l e e x p l a n a -
t i o n is' t h a t h i g h n u c l e o t i d e c o n c e n t r a t i o n s r e s u l t e d i n
higher
i o n i c s t r e n g t h a n d t h i s e f f e c t a l o n e c a u S e d t h e release o f bound
/'
P i , which w a s i n s e n s i t i v e t o lower ATP c o n c e n t r a t i o n s .
K a s a h a r a and P e n e f s k y f o u n d t h a w a l t s i n h i b i t e d P i b i n d i n g t o
F1 which was a t t r i b u t e ' d t o t h e i r e f f e c t s o n t h e enzyme a n d o n .
the i o n i c s t r e n g t h .
Z t i s a l s o p o s s i b l e t h a t a t t h e l o w ATP
c o n c e n t r a g i o n s u s e d , a p p r o x i m a t e l y 60% of t h e F1 i s n o t a c t i v e ,
b u t t h i s f r a c t i o n c a n become a c t i - v e upon l o n g e r e x p o s u r e t o ATP
-
-
o r upon b r i e f e x p o s u r e t o h i g h ATP c o n c e n t r a t i o n s .
, I n t h e e x p e r i m e n t d e s c r i b e d i n F i g u r e 1, a h i g h e r c o n c e n t r a t i o n e f AMPPNP w a s u s e d t h a n i n t h a t described i n F i g u r e 2 .
The n u c l e o t i d e s ATP and AMPPNP show b a s i c a l l y t h e same p a t t e r n
o f P i r e l e a s e f r o m F1 ( F i g u r e 1 a n d R e f .
1 2 3 ) , however t h e
s e n s i t i v i t y of P i r e l e a s e t o ATP i s g r e a t e r ( F i g u r e 1 ) .
F i g u r e 15 a n d T a b l e V I show t h a t t h e amount o f AMPPNP bound b y
F 1 i n c r e a s e s as t h e n u c l e o i i d e c o n c e n t r a t i o n i n t h e c o l u m n
increases.
A t h i g h e r AMPPNP c o n c e n t r a t i o n s
(i.e.
> 1 p ~ ) ,t h e
r e l e a s e o f P i f r o m F1 i s o b s e r v e d , t h u s t h e c o n f o r m a t i o n a l
c h a n g e w h i c h r e s u l t s . i n t h e release of P i has b e e n a c h i e v e d .
T h i s c a n be e x p l a i n e d i f it i s assumed t h a t more t h a n o n e AMPPNP
m o l e c u l e b i n d s t o F1 ( F i g u r e 2 9 ) .
I t w a s shown t h a t ATP a n d
AMPPNP b i n d t o F 1 e q u a l l y w e L l ( c . f .
T a b l e s I11 a n d V I ,
a n d see
"
F i g u r e 29
P i r e l e a s e f r o m F1 On e x p o s u r e t o h i g h AMPPNP
concentrations.
- Clb -
AMPPNP
i
2 AMPPNP
J/ slow
r
7
AMPPNP el
>
A~MPPNP>
AMPPNP
E
fast
AMPPNP)
,
-
i P @
Conversion of tightly
bound Pi to loosely
bound Pi .
a
AMPPNP
Ppi
AMPPNP.
AMppNpm
Conformational
change
Ref.
1 2 4 ) , anc3 t h a t AMPPNP d i d n o t c a h s e a r e l e a s e g r e a t e r t h a n
30% of t h e bound AMPPNP f r o m F1 ( ~ i g .l o ) , t h u s t h e n e t e f f e c t
i s t h a t AMPPNP a c c u m u l a t e s o n F1 i n t h e p r e s e n c e o f h i g h e r
AMPPNP c o n c e n t r a t i o n s .
There i s no h y d r o l y s i s of AMPPNP so i t ,
r e m a i n s bound t o F1 ( 1 2 5 , 1 2 6 ) .
C r o s s and N a l i n
( 4 8 ) h a v e demon-
s t r a t e d t h e p f e s e n c e o f t h r e e r e a d i l y e x c h a n g e a b l e AMPPNP
b i n d i n g s i t e s t h a t a r e d i s t i n c t from t h r e e v e r y s l o w l y e x c h a n g e able AMPPNP b i n d i n g s i t e s o n F1,
s o t h e F1.Pi
complex c a n b i n d
a maximum o f t w o AMPPNP m o l e c u l e s a t t h e c a t a l y t i c s i t e s .
The
s e c o n d AMPPNP m o l e c u l e b i n d i n g a t t h e t h i r d s i t e would be d o i n g
so w i t h a l o w e r a f f i n i t y ; C r o s s and N a l i n found one h i g h
a f f i n i t y s i t e , K d = 18 nM,
= 1.0
pM (48).
and t w o l o w e r a f f i n i t y sites, K d
P e n e f s k y ' s o b s e r v a C i o n t h a t AMPPNP was more
e f f e c t i v e t h a n ATP i n i n h i b i t i n g P i b i n d i n g t o kl
Ref.
7 3 ) c a n be e x , p l a i n e d as o u t l i n e d b e l o w .
(Figure 9 of
AMPPNP i s n o t v e r y
e f f e c t i v e i n p f o m o t i n g t h e release o f bound AMPPNP f r o m F1
(Figure
lo),
whereas ATP i s v e r y e f f e c t i v e i n p r o m o t i n g t h e
r e l e a s e of hound ATP ( i . e . h y d r o l y s e d ATP)
f r o m F1 ( F i g u r e 1 3 ) .
i
T h e r e f o r e , when F1 i s i n c u b a t e d w i t h AMPPNP f e w e r b i n d i n g s i t e s
are a v a i l a b l e f o r P i b i n d i n g (see F i g u r e 1 5 , and T a b l e s V and
VI).,
The o r d e r o f e f f e c t i v e n e s s o f t h e n u c l e o t i d e s i n p r o m o t i n g
t h e r e l e a s e , o f P i f r o m F1 i s ATP > AMPPNP > > ADP.
- e; -
--
T h e f i n d ' i n g t h a t ATP q u e n c h e s t h i a u r o v e r t i n f l u o r e s c e n c e ,
whereas A M P P ~ ~ P( a s t r o n g , c o m p e t i t i v e i n h i b i t o r of ATPase
_--
a c t i v i t y ) . does n o t ( 3 1 , 1 2 7 ) l e d F e r g u s o n et. al. to suggest t h e
'
p o s s i b i l i t y t h a t a u r o v e r t i n f l u o r e s c e n c e q u e n c h i n g may r e p r e s e n t
s o m e t h i n g more t h a n m e r e ATP b i n d i n g .
They s u g g e s t e d t h a t
- s u b u n i t i n t e r a c t i o n s may be o c c u r r i n g .
This proposal
sub\
may w e l l e x p l a i n why ATP is more e f f e c t i v e t h a n AMPPNP i n
$
.
p r o m o t i n g t h e release o f P i f r o m F1.
( I n theapreincubation
mode, t h e s l o p e s . of t h e P i ' r e l e a s e c u r v e s a r e 1 4 0 % . ~ ~ - l
( F i g u r e 6 ) , and 1 3 % . p ~ - 1 ( F i g u r e 7 ) , w i t h ATP a n d AMPPNP,
\ respectively.
T h u s ATP i s a b o u t 10 t i m e s m o r e e f f e c t i v e t h a n
.
AMPPNP i n p r o m o t i n g P i r e l e a s e f r o m F1 )
Release of Pi from P1:
Pulse-Chase ~ e t h o d
r
The p u l s e - c h a s e
method o f f e r e d t h e o p p o r t u n i t y t o s t u d y t w .
4
e f f e c t o f t h e n u c l e o t i d e s on t h e r e l e a s e o f P i from F1 when
ADP w a s a l s o p r e s e n t a t t h e c a t a l y t i c s i t e .
-
Chase-ATP . w a s
e f f e c t i v e i n a c h i e v i n g the c o n f o r m a t i o n a l c h a n g e which r e s u l t e d
i n t h e e x p u l s i o n o f P i and ADP from F1 ( F i g u r e s 6 , 8 , 9 ) .
F i g u r e 30 shows a s c h e m a t i c r e p r e s e n t a t i o n o f t h e p r o c e s s t h a t
may b e o c c u r r i n g :
t h e pulse-ATP
( a t e q u i l i b r i u m w i t h ADP, P i )
i s bound a t o n e s i t e , and t h e chase-ATP b i n d s a t a n o t h e r s i t e
(61,62,103,104);
-'-'-,
t h i s l a t t e r e v e n t t h e n e f f e c t s t h e re
lease o f
4.
h y d r o l y s e d pulse-ATP
( i . e . ADP,Pi) from F1.
Thus t h e p r e s e n c e
o f ADP a t t h e s a m e s i t e w i t h P i d l d n o t p r e v e n t P i release
n o r ATP b i n d i n g a t a n a l t e r n a F e s i t e .
The r e s u l t s o b t a i n e d w i t h chase-ADP
a r e e x p l a i n e d , as f o r
t h e p r e i n c u b a t i o n s t u d i e s d i s c u s s e d a b o v e , by t h e f a i l u r e o f F1
t o b i n d ADP u n d e r t h e ' s e e x p e r i m e n t a l c o n d i t i o n s ( F i g u r e 2 3 ) .
The s m a l l P i r e l e a s e d o b s e r v e d ( F i g u r e 8 ) c a n b e a c c o u n t e d f o r
by t h e p r e s e n c e o f t r a c e amounts o f ATP i n t h e s e ADP p r e p a r a t i o n s ( e .g.
i.e.
100 pM ADP s o l u t i o n may c o n t a i n a b o u t 1 . 4 8 ATP,
1 . 4 p M ATP, w h i c h a c c o u n t s f o r t h e r e s u l t s shown i n F i g u r e
8) (103,123).
rb
F i q u r e 30
and ADP release from F1 on t h e b i n d i n g o f c h a s e - A T P .
--
, - ~
(chase)
Conformational
'
Change
I
ADP Pi
Pi
.-
Conversion of tightly
bound ADP and Pi to
loosely bound ADP
a n d Pi
-
+
90
Figures 7 and 8 show t h a t AMPPNP was able t o > e f f e c t t h e
r e l e a s e of P i from F l i n t h e presence of ADP a t thy c a t a l y t i c
(
si%e.
In a d d i t i o n , AMPPNP was about equally e f f e c t i v e a s ATP i n
.f>.
promoting &he r e l e a s e of P i from F I i n t h e .presence of ADP
(F'igure 8.).
Figure 31 i l l u s t r a t e s the process t h a t may be
The -Fl now has L o
occurringwon t h e binding of BMPPNP.
.
nukleotihe-binding s i t e s f i l l e d , one with ADP p l u s P i a t
f
equiliibrium with ATP (61.62). and t h e o t h e r with AMPPNP (an
analog
bf
ATP ) ; therefore-We
!
conformational change i s achieved
a s when chase-ATP i s bound ( c . f . Figures 30 and 3 1 ) .
Since t h e
e f f e c t s of ATP and AMPPNP on t h e r e l e a s e of hydrolyzed ATP were
s i m i l a r (Figures 6 - a ) , hydrolysis of chase-ATP ( o r n u c l e o t i d e )
i s not necessary f o r . t h e r e l e a s e of hydrolyzed pulse-ATP.
i s necessary i s f o r a second A T P - b i n d i n g
What
s i t e t o be f i l l e d .
6.
Thus binding of ATP
or^ AMPPNP ( i . e . g u b s t r a t e o r s u b s t r a t e
ana16g) a t another s i t e i s s u f f i c i e n t t o e f f e c t the'conformat i o n a l change which r e s u l t s i n t h e r e l e a s e of P i from another
'
A
s i t e on. F1 which a l s o contains bound'
WP.
I n t h e presence of
L
3
ADP a t - t h e + c a t a l y t i cs i t e , t h e ' o r d e r of e f f e c t i v e n e s s of t h e
I
.-
nucleotides~in~promoting
t h e r e l e a s e of P i from F1 i s ATP =
>
AMPPNP > > ADP.
+
In Figures 6 and 13, t h e slopes ( 4 6 and 4 5 % . p ~ - 1 r, e s p e c t i v e . 1 ~ )of t h e ? P / P ~ r e l e a s e c u b e s .of t h e ATP-chase experi-
.
,
-
87a
-
F i g u r e 31
P i a n d W P release from F1 o n t h e b i n d i n g o f c h a s e
AMPPNP
.
(chase)
r:
2 AMPPNP
iDP
AMPPNP l
I
Conformational
change
ADP p i
AMPPNPm
AMPPNP
Conversion of tightly
bound ADP and Pi to
loosely bound ADP
and Pi
m e n t s w e r e approximately,,.equal:
and i n a d d i t i o n t h e y w e r e a l s o
e q u a l t o t h e s l o p e s ( 4 5 % . p ~ - 1i n b o t h c a s e s ) o f t h e A D P / P ~
release c u r v e s o f t h e jAMPPNP-chase
7 and 14.
e x p e r i m e n t s shown i n F i g u r e s
This demonstrates thfiuiva.lent.
concentrations of
ATP a n d AMPPNP i n t h e chase h a d t h e same e f f e c t ( ~ i g l i r e8 ) .
\
'
Whereas, a comparir.dn o f t h e s l o p e s ( 1 2 0 a n d l 7 0 % . ~ ~ -r e~s ,p e c t i v e l y ) o f t h e chase-ATP
b i n d i n g c u r v e ( F i g u r e 1 3 ) and t h e chase-
AMPPNP b i n d i n g c u r v e ( ~ i g u r e1 4 ) , i n d i c a t e s t h a t more AMPPNP
t h a n ATP i s b i n d i n g p e r m o l e o f F1 a t a n e q u i v a l e n t n u c l e s t i d e
c o n c e n t r a t i o n (see also T a b l e s V and V I ) .
4
t h e s l o p e s o f chase-ATP
t o chase-AMPPNP
However,
the r a t i o of
b i n d i n g - (120/170 o r 0 . 7 )
i s n o t o f s u c h a m a g n i t u d e t o s u g g e s t t h a t l a b e l l e d FI i s
i
4
1
b i n d i n g more t h a n o n e AMPPNP m o l e c u l e a t t h e s e n u c l e o t i d e .
concentrations.
S i n c e t h e r a t i o o f t h e s l o p e s o f t h e chase-ATP
b i n d i n g c u r v e t o t h a t o f t h e ATP, ( i .e. ADP, P i ) - r e l e a s e
(Figure 13) is approximately 2.8
,
( i . e . 1 2 0 % .y'bl-l
I
curve
4 3 ~ . ~ )~) ,- l
a n d n o t 1, it must be t h a t chase-ATP m o l e c u l e s ' a r e b i n d i n g t o F1
1
w i t h o u t pulse-ATP
molecules.
Grubmeyer a n d P e n e f s k y ( 6 1 , 6 2 ) h a v e f o u n d t h a t h y d r o l y z a b l e
n u c l e o t , i d e s s u c h a s ATP, GTP, and ITP a r e e x c e l l e n t promoters o f
4
h y d r o l y s i s or release of p r e v i o u s l y bounh T N P - [ ~ - ~ ~ P ] A T P where,
a s non-hydrolyzable
n u c l e o t i d e s s u c h as TNP-ADP,
g i v e lower r a t e s a n d e x t e n t s o f h y d r o M s i s .
WP,
and AMPPNP
In t h e experiments
reported here, however', the non-hydrolyzable nucleotide, A M P P N P ,
I
which bound t o F I was almost as e f f e c t i v e as t h e hydrolyzable
'
b
nucleotide, ATY, i n promoting t h e r e l e a s e of previously bound
' P i and ADP a t equilibrium w i t h ATP.
Grubmeyer and Penefsky
,;i
( 6 2 ) were using higher concentrations of nucleotides ( e . 9 . 100
p M AMPPNP,
1
mEJ1
ADP,
and 140 pM ATP-ADP
6 2 ) ) i n t h e i r experiments.
( s e e Figure 5 of Ref.
( I n t h e pulse-chase s t u d i e s reported
here, lower nucleotide concentrations (below 2 p M ) were a b l e t o
promote t h e r e l e a s e of P i and ADP (Figures 6 - 8 ) ) .
I t i s also
p o s s i b l e t h a t t h e i r observations a r e equ'ivalent t o t h e observa-
4
t i o n s made i n the l e s s s e n s i t i v e phase of r e l e a s e seen i n both
preincubation and pulse-chase modes of t h e s t u d i e s reported
herein.
-
90
-
Comparison of the Results of Preincubation and Pulse-Chase
Studies
I t a p p e a r s t h a t ATP w a s m o r e e f f e c t i v e i n p r o m o t i n g P i
r e l e a s e f r o m F1 i n p r e i n c u b a t i o n e x p e r i m e n t s t h a n i n p u l s e - c h a s e
experiments (Fig&e
b),
'Find a s c h e m a t i c d i a g r a m o f e a c h p r o c e s s
The t w o p r o c e s s e s
i s shown i n F i g u r e s 26 a n d 30, r e s p e c t i v e l y .
a r e ' s i m i l a r e x c e p t f o r t h e p r e s e n c e o f ADP a t t ' h e P i b i n d i n g
T
site i n t h e pulse-chase
experiments.
Since t h e s e n s i t i v i t y of
t h e P i r e l e a s e t o ATP i s a p p a r e n t l y g r e a t e r i n t h e p r e i n c u b a t i o n than i n t h e pulse-chase
experiments,
P i r e l e a s e is
i.e.
g k e a t e r i n t h e a b s e n c e t h a n i n t h e p r e s e n c e o f ADP a t t h e same
c a t a l y t i c s i t e , it appears t h a t ADP a t t h e s a m e c a t a l y t i c s i t e
w i t h P i i s i n f l u e n c i n g t h e P-elease
reaction.
I n F i g u r e 6 , the s l o p e s of t h e P i r e l e a s e c u r v e s are
<
1 4 0 % . p ~ - l( P r e i n c u b a t i o n
respectively;
ode) a n d 45%.pM-l
( p u l s e - c h a s e Mode),
and t h e r a t i o o f t h e s l o p e s ( ~ r e i n c u b a t i o n / ~ u - l s e -
c h a s e ) i s a p p r o x i m a t e l y 3 ( 1 4 0 % . p ~ - l t 4 5 % . p ~ - 1 ) . [N.B.
In the
p r e i n c u b a t i o n mode, t h e m o l e o f l a b e l b o u n d / m o l e o f F1 i s
a p p r o x i m a t e l y 0 . 2 t o O e . 3 ; w h e r e a s i n khe p u l s e - c h a s e
mode,
m o l e o f l a b e l b o u n d / m o l e o f F1 i s a p p r o x i m a t e l y 0 . 1 ,
before
exposure to nucleotide. }
One l i k e l y e x p l a n a t i o n w h i c h may
=Y
a c c o u n t f o r t h e l e s s e f f e c t i v e r e l e a s e of P i i n t h e p u l s e c h a s e e x p e r i m e n t s t h a n i n p r e i n c u b a t i o n e x p e r i m e n t s is
the
*
c o m p e t i t i o n between l a b e l l e d and u n l a b e l l e d F1 f o r t h e chase.
,
ATP.
T h a t t h e r e i s c o m p e t i t i o n b e t w e e n l a b e l l e d and u n l a b e l l e d
F1 f o r ch2se-ATP i s s u b s t a n t i a t e d by F i g u r e 1 3 and Table V .
The
s l o p e o f t h e chase-ATP b i n d i n g c u r v e i s 1 2 0 % . p ~ - 1 , a n d t h a t o f
t h e P i r e l e a s e c u r v e i s 43%.ph-l ; t h u s t h e r a t i o o f ATP
b i n d i n g t o P i r e l e a s e i s a p p r o x i m a t e l y 3 ( 1 2 0 % . ~ ~ -+l
43%.p ~ - \ ) , and t h e r e f o r e c h a s e - A T P
is very l i k e l y also binding
t o u n l a b e l l e d F1.
F i g u r e 7 shows t h a t AMPPNP w a s more e f f e c t i v e i n f a c i l i t a t i n g t h e r e l e a s e of P i i n t h e p u l s e - c h a s e
mode t h a n i n t h e
t.
p r e i n c u b a t i o n mode, t h i s i n s p i t e o f t h e g r e a t e r c o m p e t i t i o n by
u n l a b e l l e d F1 f o r C ~ ~ S ~ - A M P P Ni nP t h e p u l s e - c h a s e
experiment.
I n t h e p r e i n c u b a t i o n e x p e r i m e n t s , r e l a t i v e l y l o w amounts o f
-
1
2
AMPPNP w e r e n o t e f f e c t i v e i n p r o m o t i n g P i release from F1
-i
( F i g u r e 2 ) , w h e r e a s h i g h e r amounts o f AMPPNP were e f f e c t i v e
B
(Figure 1).
,
These r e s u l t s a r e d i a g r a m e d i n F i g u r e s 27 and 2 9 ,
r e s p e c t i v e l y , ' a n d t h e e f f e c t o f AMPPNP i n p u l s e - c h a s e
ments i s diagr,amed i n F i g u r e 3 1 .
experi-
A comparison o f F i g u r e s 29 and
31 r e v e a l s t h a t t h e t w o processes are th,e s a m e e x c e p t t h a t i n
o n e case ( p r e i n c u b a t i o n mode) t w o AMPPNP m o l e c u l e s a r e r e q u i r e d ;
w h e r e a s i n t h e o t h e r case ( p u l s e - c h a s e mode) o n e AMPPNP m o l e c u l e
i s r e q u i r e d ; and i n a d d i t i o n , ADP i s a b s e n t a t t h e P i b i n d i n g
,
.
s i t e i n F i g u r e 29 and p r e s e n t 'at ,the P i b i n d i n g s i t e i n F i g u r e
31.
Thus t h e d i f f e r e n c e shown by t h e two p r o c e s s e s may be
a t t r i b u t e d t o t h e ADP a t t h e F i b i n d i n g s i t e .
I n F i g u r e 7,
t h e s l o p e s of t h e P i r e l e a s e curvek of t h e p r e i n c u b a t i o n and
pulse-chase modes a r e 1 3 and 4 S % . p ~ ' 1 r
r e s p e c t i v e l y ; hence from
t h e r a t i o of t h e s l o p e s (Pulse-chase:Preincubation, i . e .
43:13),
t h e r e l e a s e i s about 3 times g r e a t e r i n t h e pulse-chase mode
than i n t h e p r e i n c u b a t i o n mode.
I t is therefore easier for
AMPPNP t o e f f e c t t h e confoYmationa1 change (which
Xs u l t s
i n the
r e l e a s e of P i ) , i f another n u c l e o t i d e ( i . e . ADP) i s p r e s e n t a t
t h e P i binding s i t e .
1
Helease of AHPPHP from F1:
Pulse-Chase Methods
Chase-AMPPNP w a s n o t v e r y e f f e c t i v e
pulse-AMPPNP
( < 3 0 % ) from F1
(Figure 10).
i h
e x p e l l i n g t h e bound
AMPPNP i s n o t
h y d r o l y z e d by F 1 , h e n c e a l l t h e AMPPNP bound i n b o t h p u l s e a n d
c h a s e a c c u m u l a t e s on F 1 .
( A s mentioned b e f o r e ,
F1 c a n c o n t a i n a
maximum o f t h r e e AMPPNP m o l e c u l e s a t t h e e x c h a n g e a b l e n u c l e o t i d e
binding sites (48)).
Table V I supports t h e hypothesis t h a t
?
chase-AMPPNP i s bound and a c c u m u l a t e d on F1 as t h e chase-AMPPNP
concentratioh increases.
non-release
AMPPNP:
F i g u r e 32 A f f e r s a scheme f o r t h e
of pulse-AMPPiqP
t h e pulse-AMPPNP
f r o m F1 on t h e b i n d i n g o f chase-
i s n o t h y d r o l y z e d a n d it i s n o t
r e a d i l y c h a n g e d from b e i n g t i g h t l y bound t o l o o s e l y - b o u n d
these experimental conditions).
(under
T h i s r e l a t i v e i n a b i l i t y of
AMPPNP t o p r o m o t e t h e r a p i d d i s s o c i a t i o n o f AMPPNP f r o m
F1 .AMPPNP
complex was a l s o f o u n d by N a l i n a n d C r o s s ( 1 0 2 ) .
f i n d i n g l e n d s s u p p o r t t o t h e p r o p o s a l of , F e r g u s o n et. al.
This
(127)
t h a t t h e b i n d i n g o f AMPPNP i s d i f f e r e n t f r o m t h a t o f ATP i n t h a t
ATP i s more r e a d i l y a b l e t o e f f e c t s u b u n i t - s u b u n i t
interactions.
e
Fiqure 32
The binding of pulse-AMPPNP and chase-AMPPNP to F 1 .
A:K
(pulse)
, ,
(chase)
A
p
N
{:i
>AMPPNP
AMPPNP<
< AMPPNP
Slow
Conformational
Change
PPNP
AMPPMP
S e v e r a l c o n c l u s i o n s w e r e made from t h i s wbrk.
included:
These
(1) The o r d e r o f e f f e c t i v e n e s s o f t h e n u c l e o t i d e s
(ATP, ADP, a n d AMPPNP) i n p r o m o t i n g t h e r e l e a s e o f a p p r o x i m a t e l y
70% ( i . . e . ,
t h e s t e e p p h a s e o f t h e b - i p h a s i c r e l e a s e ) o f t h e bound
P i ( 0 . 2 mole P i / m o l e F1 ) f r o m F1 was ATP > AMPPNP > > ADP.
( i i ) O n l y b i n d i n g o f ATP or AMPPNP ( s u b s t r a t e or s u b s t r a t e
a n a l o g ) a t an a l t e r n a t i v e s i t e ( h y d r o l y s i s o f incoming nucleot i d e was n o t n e c e s s a r y ) w a s e s s e n t i a l t o p r o d u c e t h e c o n f o r m a v
t i o n a r c h a n g e
lp
_
be h y d r o l y z e d and r e l e a s e d i n p r e f e r e n c e t o t h e r e l e a s e o f t h e
p r e v i o u s l y bound n o n - h y d r o l y z a b l e
a
C'
AMPPNP.
T h i s may b e l o o k e d a t
by d o i n g t h e ' a p p r o p r i a t e c o n t r o l s a n d / o r t e s t s , e . g . ,
funning
columns w i t h o u t AMPPNP i n t h e p u l s e f o r , e a c h ATP c o n c e n t r a t i o n
.
u s e d t o see hoy much ATP i s bound,
,
and examining t h e bottom p a r t
A3
o f t h e 'column f o r ~
*
Ref
., 1 1 4 ) .
'
~
and
p ~[ yp - ~3 2 ~ (see
~ ~ Methods
~ ~
and
~ l t e r n a t i v e l y ,comparable q u a n t i t i e s o f l a b e l l e d
AMPPNP c a n , b e p l a c e d i n t h e c h a s e ( u n l a b e l l e d AMPPNP i n t h e
I
p u l s e ) t o d e t e r m i n e haw much AMPPNP is bound unde,r t h e s e c o n d i tions.
ASsuming t h a t ATP 'and AMPPNP b i n d e q u a l l y w e l l t o F 1 ,
i,;, c a n be d e t e r m i n e d how much ATP s h o u l d b i n d i n t h e c h a s e .
... ATP t h a n e x p e c t e d i s f o u n d on F 1 , it c a n b e assumed
~ h u si f less
-
'
** 6
t h a t t h e I ~ wPa s bound b u t w a s h y d r o l y s e d a n d r e l e a s e d p r e f e r e n -
.
,
m
-
99
-
>
a
2.
T h e s e n s i t i v i ' t y o f r e l e a s e o f * P i a n d AOP ( b o t h f r o m
h y d r o l y z e d ATP) f r o m . F1 by' i n c o m i n g ATP w e r e shown t b . ' b e s i m i l a r
r
i n these' s t u d i e s ( F i g u r e 9 ) .
However, t h e r a t e o f releaqe o f
C
.
b o u n d ADP ( t h e o t h e r p r o d u c t o f ATP h y d r o l y s i s by Fl .t was n o t
e x t e n s i v e l y s t u d i e d as w a s d o n e w i t h bound P i .
~ h e sit i s
p r o p o s e d t o i n v e s t i g a t e t h e r e l e a s e o f bound ADP ( i . e z i n t h e
a b s e n c e o f P i ) f r o m F1 o n e k p o s u r e t o ATP a n d APlPPNP.
-
In
P
preincubation studies:
t h e F l w i l l be p r e i n c u ' b a t e d w i t h
before a p p l i c a t i o n t o c o l u m n s w i t h A'I'P,
1abelled'AUP
d
o r AMP&
i n t h e middle s e c t i o n s ( c f . Figures 1
.
*
'-
( P r e c a u t i o n s w i l l be t a k e n t o ' e n s u r e t h a t t h e s m a l l amount o f
ATP i s removed f r o m t h e AUP s o l u t i o n s ( 3 1 ) ) .
The > e s u l t s of
t h e s e f i n d i n g s w i l l be compared w i t h t h o s e i l l u s t r a t e d i n
F i g u r e s 1-5.
I f t h e r e s u f t s are s i m i l a r , t h i s would i n d i c a t e '
t h a t W P and P i d o b i n d a t t h e " s a m e s i t e , s i n c e t h e
c o n f o r m a t i o n a l 'change e f f e c t e d by t h e incoming n u c l e o t i d e should
t
s i m i l a r i n b o t h cases.
T h e s e r e s u l t s would a i s o t e l l of t h e r e l a t i v e e f f e c t i v e n e s s
t h e t w o n u c l e o t i d e s (ATP a n d A M P P N P ) i n p r o m o t i n g t h e r e l e a s e
ADP f r o m F1.
I t w a s p r o p o s e d i n F i g u r e 31 t h a t AMPPNP was
j u s t as e f f e c t i v e as ATP, i n p r o m o t i n g t h e r e l e a s e o f P i f r o m
F1 i n t h e p u l s e - c h a s e
mode ( F i g u r e s 6 ;a n d 7 ) b e c a u s e ADA?
--
(another nucleotide) w a s present on F 1 6
/
T h e r e f o r e i n these
* ,
e x p e r i m e n t s , , it i s r e a s o n a b l e t o e x p e c t t h a t AMPPNP be' j u s t A s
i n p r o m o t i n g t h e r e l e a s e o f ADP f r o m F 1 .
e f f e c t i v e a s ATP
,
~ h u s
'
t h e p r o p o s e d mechanism of P
I
li release i n
t h e p r e s e n c e o f ADP b y
&MPPNP ( F i g u r e 3 1 ) c a n be ' f u r t h e r e n h a n c e d . o r be c a l l e d i n t o
question.
using pulse-chase
experiments, the r o l e of P i on the
r e l e a s e o f ADP i n t h e p r e s e n c e o f i n c o m i n g h u c l e o t i d e s w i l l be
investigated.
i.e.
( l e e *
[
3
~
The - E l w i l l be p r e i n c u b a t e d w i t h l a b e l l e d ADP,
and
]
~a p p~l i e~d t o columns c o n t a i n i n g l a b e l l e d P i
C 3 * p I p i ) of varied concentrations i n
the p u l s e s e c t i o n s ,
a n d e i t h e r ATP o r AMPPNP ( f i x e d c o n c e n t r a t i o n ) i n t h e c h a s e
sections.
I f , as t h e P i c o n c e n t r a t i o n i n t h e p u l s e i n c r e a s e s ,
t h e r e l e a s e o f l a b e l l e d ADP on e x p o s u r e t o chase n u c l e o t i d e
d e c r e a s e s , t h i s would s u g g e s t ;;hat
>
t h e P i is binding a t e
c a t a l y t i c s i t e and t h u s p r e v e n t i n g the b i n d i n g o f c h a s e nucleotide.
The amount o f n u c l e o t i d e bound i n t h e chase i n t h e
a b s e n c e and p r e s e n c e of t h e P i p u l s e c a n be d e t e r m i n e d by
b
doing the a p p r o p r i a t e c o n t r o l experiments, i.e.
F1 i s n o t
p r e i n c u b a t e d w i t h l a b e l l e d ADP, b u t l a b e l l e d chase n u c l e o t i d e s
( C ~ H I A oTrP [
1
3
~
]
a ~r e u~ s e d~.
~Thus~ t h~e
e)x p e r i m e n t s o u t l i n e d
i,, t h i s s e c t i o n s h o u l d h e l p t o c l a r i f y t h e r o l e of ADP a t t h e
-
,.-,
-
c a t a l y t i c s i t e s a n d what i n f l u e n c e s i t s release f r o m F1.
-
3.
To show t h a t c a t a l y t i c s i t e s a r e i n v o l v e d i n t h e b i n d l
i n g and r e l e a s e o f n u c l e o t i d e s t o a n d f r o m F1, p u l s e - c h a s e
2
e x p e r i m e n t s w i l l be p e r f o r m e d as o u t l i n e d b e l o w .
The F1 w i l l be
vr
preincubated with l a b e l l e d ADP
and then a p p l i e d t o
'
columns which c o n t a i n l a b e l l e d ATP ( [ y - 3 2 ~ ] ~ ~ (Pf i) x e d concent r a t i o n ) i n t h e pulse-section
and unlabelled ATP ( v a r i a b l e
c o n c e n t r a t i o n ) i n t h e chase s e c t i o n .
The amount of each type of
- L-pulse-ATB-is expected
A
-
---
l a b e l on t h e F 1 w i l l be monitored.
The
e f f e c t t h e r e l e a s e of l a b e l l e d ADP;
and t h e chase-ATP i n t u r n i s
___---
/
-
-
expected- t o promote t h e r e l e a s e of
--
l a b e l l e d ADP )
.'
1
on ,twpdzyme
,
-L
.
--
pulse-^^^
to
( a s well a s
This experiment would demonstrate t h a t what got
can g e t o f f , i . e .
t h e pulse-ATP,
which e f f e c t e d
t h e r e l e a s e of p r e v i o u s l y bound ADP, can i n t u r n be released ( a s
I
ADP and P i ) from i t s binding s i t e by chase-ATP.
Thus i f
chase-ATP can e f f e c t t h e r e l e a s e of p u l s c f ~ ~t h~e,r e f o r e most
'I
l i k e l y binding of pulse-ATP and r e l e a s e of hydrolyzed pulse-ATP
( A D P and P i ) i s o c c u r r i n g a t t h e same s i t e ,
i.e.
a catalytic
site.
4.
S i m i l a r s t u d i e s a s those performed h e r e ( b o t h preincu-
b a t i o n and pulse-chase modes) can be c a r r i e d out w i t h chlorop l a s t , b a c t e r i a l , o t h e r mitochondria1 ATPases, and o t h e r
ATPases.
Since t h e s e enzymes a r e s i m i l a r or simi4ar mechanisms
I
may be involbed, t h e s t u d i e s w i l l be h e l p f u l i n r e v e a l i n g s i m i l a r i t i e s o r diffierences
.
which subunit-subunit
i n t e r a c t i o n s a r e thought t o be involved i n
Other complex multisubunit enzymes i n
t h e c a t a l y t i c p r o c e s s may be s i m i l a r l y i n v e s t i g a t e d .
Examples
_--
-
of such enzymes include Alkaline Phosphatase, Alcohol
Dehydrogenase, Succinyl-CoA Synthetase, Glyceraldehyde-3Phosphatase Dehydrogenase, and Malate Dehydrogenase (110,111).
APPENDIX I
--r
Development of the Modified Sephadex Centrifuqe Column Technique
Introduction
I n a n i n v e s t i g a t i o n o f t h e e f f e c t o f n u c l e o t i d e s (ATP and
ADP) o n t h e r e l e a s e o f bound P i f r o m F 1 , t h e e x p e r i m e n t o f
H u t t o n a n d Boyer w a s r e p e a t e d .
I
The r e s u l t s shown i n F i g u r e 25
w e r e s i m i l a r t o t h e i r s ( F i g . 2 o f Ref. 9 5 ) .
It w a s c o n c l u d e d
3
t h a t both,- ATP and ADP f a c i l i t a t e d t h e d i s s o c i a t i o n of P i f r o m ,
1F l ,
a n d t h a t t h e e f f e c t s o f ATP and ADP w e r e n o t d i s t i n g u i s h a b l e
*
b e l o w 15 seconds.
P e n e f s k y had p r e v i o u s l y f o u n d t h a t P i b i n d s
,
r e v e r s i b l y t o F1 w i t h a h a l f - l i f e
whereas the h a l f - l i f e
9
of about 2 minutes ( 7 3 ) ,
f o r release of P i i n t h e p r e s e n c e of
n u c l e o t i d e s i s s h o r t e r ( R e f . 96 a n d F i g . 2 5 ) .
f
Thus i n o r d e r t o d i s t i n g u i s h t h e e f f e c t s o f ATP a n d ADP o n
t h e r e l e a s e of P i from F1, a n o t h e r approach w a s used.
Some o f
the experiments performed i n the m o d i f i c a t i o n of t h e convention+
a1 S e p h a d e x ' c e n t r i f u g e column t e c h n i q u e a r e r e p o r t e d here.
Results and Discussion
The Effect of ATP-Containinq Gels in the Middle of the Columhs
F i g u r e 1 A o u t l i n e s t h e p r e p a r a t i o n o f a column w i t h a n ATPcontaining s e c t i o n i n the c e n t r e .
,
-
The r a t i o n a l e w a s t h a t t h e F1
i n moving t h r o u g h t h e column d u r i n g c e n t r i f u g a t i o n would have
-
Figure 1 A
O u t l i n e o f t h e m o d i f i e d ' s e p h a d e x c e n t r i f u g e column
technique:(a)
Preparation of t h e g e l s .
prepared (28,73).
I n o n e s e t t h e e q u i l i b r a t i o n b u f f e r of t h e ,
Sephadex c b n t a i n e d :
and 47 pM P i .
Two s e t s o f Sephadex columns w e r e
90 mM T r i s - a c e t a t e ,
1 . 6 mM MgSO,,
,
The o t h e r s e t had ATP added t o t h e e q u i l i b r a -
t i o n buffer.
(b)
p H 7.5,
Removal o f g e l s .
The g e l s w e r e removed 6rom t h e column
b a r r e l by $ e c a n t i n g g e n t l y .
two h a l v e s @ 1 . 5 c m ) ,
-
The g e l w i t h o u t ATP was c u t i n t o
and from t h e ATP-containing
gel was
zi~.t
a measured l e n g t h .
(c)
Assembly o f co1urr.n.
One o f t h e
gels
was p l a c e d i n s i d e t h e column b a r r e l .
i
p l a c e d on t o p o f t h e p r e v i o u s l y i n s e r t e d g(l. L a s t l y , t h e o t h e r
\
b u f f e r e q u i l i b r a t e d g e l w a s p l a c e d on t o p
I
I
gel. .
( d ) .Column a s s e m b l y .
/
the ATP-containing
u
The r e a s s e m b l e d c o l u h d w a s p l a c e d i n a 1 5
mL c o n i c a l c e n t r ' i f u g e t u b e , and w a s t h e n r e a d y f o r u s e .
,
Column 1 is equilibrated with the
appropriate buffer
~ & m n 2 is equilibrated with
nucleotide in buffer
The pieces of gels are arranged
as shown above and then
reassembled in a column
Gels are removed from columns
Gel 1 is cut into halves
From gel 2, x cm is cut
The reassembled column is
placed in a centrifuge tube
-3
o n l y b r i e f c o n t a c t w i t h t h e ATP o f the A T P - c o n t a i n i n g
It
gel.
w a s e x p e c t e d t h a t t h i s r e l a t i v e l y b r i e f e x p o s u r e would be enough
f o r t h e ATP t o e x e r t i t s e f f e c t ( s e e F i g .
25).
F i g u r e 2A shows t h a t as t h e c o n c e n t r a t i o n o f ATP i n t h e 1 . 0
-
c m middle ATP-containing
s e c t i o n o f t h e column i n c r e a s e d , s o d i d
t h e r e l e a s e o f bound P i from F1.
T h i s c l e a r l y demonstrated
t h a t t h e F1 h a d a c c e s s t o t h e ATP o f t h e A T P - c o n t a i n i n g
section.
'.
middle
However, a l l o f t h e bound P i w a s n o t removed from F1
d e s p i t e t h e h i g h c o n c e n t r a t i o n (400 mM) o f ATP u s e d .
F.igure 3A shows ' t h a t on i n c r e a s i n g t h e l e n g t ' h o f t h e ATPc o n t a i h i n g m i d d l e s e c t i o n , t h e r e l e a s e o f bound P i from F1
a l s o increased.
From t h e r e s u l t s shown i n F i g u r e s 2A and 3A, it
$,
w a s i n f e r r e d t h a t t h e r e m o v a l o f a l l bound P i f r o m F1 m i g h t be
possible i f
( i ) t h e amount o f ATP i n t h e m i d d l e . s e c t i o n w a s
%
. ,lir
h i g h e r , o r (ii) t h e A T P - c o n t a i n i n g m i d d l e g e l w a s l o n g e r , o r
4%
( i i i ) t h e t i m e o f c o n t a c t b e t w e e n F1 and t h e ATP w a s l o n g e r .
Experiments w e r e performed w i t h t h e r e a s s e m b l e d columns
c o n t a i n i n g v a r i o u s f i x e d l e n g t h s of middle g e l s , w i t h each f i x e d
l e n g t h h a v i n g b e e n e q u i l i b r a t e d w i t h b u f f e r co-ng
c o n c e n t r a t i o n s o f ATP.
different
F i g u r e 4A shows t h e r e s u l t s o f t h e f i r s t
such e x p e r i m e n t . , ' i n which a 1 . 5 cm A T P - c o n t a i n i n g m i d d l e s e c t i o n
w a s used.
I t s h o u l d be n o t e d t h a t a v e r y h i g h c o n c e n t r a t i o n
( 4 5 8 rnM) o f ATP w a s u s e d ; and t h a t it w a s i m p r a c t i c a b l e t o u s e a
higher concentration i n t h
e e q u i l i b r a t i o n b u f f e r s i n c e t h e maxi,#
i
F i g u r e 2A
The e f f e c t o f ATP o n t h e release o f bound P i frorn F 1 .
T h e reassembled c o l u m n s w e r e p r e p a r e d as d e s c r i b e d i n F i g u r e
LA.
T?le c o n c e n t r a t i o n o f ATP i n t h e e q u i l i b r a t i o n b u f f e r u s e d
t o p r e p a r e the 1 . 0 c m middle ATP-containing
1 0 0 , a n d 400 mM).
ing:
3.7
gels was varied (0,
80 p L a l i q u o t s oS, a r e a c t i o n m i x t u r e c o n t a i n -
yM F 1 , ' 9 0 mM T r i s - a c e t a t e ,
47 yM P i w i t h [ 3 2 ~ ] ~ i( 1 . 3
x
lo6
pH 7 . 5 ,
1 . 6 mM MgS04r a n d
c p m / n m o l e ) . w h i c h h a d bie'n
a l l o w e d t o s t a n d f o r 30 m i n u t e s a t 23OC, w a s a d d e d t o t h e reasb
sembled c o l u m n .
C e n t r i f u g a t i o n (1050
immediately f o r 2 minutes.
control,
x
g ) was carried out
+
F o r e a c h ATP c o n c e n t r a t i o n a
i. e. n o ATP i n t h e m i d d l e g e l , w a s a l s o r u n s i m u l t a n e -
ously.
The 1 0 0 % P i bound c o r r e s p o n d e d t o a r a t i o o f 0 . 3 5 mo! e
pi/mole
F1.
ments.
Each p o i n t is the a v e r a g e o f d u p l i c a t e e x p e r i -
F i g u r e 3A
-
-
- F1.
The e f f e c t o f ATP on t h e r e l e a s e of bound P i from
.---ir
__
The r e a s s e m b l e d columns w e r e p r e p a r e d a s d g p i b e d i n F i g u r e
---
d
1A.
T h e c o n d i t i o n s and c o n c e n t r a t i o n s w e r e , t h e same as
t
d e s c r i b e d i n F i g u r e 2A, e x c e p t t h a t a c o n c e n F r a t i o n o f 100 mM
ATP was i n t h e e q g i l i b r a t i o n b u f f e r u s e d t o p;?epare
the differ-
.\
e n t l e n g t h s of t h e ATP-containing
middle g e l s .
\She s p e c i f i c
t
a c t i v i t y o f t h e C3*p1pi added t o t h e r e a c t i o n nfixthqe was 1 . 2
\
x
lo6
cpm/nmole.T
Fqr each l e n g t h a c o n t r o l ,
i.e.
a c'h-umn w i t h
\
' i
no ATP I n t h e m i d d l e g e l , w a s a l s o r u n s i m u l t a n e o u s l y .
he
\
P i bound c o r r e s p o n d e d t o a r a t i o . o f 0 . 1 7 mole ~ i / m o l eF I .
\
6
Each p o i n t i s t h e a v e r a g e o f d u p l i c a t e e x p e r i m e n t s .
100%
F i g u r e 4A
Y
The e f f e c t o f ATP on t h e r e l e a s e of bound P i from F 1 .
Th,e reassembled c o l u m n s were p r e p a r e d as d e s c r i b e d i n F i g u r e
>
1A.
~ 6 ceo n d i t i o n s
a n d c o n c e n t r a t i o n s w e r e t h e s a m e as d e s c r i b -
ed i n F i g u r e 2A, e x c e p t t h a t t h e l e n g t h o f t h e m i d d l e s e c t i o n
was k e p t c o n s t a n t a t 1 . 5 c m ,
a n d t h e ATP c o n c e n t r a t i o n was
.r
v a r i e d ( 0 , 1 0 0 , 458 mM) i n t h e e q ~ i l i b r a t h b- u f ~ * e o
rf the
.
*
middle g e l s .
The s p e c i f i c a c t i v i t y of t h e C3'F']pi
in the
i
/
r e a c t i o n mixture w a s 1.23
x
lo6 cpm/nmole.
150 pL a&
t h e r e a c t i o n m i x t u r e w e r e added t o the reassembled columns.
-b
1 0 0 8 P i bound c o r r e s p d n d e d t~ 0.18 m o l e ~ i / m o l e F 1 .
, •’
point. is t h e averageQ
'
d u p l i c a t e experiments.
Each
The
:@
mum s o l u b i l i t y o f ATP was b e i n g a p p r o a c h e d .
A c o m p a r i s o n of
L
3
F i g u r e s 2 A a n d 4 A shows t h a t as t h e l e n g t h of t h e A T P - c o n t a i n i n g
middle s e c t i o n i n c r e a s e d ,
s o d i d t h e amount o f P i r e l e a s e d
?,
\
\
c,
from F1.
I
I
-- +
E x p e r i m e n t s w i t h 2.0 c m A T P - c o n t a i n i n g
middle s e c t i o n s gave
r e s u l t s ( n o t shown) s i m i l a r t o those o b t a i n e d p r e v i o u s l y
4A).
H e r e even w i t h the i n c f e s e d
(Fig.
l e n g t h ( 2 . 0 c m ) o f t h e ATP-
m n t a i n i n g m i d d l e g e l ( 3 0 0 rnM ATP i n t h e e q u i ' l i b r a t i o n b u f f e r ) ,
n o t a l l t h e bound P i w a s r e l e a s e d f r o m F 1 .
Experiments w e r e
also performed w i t h a l o n g e r ATP-containing mid-section g e l ( 3 . 0
cm,
3,00 mM ATP i n t h e e q u i l i b r a t i o n b u f f e r ) , a n d a g a i n n o t a l l
t h e bound P i w a s removed f r o m F1 e q u i l i b r a t e d w i t h F 1 .
\
The r e a s s e m b l e d c o l u m n s w e r e s u b j e c t e d t o v a r i o u s c e n t r i f u g a l f o r c e s ( f r o m 420
No.
5, 'I.E.C.
g t o 1050
x
x
g , i.-e., S e t t i n g N o .
C l i n i c a l C e n t r i f u g e , Rotor 2 2 1 ) .
2 to
The o b j e c t i v e o f
;cr
t h i s e x p e r i m e n t w a s t o a l l o w l o n g e r e x p o s u r e o f t h e F1 t o ATF i n
the piddle section.
The e x p e r i m e n t a l c o n d i t i o n s a n d p r o c e d u r e s
were as d e s c r i b e d i n F i g u r e s 1 A a n d 2 A ,
mid-section
e x c e p t t h a t t h e 1.0 crn
g e l w a s e q u i l i b r a t e d , i n b u f f e r c o n t a i n i n g . 10'0 mM
For eachLspeed of c e n t r i f u g a t i o p i n v e s t i g a t e d , t h e t i m e of
ATP.
c e n t r i f u g a t i o n was kept constant (2 minute&).
I t was f o u n d t h a t
/
c e n t r i f u g a t i o n s a t 420
x
g , 630
w h i c h were d i f f i c u l t t o h a n d l e ,
x
g , a n d 840
i.e.,
x
/
-
-'
t h e s e g e l s were d i f f i c u l t
t o remove and r e a s s e m b l e w i t h o u t b e i n g b r o k e n .
6
g produced g e l s
Centrifugations
f
of r e a s s e y d i e d
I /
arnounf-if of
t
?
coluhins a t 630
x
g and 840 1( g g a v e n e g l i g i b l e
\ -\
>'
c e n t r i f u g a t ' e l - . The c e n t r i f u g a t i o n s a t 1 0 5 0
d u c e d g e l s which were e a s y t o work w i t h ,
x
g pro-
and w i t h t h e reassemblb
b
ed columns g a v e enough c e n t r i f u g a t e .
Thus a l l c e n t r i f u g a t i o n s
( p r e p a r a t i o n o f g e l s and i n v e s t i g a t i o n s w i t h F l ) w e r e a t 1050
x
t
I
I
g , h e n c e s p e e d o f c e n t r i f u g a t i o ~w a s e l i m i n a t e d as a v a r i a b l e t o
i n c r e a s e t h e c o n t a c t t i m e b e t w e e n F1 and ATP o f t h e m i d - s e c t i o n
'
i
\
c-1
gel.
D e t e r m i n a t i o n of the O p t i m u m L e n q t h s of G e l s for the T o p and
B o t t o m Sections of the Reassembled C o l u m n s
*
T h e t o p s e c t i o n o f t h e reassembled column is t o e n s u r e t h a t
o n l y P i bound t o F1 r e a c h e s t h e n u c l e o t i d e - c o n t a i n i n g m i d section gel.
F i g u r e 5A shows t h e r e s u l t s o f a n e x p e r i m e n t w i t h
v a r i o u s l e n g t h s o f b u f f e r - e q u i l i b r a t e d g e l s a l o n e i n t h e -column
(bottom g e l a d n u c l e o t i d e - c o n t a i n i n g m i d d l e g e l w e r e n o t
ff
used).
E v i d e n t l y , as t h e l e n g t h s o f t h e g e l s i n c r e a s e d , t h e
amount o f l a b e l i n t
'centrifugate
7
decreased.
Thus t h e L e f f g t h
o f t h e t o p s e c t i o n q n f l u e n c e d t h e removal o f f r e e , l o o s e l y , a n d
h
n o n - s p e c i f i c a l l y bou d l a b e l f r o m t h e a p p l i e d s a m p l e .
\
concluded t h a t a t o p
It was
t le3st 2 . 5 c m w a s r e q u i r e d .
h--""i
Table
IA, shows t h a t tpe [ " ~ ] P ~ - e ~ u i l ~ r % / ~
P1e ds a m p l e s g a v e c e n t r i -
-.
f u g a t e s w i t h h i g h e r amounts o f l a b e l q h a n t h e
C32~I~i-
v VT.
-
e q u i l i b r a t e d b u f f e r w i t h o u t F1.
c 32
~ ] ~bound
i
The d i f f e r e n c e was d u e t o t h e
t i g h t l y t o F l w h i c h w a s n o t removed by t h e g e l .
-
llla
-
Figure 5A
The e f f e c t of d i f f e r e n t l e n g t h s of b u f f e r - e q u i l i b r a t e d g e l s
-
---I
on t h e remov+'
f
of l a b e l from t h e r e a c t i o n mixtures.
The experi-
i
mental c o n d i t i o n s and procedures were t h e same a s described i n
Figures 1 A and 2A. except t h a t no bottom g e l nor ATP-confainitly
g e l was used.
.80 pL a l i y u o t s of each r e a c t i o n mixture ( i . e .
with and without F 1 ) was applied t o each column.
a c t i v i t y of t h e c 3 * p ] p i
cpm/nmole.
in
The s p e c i f i c
each . r e a c t i o n mixture was 1 . 4
and t h e c o n c e n t r a t i o n of F l was 1.85 p M .
x
lo6
A ' 25 (rL
a l i q u o t of each c e n t r i f u g a t e was counted t o determine t h e amount
of l a b e l t h a t passed through t h e column.
13*p1pi
t h a t passed through t h e col;mn
The 100% cpm of
cprresponded t o t h e
amount of l a b e l i n 25 pL of rea.ction miAture.
1
was performed i n d u p l i c a t e .
,
and
c 3 * p ] p i reaction
(
0 c 3 2p,Jpi
mixture).
E a c h experiment
.
e q u i l i b r a t e d buffer.
,
Table %A
The effect of different lengths of buffer-equilibrated gels on
the removal of label from the reaction mixtures.
Length of Gel
in Column
(cm)
3 cpm passed
through colurnry\
(25 V L c09'd)
~a.m~l,e'~p~l ied
t o c6lumn
f
\
'Pi
Pi
+
F1
Pi
P i + F1
Pi
Pi
\
+
F1
Experimental conditions and procedures were as described in
Figure 5A.
The bottom gel of t h e column is involved i n t h e uptake of
previously bound molecules which a r e released by Fl* during o r a f t e r t h e binding of nucleotide from t h e middle-section.
Figure
6 A and Table I I A show t h a t as t h e t o t a l length of t h e gel i n t h e
column increased ( i . e . ,
as t h e le,ngth of t h e bottom s e c t i o n
i n c r e a s e d ) , t h e amount of P i going through t h e column decreased.
I t was concluded t h a t a m i n i m u m length of 2.0 cm was
required f o r t h e bottom s e c t i o n , s i n c e with s h o r t e r bottom sect i o n s , the amount of l a b e l i n t h e c o n t r o l s ( i . e . ,
out F 1 ) was high.
samples with-
Even with t h e maximum 3.0 cm bottom s e c t i o n ,
4
a l l t h e bound P i was not removed from F 1 ; t h i s suggested t h a t
a bottom s e c t i o n g r e a t e r than 3.0 cm was needed t o allow t h e
complete removal of P i from F 1 .
However, t h e length of t h e
1.0 rnL t u b e r c u l i n syringe which was used a s t h e column b a r r e l
could not a c c o ~ o d a t emore than 6 . 5 cm t o t a l length of reassembled g e l s .
Use of a Lonqer Reassembled Column and the Determination of the
Parameters under which the Column Functions Best
A longer c
umn b a r r e l was made ( a s described under ~ e t h s & h )
I
t o accommodate the optimum lengths of t h e t h r e e s e c t i o n s , viz.1:
( i ) a 2 . 5 cm top g e l ,
cm bottom g e l .
li
/'
that
( i i ) a 1.0 cm middle g e l , and ( i i i )& 4.0
The 4.0 crn bottom s e c t i o n was t h e maxi&
/
could be used without any a l t e r a t i o n i n t h e lengths of t h e o t h e r
.
0
I
Figure 6A
The e f f e c t o f d i f f e r e n t l e n g t h s o f b o t t o m g e l s ;on t h e r e m o v a l
L
' o f label from t h e r e a c t i o n m i x t u r e s . '
The e x p e r i m e n t a l c o n d i -
t i o n s a n d c o n c e n t r a t i o n s u s e d w e r e as d e s c r i b e d i n F i g u r e 3 A ,
e x c e p t t h a t the l e n g t h s o f t h e t o p and middle g e l s w e r e 2 . 5 c m
d
and 1 . 0 cm,
respectively.
The c o n c e n t r a t i o n o f F1 i n t h e r e a c -
t i o n m i x t u r e w a s 1.89 pM.
For b o t t o m sec;ions
2 . 0 cm a n d
.
s h o r t e r , t h e s p e c i f i c a c t i v i t y o f t h e L3*p]pi i n t h e r e a c t i o n
m i x t u r e w a s 1.42
t h a n 2.0 c m ,
\x
lo6
cpm/nmole;
and f o r bottom s e c t i o n s l o n g e r
t h e s p e c i f i c a c t i v i t y of the L 3 * p I p i i n t h e r e a c -
t i o n m i x t u r e w a s 1.35
x
lo6
cpm/nmole.
average of d u p l i c a t e experiments.
XI
which p a s s e d t h r o u g h ,
0%
which p a s s e d t h r o u g h ,
and
c 0%
Each p o i n t i s t h e
.C3 * p d p i i n b u f f e r
[ 3 2 ~ ] ~ i n FI-reaction
a
r a t i o o f mole Pi/mole
mixture
F1 3
% CPM PASSEDTHROUGHOR (mole P,/ mole F,) BOUND
,/
Table I I A
--
The e f f e c t of t h e t o t a l length of the column on t h e lremoval of
l a b e l from t h e r e a c t i o n mixture.
Total
Length
of
Column
*
Length of
Bbttom
Section
(cm)
sample
Applied
t o Column
Label i n
2 5 pL
Centrifugate
( cpm
% of
mole P i
label*
i n 2 5 pL
Centrifugate
mole F k
100% l a b e l was t h e amount of l a b e l i n 2 5 p L r e a c t i o n mixture.
Experimental conditions and
were, as described i n
-----
Figure bA.
8
'\
C
,
two s e c t i o n s , because some space i n t h e column b a r r e l above t h e
top gel was required t o allow f o r the i n s e r t i o n of a cut 1.0 rnL
pipette t i p .
Thraugh t h e t i p t h e measured volume of t h e p a r t i c -
u l a r reaction mixture was added..
I t was found t h a t adding t h e
sample of the r e a c t i o n mixture d i r e c t l y t o t h e gel (immediately
before c e n t r i f u g a t i o n ) caused t h e r e s u l t s t o be somewhat l e s s '
i
reproducible.
Thus using t h e cut t i p on top of t h e column,
besides g i v i n g more reproducible r e s u l t s , allowed s i x ,columns t o
be prepared for simultaneous c e n t r i f u g a t i o n .
*
A n experiment was performed w i t h t h e optimum lengths ( 2 . 5 ,
1.0,
and 4.0 cm) of t h e t h r e e s e c t i o n s i n t h e longer column
barrel.
The r e a c t i o n mixtures and procedures were t h e same a s
described i n Figures 1A and 6A, except t h a t t h e volume of t h e
reaction mixture applied t o each via t h e cut p i p e t t e t i p was 100
pL.
The concentration of ATP i n t h e b u f f e r used i.n t h e prepara-
t i o n of t h e middle gel was 50 mM; and t h e concentratiqn of F1
-
and s p e c i f i c a b t i v i t y of t h e C 3 * p J p i i n t h e r e a c t i o n mixture
-
were 1.29 m g . m ~ - l
( o r 3.7 p M ) and 1.0
x
lo6
cpm/nmole, respect-
ivel&--- Without ATP i n t h e middle s e c t i o n of t h e column, t h e
experiment gave a r a t i o of 0.187 mole ~ i / m o l eF1.
I t showed
t h a t it was p o s s i b l e t o remove almost a l l of t h e bound P i from
F1.
This was s i m i l a r t o t h e r e s u l t s seen e a r l i e r when 2 . 5 and
3.0 cm bottom s e c t i o n s were used (Fig.. 6 A ) .
I t was decided t o
u s e t h e l e n g t h e ~ e dcolumn b a r r e l i n f u t u r e i n v e s t i g a t i o n s o f ' the
bound s p e c i e s o f F 1 .
However, w i t h t h e i h c r e a s e d l e n g t h o f t h e reassembled g e l s
i n t h e column, t h e o t h e r v a r i a b l e s w e r e i n v e s t i g a t e d t o d e t e r mine w h e t h e r o t h e r c h a n g e s w e r e r e q u i r e d .
were c o n s i d e r e d p r e v i o u s l y i n c l u d e d :
t h e r e a s s e m b l e d column,
T h e parameters t h a t
( i ) t h e t o t a l l e n g t h otf
( i i ) the d i f f e r e n t l e n g t h s of t h e w r e e
s e c t i o n s , and ( i i i ) t h e c e n t r i f u g a l f o r c e a n d t h e t i m e it was
applied.
A d d i t i o n a l f a c t o r s t h a t had t o be c o n s i d e r e d i n c l u d e d :
( i ) t h e amount o f F1 a p p l i e d t o t h e column,
( i i ) t h e volume o f
9
t h e r e a c t i o n i n i x t u r e ' a p p l i e d t o t h e column,
(ii'i)t h e amount of
n u c l e o t i d e and/or P i i n the middle s e c t i o n ,
(iv) the specific
f
a c t i v i t y o f t h e r a d i o l a b e l l e d c o m ~ d u s e d ,and ( v ) t h e i o n i c
c o m p o s i t i o n , pH, a n d t e m p e r a t u r e of t h e e q u i l i b r a t i o n b u f f e r s
a
and g e l s .
d
T a b l e I I I A s h o w s t h e e f f e c t o f u s i n g v a r y i n g amounts o f F 1
i n t h e m o d i f i e d S e p h a d e x c e n t r i f u g e column t e c h n i q u e .
The
volume o f t h e F1 r e a c t i o n m i x t u r e added t o t h e colurrir-wps k e p t
c o n s t a n t , w h i l s t t h e c o n c e n t r a t i o n (mg p r o t e i n . m ~ - l ) w a s v a r /
\ W i t h ATP i n t h e c o l u m n s ,
ied.
no g r e a t d i f f e r e n c e i n t h e amount
I
o f [ ! 2 ~ ] ~ i bound t o
%I
was o b s e r v e d w i t h the d i f f e r e n t c o n c e n -
I
t r a p i o n s o f F1 i n t h e r e a c t i o n m k x t u r e s ( T a b l e I I I A ) .
tpe
Values of
c o n c e n t r a t i o n s o f F1 l o w e r t h a n 0.63 mg p r 0 t e i n . m ~ - 1 d i d n o t
I
, j g i v e i r e p r o d u c i b l e r e s u l t s - ( t h e s e are n o t shown i n
i
/
.--
L
i
~ a b l kI I I A ) ;
Table I I I A
The e f f e c t O•’"ATP
on t h e rel!ease of P i from F1 when d i f f e r e n t
1
--. - i
mixture.
a m o u n k f p r o t e i n w e r e used i n
I
F1
(mg.m~-l)
B3A
(mg.rn~-l)
CATP 1
L a b e l i n '20 pL
i n Column C e n t r i f u g a t e
(mM)
"
(c.p.m.)
.
mole Pi
mole F1
7
The e x p e r i m e n t a l c o n d i t i o n s and p r o c e d u r e s w e r e as d e s c r i b e d
i n F i g u r e s 1 A and 6Al e x c e p t t h a t i n t h e re ssembled column o n l y ,
optimum l e n t h s e
used.
9.
l 2.5, 1
and 4.0 c m ,
respectiyely) were
The c o n c e n t r a t i o n o f ATP i n t h e e q u i 3 1 i b r a t i o n b u f f e r o f
m i d d l e g e l w a s 50 mM.
The volume o f r e a c t i o n m i x t u r e s a p p l i e d
J
t o t h e columns w a s 100 pL, and the volume o f c e n t r i f u g a t e
c o u n t e d was 20 pL. The s p e c i f i c a c t i v i t y of t h e C3*p]pi i n
t h e r e a c t i o n m i x t u r e s was 1 . 0 ~ ' 1 cpm/nmole.
6 ~
The e x p e r i m e n t
w a s performed i n d u p l i c a t e .
7
whereas w i t h 1 . 2 6 a n d ' 0.. 95 mg p r o t e $ n : m ~ ~ l F1
+ reaction mixtures,
I
*
t h e r e s i l t s w e r e* soi8ewhat m o r e ' r e p r p d u c i b l e .
It w a s d e c i d e d
t h a t t h i s lower, c o n c e h t r a t ' i o n of: F1 ( 0 . 9 5 rng p r o t e i n . mL-1 )
,,I
1.
Lu'
~ a b i eI I I A a l s o shows t h e
w o u i d be u s e d i n s u b s e q u g n k s t u b i e s .
*"
r e s u l t s when t h e t o t a l .B..
67..
<
21,
-
(1982). Biochemistry,
i
r-7
w
Schafer, G.,
(1982)
and S c h a p , G .
1 2 7 , ?91-299.
-
4073-4082.
"
J.,
,
!
and p e b e w T ( 1 9 8 2 ) J . B i o e n e r g .
E?.
Biomembr.,
L
-5
.
I
1 4 , 479-498.
68.
Tamura, J . K . ,
69.
Gresser, M.J.,
Rosen, G . ,
Matsuoka,
--
Chem.,
Chem.,
Vinkler, C.,
and Boyer, P . D .
- 10654-10661.
254,
-
Takeda, K . ,
Futai, M.,
and Novikova,
K O Z ~ O V I~. A . ,
(1'982) FEBS L e t t s . ,
I.Y.
!
73;
P e n e f s k y , H.S.
74.
Kasahara, M.,
and Tonumura, Y.
92, 1383-1398.
-
381-384.
'
and ,Boyer, P . D .
G.,
1
I.,
( 1 9 8 2 ) J . Biochem.,
72.
(1977) J. B i o l .
Chem.,
and Per?efsky, H.S.
Van G e l d e r , B. F . ,
Eds. ) pp.
252,
2891-2899.
( 1 9 7 7 ) i n " S t r u c t u r e and
295-305,
and P e n e f s k y , H.'s.
Kasahara, M.,
Lauquin, G.,
Pougeois,
Y.,
(1,978) J . B i o l .
Chem.,
?
R.,
and V i g n a i s ,
Biochemistry, 1 9 , 4620-4626.
...
and
E l s e v i e r , Amsterdam-.
--.
253, 4180-4187.
76.
150,
1
F u n c t i o n o f Energy T r a n s d u c i n g Membranes" ( v a n Dam,
75.
k
254, 10649-10653.
-
Gresser, M . J . ,
(1979) J. B i o l .
71.
( 1 9 8 3 ) ~ i o c h e m i s t r y , 22,
Cardon, J . , Rosen.
( l 9 7 9 ) , J. B i o l .
70.
-
and Wang,+ J . H .
P.V.
(1980)
,'
Maloney, P.C. .(1982)-, J. -Membr. Biol., 67, 1-12.
Papa, S. (1982) J. Bioenerg. Biomembr., 14, 69-86.
Ovchinnikov, Y.A., Abdulev, N.G. ,' and Modyanov, N.N.
(1982)
Ann. Rev. Biophys. Bioer~erg.,11, 445-463.
'
-
'80. Elthon, T.E.,
81.
and Stewart, 'c.R.
(1983) ~ioscience,11,
Nagle, J.F., and Tristam-Nagle, S. (1983) J. Membr. Biol.,
74, 1-14.
-
82.
Skulachev, V.P.
<
(1984) Trends. Biochem. Sci., 9, 182-185.
83. TGelson, N., and Cidon, S. (1984)
J. Bioenerg. Biomembr.,
* +
84.
Boyer, P.D., Cross, R.L., and Momsen, W. (1973) Proc.
I
i
Natl. Acad. Sci. USA, 70, 2837-2839.
i
\\
'L,
85.
Boyer, P.D. (1974) in "Dynamics of ~nergy-~ransducing
Membranes" (~rnster,L., Estabrook, R., and Slater, E.C.,
\~
-\
Eds. )
.-
i
.
86.
pp. 289-301, Elsevier, Amsterdam.
-.\
sLater,- E.C. (1974) in "Dynamics of Energy Transducing
'
.
Membranes" (Ernster, L., Estabrook, R., and Slater, E.C.,
Eds.) pp. 1;20,
87.
Elsevier, Amsterdam.
Cross, R.L., and Boyer, P.D. ~(1975)Biochemistry, 114,
4
*
/
7
I-'
'
--..
-88:
,
392-398.
-
9
Rosing, Je.1 Xayalar, C., . and Boyer, P.D. (1.917) J. Biol.
Chem., 252. 2478-2485.
*
,
-
89.
-
Boyer, P.D., Smith7 D.J., Rosing, J., and Kayalar, C.
i
(1975) in "Electron Transfer Chains 'an@ oxidative
Phosphorylation" (Quagliarello, E., Papa, S., Palmieri, F.,
-
Slater, E.C., and Siliprandi, N , ,
Eds.) pp. 361-372.,
/-
',
Norgh-Holland, Amsterdam,.'
i
90.
Jagendorf, A.T.
(1975) Fed. Proc., 34, 1718-1722.
91.
~ a ~ a l a rC.,
, Rosing, J., and Boyer, P.D.
I
(1476) Biochem.
,
Biophys. Res. Comrnun., 72, 1153-1159.
92.
Adolfsen, A., and Moudrianakis, E.N. @976)
Biophys., 172, 425-433.
93.
Kayalar, C.,
Arch. Biochem.
4t
Rosing, J., and Boyer, P.D.
\
(1977) J. Biol.
'
&
Chem., 252, 2486-2491.
94.
Hackney, D., and Boyer, P.D (1978) J. Biol. Chem., 253,
95.
Boyer, P . D . ,
Gresser, M.J., Vinkler,
G.,
Hackney, D . ,
and
Choate, G. (19'77) in "Structure and Function of
Energy-Transducing Membranes" (van Dam, K. and Van Gelder,
B.F., Eds. ) ,
96.
pp. 261-274, ~lsevier/~orth-~olland,
Hutton, R.L.,
and Boyer, P.D. (1979) J. Biol. Chem., 254,
\
9990-9993.
$
97.
Boyer, P.D., Kohlbrenner, W.E.,
L.T., and
0'
P
McIntosh, D . B . ,
Smith,
Neal, C.C. (1982) Ann. New York Acad. Sci.,
98.
C r o s s , R.L.,
!
t
Top.
Cunnlng.ham, D . ,
C e l l . Reg.,
and Tamura, J . K .
8,
335-344.
L
-
I
99.
Gresser, M . J . ,
Biol.
Myers, J . A . ,
'Chem.,
257:';
1 0 0 . K o h l b r e n n e r , W.E.,
102. Nalin,
Chem.,
257,
-
104. C r o s s , R.L.,
Biol.
''L
Chem.
Cross,
I
(1982) J.
R.L.,
( 1 9 8 3 ) J. B i o l .
(1982) J;-.Biol.
an2 Penefsky,
Chem.,
Chem. 259,
(1984) J. B i o l ;
R.L..
Chem.,
257,
-
H.S.
(19821, J -
H.S.
(1982). J .
12092-12100.
~ r u b m e ~ e kC,. ,
aqd P e n e f s k y ,
\
,, 257,
105. F i l l i n g a m e , ' R . H .
P.D.
P.D.
and C r o s s ,
1 0 3 . Grubme-y-er, C . ,
Biol.
and Boyer,
and Boyer,
C.M.,
a n d B o k , P.D.
12030-12038.
*8-'4-
101. 0 8 N e a l , C.C.,
( 1 9 8 4 ) CU&.
12101-1'2105.
( 1 9 8 0 ) Annu.
Rev.
Biochem.,
106. S h a v i t , N .
( 1 9 8 0 ) Annu.
Rev.
Biochem.,
1 0 7 . Wang, J . H .
( 1 9 8 3 ) Annu.
Rev.
Biophys.
49,
-
49,
-
1079-1113.
111-138.
Bioenerg.
(1983), 12,
21-34.
1 0 8 . F e r s h t , A.
( 1 9 7 7 ) i n "Enzyme S t r u c t
nd Mechanism" pp.
+-
103-133, and 156-172,
109. Penefsky, H.S.
110. C a r d o n , J.W.,
7615-7622.
111. Cardon, J . W .
San F r a n c i s c o .
( 1 9 7 9 ) Methods Enzymol.~, 5 6 , 527-530.
and , B o y e r , P.D.
(1982) J. B i o l .
Chem.,
257,
-
B
( 1 9 7 9 ) Ph.D.
I*
U.S.A.
Freeman,
T h e s i s , Univ. C a l . ,
Los A n g e l e s ,
112.
Lowry,
R.J.
( 1 9 5 1 ) J. B i o l .
113. D a v i s ,
R.J.
N.Y.
"
193,
-
Chem.,
( 1 9 6 4 ) Ann.
Farr, A.L.,
N. J . ,
Rosebrough,
O.H.,
and - R & d a l l ,
265-275.
Acad.
S c i . U.S.A.,
121,
-
,.-
404-427.
114. R a n d e r a t h ,
115. H a c k n e y ,
Minkov,
A . D.
and R a n d e r a t h , E .
K.,
D.D:
(1979)
Biophys Res.
F i t i n , A.F.,
I.B.,
(k979), Biochem.
Vasilyeva,
Biophys
.,
16,
-
( 1 9 6 4 ) J. C h r o m a t o g r . ,
Res.
Comrnun.*
91,
-
and ~ i h o ~ r a d o v ,
E.A.,
Comrnun.
.,
89,
-
1300-1306.
F i t i n , A.F.,
Vasilyeva,
~ i o c h b , m .B i o p h y s
Vasi,Lyeva,
and V i n o g r a d o v ,
E.A.,
. R e s . Comrnun,,
(1979)
434-439.
Minkov,
F i t i n , A.F.,
E.A.,
-86,
-
A.D.
I.B.,
and V i n o g r a d o v ,
A.F.,
and V i n o g r a d o v ,
/"
A.D.
(1980) Biochem.
Vasilyeva,
A.D.
( 1 9 8 2 ) Biocheni.
R.C.,
Kohlbrenner,
Gresser,
W.E.,
, and
Beharry
Curr.
Minkov,
E.A.,
Steinmeier,
M.J.,
Top.
188,807-815.
J.,
202,
J.,
9-14.
and Wang, J . H .
and B o y e r ,
Gresser,
Beharry,
Cell.
Fitin,
I.B.,
Reg.,
M.J.
S.,
24,
(1979) Biochemistry,
P.D.
( 1 9 8 2 ) J. B i o l .
( 1 9 8 3 ) - Fed.
Proc.,
and Moennich, D.M.C.
365-378.
18,
-
Chem.,
42,
-
1937.
(1984)
124. Beharry,
S.,
and G r e s s e r , M . J .
( 1 9 8 5 ) Fed. P r o c . ,
+(4),
4
q
1 2 5 . Phi105 R . D . ,
and Selwyn, M . J .
(1974) ~ i o c h e m .J . ,
143,
-
745-749.
. 126.
belda,
F.J.F.,
Carmona, F.G.,
Gomez-Fernandez,
. 127.
J.C.,
Canovas, F.G.,
and Lozano, J . A .
Ferguson, S . J . ,
Lloyd, W . J . ,
Raada, 6 . K : ;
( 1 9 7 6 ) Biochim.
Biophys. Acta,
430,
( 1 9 8 3 ) Biochem. J . ,
'.
and S l a k e r , E.C.
189-193.
Source Exif Data:
File Type : PDF File Type Extension : pdf MIME Type : application/pdf PDF Version : 1.5 Linearized : Yes Encryption : Standard V2.3 (128-bit) User Access : Extract Page Count : 204 XMP Toolkit : XMP toolkit 2.9.1-13, framework 1.6 About : uuid:621bc7d8-4b97-44b2-980f-a60b8ea6cc08 Producer : Adobe Acrobat 6.0 Paper Capture Plug-in Modify Date : 2006:02:28 11:27:08-08:00 Create Date : 2006:02:28 11:13:56-08:00 Metadata Date : 2006:02:28 11:27:08-08:00 Creator Tool : Adobe Acrobat 6.0 Document ID : uuid:3a4fb07b-a69e-4966-8563-d9fbe6b22de4 Format : application/pdf Title : Nucleotide-facilitated release of inorganic phosphate and hydrolysed adenosine triphosphate from beef heart mitochondrial adenosine triphosphatase Description : Phosphates; Nucleotides -- Separation; Mitochondria Creator : Beharry, Seelochan Author : Beharry, Seelochan Subject : Phosphates; Nucleotides -- Separation; MitochondriaEXIF Metadata provided by EXIF.tools