Binding of ADP to beef-heart mitochondrial ATPase (F1)

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Binding of ADP to beef-heart mitochondrial ATPase (F1)

Citation for published version (APA):

Wielders, J. P. M., Slater, E. C., & Muller, J. L. M. (1980). Binding of ADP to beef-heart mitochondrial ATPase (F1). Biochimica et Biophysica Acta, Bioenergetics, 589(2), 231-240.

https://doi.org/10.1016/0005-2728(80)90040-7

DOI:

10.1016/0005-2728(80)90040-7

Document status and date: Published: 08/02/1980

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Biochimica et Biophysica Acta, 5 8 9 ( 1 9 8 0 ) 2 3 1 - - 2 4 0 © E l s e v i e r / N o r t h - H o l l a n d B i o m e d i c a l Press

B B A 4 7 8 0 0

BINDING OF ADP TO BEEF-HEART MITOCHONDRIAL ATPase (F~)

J.P.M. W I E L D E R S a , . , E.C. S L A T E R b a n d J . L . M . M U L L E R b , * *

a Laboratory o f Instrumental Analysis, Eindhoven University o f Technology, P.O. B o x 513, 5600 MB Eindhoven (The Netherlands) and b Laboratory o f Biochemistry, B.C.P. Jansen Institute, University o f Arnsterdarn, Plantage Muidergracht 12, 1018 T V A msterda m (The Netherlands)

( R e c e i v e d A p r i l 1 9 t h , 1 9 7 9 )

Key words: Mitochondrial A TPase ; F 1 -A TPase ; A D P binding; Isotachophoresis; (Beef heart)

Summary

1. ADP binding to beef-heart mitochondrial ATPase (F~), in the absence of Mg 2÷, has been determined by separating the free ligand by ultrafiltration and determining it in the filtrate by a specially modified isotachophoretic procedure.

2. Since during the binding experiments the 'tightly' bound ADP (but not the ATP) dissociates, it is necessary to take this into account in calculating the binding parameters.

3. The binding data show that only one tight binding site (Kd about 0.5 #M) for ADP is present.

4. It is not possible to calculate from the binding data alone the number of or the dissociation constants for the weak binding sites. It can be concluded, however, that the latter i's not less than about 50/~M.

Introduction

Several papers have appeared reporting the determination of the binding parameters for radioactively labelled ADP or ATP to mitochondrial ATPase F1 [1--6]. However, most of these studies were made before it was found that isolated F1 contains firmly bound ATP and ADP that exchange slowly and incompletely with added nucleotide [7,8], which makes it difficult to inter- pret the binding data. Since no satisfactory procedure could be developed to correct for this exchange, in this study the binding of ADP to FI has been determined directly by adding known amounts of unlabelled ADP to FI, * Present address: Development Laboratory, Chefazo International bv, Kloosterstraat 46, 5349 AB Oss,

T h e Netherlands.

** To w h o m reprint requests should be addressed.

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232

separating the free ligand by ultrafiltration and determining it in the filtrate by isotachophoresis [9,10].

Methods

A TPase F~

F~ was prepared according to Knowles and Penefsky [11]. The preparation was stored in liquid N~ in a solution containing 0.25 M sucrose, 10 mM Tris- acetic acid buffer (pH 7.5), 2 mM ATP and 2 mM EDTA. Prior to use, the protein was precipitated from the thawed solution by adding an equal volume of neutralized satd. (NH4)2SO4, centrifuged and dissolved in the solution used in the binding experiments (see below). This washing procedure was twice repeated and the solution finally dialysed for 3 periods of 50 rain each against 100 vols. of the solution used in the binding experiment. The residual concentration of (NI-h)2SO+ was 0.5 mM. Denatured protein was removed by centrifugation and the protein concentration determined by the Lowry method, using bovine serum albumin (A~79nm, 0.667 cm 2 • mg -~) as standard. The mo- !ecu!ar weigh_t was assumed to be 319 000 [12].

The specific activity of the ATPase, measured as described by Wagenvoord etal. [13], was 111 pmol/min per mg protein. Tightly bound nucleotides were determined in a neutralized perchloric acid extract as described by Harris et al. [7], employing pyruvate kinase to convert ADP to ATP and measuring the latter by the luciferase method.

A D P

ADP (free acid) from Boehringer contained less than 1% impurities detect- able by isotachophoresis. The concentration of the stock solution was determined spectrophotometrically with pyruvate kinase and lactate dehydrogenase [14]. The concentration of dilutions of this stock solution that were added to the F1 solution was calculated by the dilution factor and checked by isotachophoretic analysis. The values found agreed within 1--2% with the calculated value.

Ultra filtration

A special ultrafiltration cell was constructed for these studies, designed for 0.3- to 5-ml volumes and with a small dead volume (50 #1) between membrane and filtrate output [15].

Before use, the membranes (Amicon Diaflo PM 10) were soaked in distilled water overnight to remove glycerol and azide. After mounting in the cell, the membrane was flushed with 1--2 ml distilled water (at 2 atm N2 pressure) and with about 0.5 ml of the binding medium containing ADP at a concentration equal to that added to the binding medium. The filter was then removed and blotted with paper tissue on both sides, the liquid container of the cell wiped dry and the filtrate channel emptied by a stream of nitrogen.

After reassembling the cell, the FI solution and ADP solution were mixed and brought on to the cell, the total volume being 0.7 ml. After 5 rain standing at 18 ° C, filtration was started with N2 at 2 atm, and four filtrate fractions, each of about 30 ~1, were collected in polythene tubes and immediately frozen

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in liquid N2 until analysis. Since t h e first filtrate fraction contained some ADP present in residual fluid ( a b o u t 5 #1) in the filter, this was n o t used in the calculations. The a m o u n t s o f ADP in the other three fractions did n o t differ appreciably, indicating that equilibrium was reached, and that ADP was not converted to o t h e r c o m p o u n d s during the ultrafiltration. The mean of the values for the second and third filtrates were used in the calculations.

Binding experiments

0.35 ml o f a solution containing 0.25 M sucrose, 25 mM Tris-HC1 buffer, 0.75 mM sodium acetate and 32 (Expt. 1) or 27.4 (Expt. 2)/aM F1 was mixed with 0.35 ml of a solution containing 0.25 M sucrose, 25 mM Tris-HCl buffer, 0.75 mM sodium acetate, 2 mM trisodium citrate and various a m o u n t s o f ADP. The final pH o f b o t h solutions was 8.0. Demineralized and distilled (carbonate- free) water was used. All solutions were checked for the presence o f anionic impurities b y isotachophoresis. The citrate was added as a check that ions were not retained during ultrafiltration and as an internal standard to correct for concentration or dilution effects in handling t h e small sample volumes and for injection errors. Acetate was present as carrier for ADP in the isotachophoresis. E D T A was o m i t t e d since it binds to FI [ 1 5 , 1 6 ] .

Isotachophoretic analysis

A n e w isotachophoretic p r o c e d u r e [9,10], using steady-state mixed zones, was developed for this investigation, since micromolar concentrations o f ADP cannot be d e t e r m i n e d b y usual isotachophoresis with 'pure' zones. A carrier and the operational system were chosen such that ADP and t h e carrier migrate

i

o.ot iu. J

i J I 1 J I i j ~ ' I N =" i -t =i i I - - ! | / / I i IIi ... ,~ . . . z _ ~ = _ . . . . - ; ~ - A C ~ - ~ . - J_.~,. . . II HCO ₃ I A l P ! P r o p " G l u t _', i l , i i j I i I = I ' E P P S t F i g . 1. P a r t o f a n i s o t a c h o p h o r e t o g r a m ( o n l y a b s o r b a n c e s i g n a l is s h o w n ) o f a n u l t z a f i l t ~ a t e s a m p l e f r o m t h e e x p e r i m e n t given i n F i g . 3 A . T h e a r r o w i n d i c a t e s w h e r e t h e p r e s e n c e o f A T P w o u l d b e n o t i c e d b y t h e a p p e a r a n c e o f a s p i k e . T h e s p i k e w i t h t h e a s t e r i s k e n l a r g e s i n t h e p r e s e n c e o f A M P . T h e A D P c o n t e n t o f t h ] s s a m p l e w a s 3 8 . 4 p m o L A c , a c e t a t e ; P r o p , p r o p i o n a t e ; G l u t , g l u t a m a t e ; E P P S , 4 - ( 2 - h y d r o x y c t h y l ) - l - p i p e r a z i n e p r o p a n e s u l f o n l c a d d .

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2 3 4

with the same effective mobility t h e r e b y forming a steady-state mixed zone. Since ADP can be measured sensitively b y ultraviolet s p e c t r o p h o t o m e t r y , it can be d e t e c t e d and measured in concentrations 2-3 orders o f magnitude lower than that o f o t h e r anions in the reaction mixture that appear in adjacent 'pure' z o n e s .

Part of a typical isotachopherogram of an ultrafiltrate sample is shown in Fig. 1. Only the absorbance trace is shown (the isotachopherogram also records the electrical conductivity}. The first and last spikes (from left to right} are d u e to minor impurities, the broad asymmetric 'peak' is due to ADP migrating together with acetate in the steady-state zone. The spike with the asterisk is partly due to AMP, present as a trace in t h e ADP preparation. Enlargement o f this spike is an indication for AMP f o r m a t i o n in the F, solution. The propionate and glutamate were n o t present in the filtrate sample b u t were injected separately. 4-(2-Hydroxyethyl)-l-piperazine propane sulfonic acid is t h e terminating ion. The a m o u n t of ADP is given b y t h e area o f the mixed zone. In this experiment, it is 38.4 pmol. If any ATP had been present (more than 5% o f t h e ADP) it w o u l d have been d e t e c t e d b y a spike at the position o f the arrow. No ATP was d e t e c t e d in any o f the filtrates from F, solutions, nor did the a m o u n t o f AMP exceed that introduced with the ADP solution.

Results

Stirring artefact

Fig. 2 shows the results o f a binding experiment when the solution above t h e m e m b r a n e was stirred during ultrafiltration. It is clear that ADP is retained even in t h e absence of F,, a n d there appears to be a large a m o u n t of non- specific binding. Other experiments [15] showed that E D T A is also retained, b u t there is little retention of Tris, whereas chlorate is excluded rather than retained. 6( 4o a. o < 20 ADP, dd~ ('OM) F i g . 2 . T i t r a t i o n o f F i w i t h A D P i n a sthTed u l t w ~ l l t m t i o n e x P e x t m e n t . •

b i n d i n g m i x t u r e w i t h o u t F 1 ; ~ - - - @ , A D P i n filtrate after b l a n k filtration; •

p r e s e n c e o f 1 4 . 2 NM F ! .

Ao A D P d e t e r m / n e d i n t h e m, ADIP i n f i l t r a t e i n

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Table I shows that, in the absence of stirring, ions were n o t retained.

The cause of t h e stirring artefact [15] is n o t known. It is m e n t i o n e d here in order to draw a t t e n t i o n to a possible source of error, since stirring is o f t e n used in ultrafiltration cells.

Fig. 2 shows also that an appreciable a m o u n t o f ADP was f o u n d in the filtrate even when none was added. This was u n e x p e c t e d , and it indicates that the 'firmly' b o u n d ADP is less firmly b o u n d than was believed, at least in this medium. In s u b s e q u e n t experiments, the a m o u n t o f ADP present in the FI preparation was carefully determined and added to the a m o u n t of added ADP to give the total a m o u n t o f ADP present. The a m o u n t f o u n d in the F, (0.7 m o l / m o l F) was appreciably less than 1 mol per mol F1, presumably due to the extensive washing and dialysis in order to remove ATP present in the solution in which FI was stored.

Binding of ADP to F1

In the lower curves o f Figs. 3A and 3B is plotted the concentration o f ADP in t h e ultrafiltrate for 10 and 8 different ultrafiltrations, respectively, with different a m o u n t s o f ADP, p l o t t e d against the total a m o u n t o f ADP in the solution b e f o r e filtration, taking a c c o u n t o f the a m o u n t o f ADP initially present in the F1. The straight line shows the a m o u n t o f ADP measured in the absence o f F1 (open circles) or after a blank filtration (closed circles). This line coincides with the theoretical line with unit slope, assuming no retention in the filtrate in the absence o f F~. The difference b e t w e e n this line and the corre- sponding points representing free ADP in the ultrafiltrate in the presence of F~ gives the a m o u n t o f b o u n d ADP. In Fig. 4 the a m o u n t o f b o u n d ADP is set o u t against the a m o u n t of free and in Fig. 5 the data are p l o t t e d in the form o f Scatchard plots.

F r o m b o t h Fig. 3 and Fig. 4 it is clear that the binding o f ADP is n o t homo- geneous. Assuming that the F1 preparation is h o m o g e n e o u s and that the molar c o n c e n t r a t i o n is correctly calculated, one molecule o f ADP binds much more strongly than subsequent molecules. It is n o t possible directly from these experiments to determine the n u m b e r o f weak binding sites and their respective affinities. However, additional information m a y be used for the selection o f a most probable binding model (see Discussion). The fitting o f binding models to the data must take the analytical errors into account. A numerical p r o c e d u r e

T A B L E I T E S T O F T H E P A S S A G E O F I O N I C C O M P O N E N T S T H R O U G H T H E M E M B R A N E T h e t e s t s o l u t i o n s c o n t a i n e d 3 . 9 ~ M F I i n 2 5 0 m M sucrose~ 2 5 m M T r l s / 4 - ( 2 - h y d r o x y e t h y l ) l - p i p e r a z i n e p r o p a n e s u l f o n i c a c i d b u f f e r . 1 . 5 m M s o d i u m a c e t a t e , 3 m M s o d i u m c h l o r a t e a n d t h e s p e c i f i e d a n i o n s ( s o d i u m s a l t s ) i n t h e c o n c e n t r a t i o n s given. T h e f i n a l p H o f t h e s e s o l u t i o n s w a s 8 . 0 . I n t h e a b s e n c e o f stir- r i n g . f i l t r a t i o n s w e r e p e r f o r m e d as d e s c r i b e d u n d e r M e t h o d s . Q u a n t i t a t i v e a n a l y s i s w a s d o n e b y i s o t a c h o - p h o r e s i s . C o m p o n e n t E D T A P P i / E D T A P i / E D T A C i t r a t e / E D T A In c e l l ( r a M ) 0 . 7 4 0 . 4 1 / 1 . 7 5 1 . 2 7 / 1 . 8 0 0 . 8 9 / 1 . 5 1 I n f i l t r a t e ( r a M ) 0 . 7 4 0 . 4 2 / 1 . 7 2 1 . 2 4 / 1 . 7 4 0 . 9 0 / 1 . 5 2

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236 8C A o. 0 E ~ x i 0 20 4'0 60 ADPtote I (juM) / 8O / Y / 6C " A / " :E / " ~ 4C ~ 2e E 8O ~do o

B

/

/ /// i i . 1 ~ i ADPtote I (juM) F i g . 3. T i t r a t i o n o f F 1 w i t h A D P w h e n f i l t r a t i o n w a s p e r f o r m e d w i t h o u t s t i r r i n g , o 0 . A D P d e t e r - m i n e d i n t h e b i n d i n g mixtv_re w i t h o u t F I ; • e , A D P d e t e r m i n e d i n b l a n k f i l t r a t i o n w i t h o u t F ! ; ~ - - - ~ , A D P d e t e r m i n e d i n f i l t r a t e w i t h 1 6 / ~ M ( A ) o r 1 3 . 7 / ~ M ( B ) F 1 p r e s e n t . 40 ADPbound 30 (,uM) 20 10 I ; I 410 I I I 0 0 60 ADPfree ( #u M ) F i g . 4 . R e p r e s e n t a t i o n o f d a t a i n F i g . 3 A (& A) a n d 3B ( e -') b y p l o t t i n g t h e A D P b o u n d as f u n c t i o n o f t h e A D P h ~ e. T h e c u r v e s a r e c a l c u l a t e d f r o m t h e d a t a ~ v e n i n T a b l e II f o r a m o d e l w i t h o n e s t r o n g a n d t w o w e a k ( i d e n t i c a l ) s i t e s . 2.0 ADP b AOPf-~ 1.0 I 1 i 0.0 3.0" A A 110 ' =I0 ADP b F i g . 5. R e p r e s e n t a t i o n o f t h e d a t a o f e x p e r i m e n t s g i v e n i n F i g . 3 A (& A) a n d 3 B (e e ) a s a S c a t e h a r d p l o t . T h e curVeS a r e c o n s t r u c t e d f r o m t h e d a t a g i v e n i n T a b l e II f o r t h e m o d a l w i t h o n e s t r o n g a n d t w o w e a k ( i d e n t i c a l ) s i t e s . T h e d a t a f o r t h e l o w e s t A D P b o u n d a r e n o t t a k e n i n t o a c c o u n t .

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T A B L E II D I S S O C I A T I O N C O N S T A N T S O F A D P B O U N D T O F 1 I N T H E A B S E N C E O F M g 2+ K d l is t h e d i s s o c i a t i o n c o n s t a n t o f A D P . b o u n d t o t h e s i n g l e s t r o n g s i t e , K d 2 t o t h e w e a k sites. T w o cal- c u l a t i o n s h a v e b e e n m a d e a s s u m i n g 2 a n d 4 h o m o g e n e o u s w e a k s i t e s , respectively. E x p e r i m e n t 2 w e a k s i t e s ( m o d e l A ) 4 w e a k s i t e s ( m o d e l B) K d t ( # M ) K d 2 (DM) K d l (/~M) K d 2 ( # M ) 1 0 . 6 1 _+ 0 . 0 6 * 6 5 ± 5 0 . 5 4 ± 0 . 0 7 1 6 2 ± 1 5 2 0 . 3 1 ± 0 . 1 1 4 1 ± 3 0 . 0 9 ± 0 . 1 2 1 1 4 ± 1 1 • E s t i m a t e d s t a n d a r d d e v i a t i o n .

that can effectively handle the error structure in the concentration of free and bound ligand has been developed by Linssen [17,18]. This program, which is suitable for non-linear regression with mutually dependent parameters, such as the free and bound ligand, has been used to determine the binding affinities presented in Table II. The fitting function used can be written as

[ B ] _ ~ (

nKd:[E]

[F] i + [ F ] /

where B is the bound ADP, F the free ADP and E the enzyme,

ni

the number

of sites (of kind i) per enzyme molecule and Kd the intrinsic dissociation constant. The error in the Kd values presented is calculated from the error in the determination of the concentrations bound and free. Other sources or error are briefly considered in the discussion.

The drawn lines in Figs. 3--5 are calculated on the assumption that F1 contains one strong binding site and two weak sites with equal intrinsic binding constants (see Discussion).

Discussion

The simplest model fitting the binding data consists of one relatively strong site (Kd about 1 #M) and an infinite number of weak aspecific binding sites [15]. Although aspecific binding may well be taking place, other data indicate that specific ADP-binding sites are, in any case, present (s.ee, e.g. Refs. 19 and 20). Moreover, a control experiment with bovine serum albumin showed that ADP does not bind aspecifically to proteins in general. No significant binding could be detected when up to 82/~M ADP was incubated with 46/~M albumin, under the same conditions as in the experiments with F1.

In considering other possible binding models, the following properties of FI were taken into account. In most respects it behaves as a dimer (~fi~Se)~. One molecule contains 2 molecules tightly bound ATP [ 7 ], binds two molecules of aurovertin [12] (each to a fi subunit), and is inhibited when two molecules of azido-ATP or azido-ADP are covalently bound, either to the ~ or ~ subunits [13,21]. Exceptions are inhibition by 4-chloro-7-nitrobenzofurazan chloride [22], which requires only one molecule bound to the ~ subunit, and the single tightly bound ADP (see below).

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238

Wagenvoord et al. [ 1 3 , 2 1 ] , have shown that, in the absence of Mg 2+, azido- ADP is b o u n d equally strongly to t w o sites on F1 (already containing one molecule o f firmly b o u n d ADP), one on an a subunit and one on a ~ subunit. Although binding o f 2 molecules of azido-ADP is sufficient c o m p l e t e l y to inhibit the ATPase, it is likely t h a t prolonged t r e a t m e n t with azido-ADP w o u l d eventually label b o t h a and ~ subunits. In t h e absence of Mg 2+ one molecule of azido-ATP is b o u n d to each o f the t w o ~ subunits and ADP prevents this binding, showing that the latter also binds to the ~ subunit. Two molecules of azido-ADP are also b o u n d in the presence of Mg 2+, b u t n o w only to the t w o ~ subunits. ADP also prevents this binding. When the ~ subunits are first labelled with azido-ATP, in the absence o f Mg ~+, 2 molecules o f azido- ADP b e c o m e b o u n d to the ~ subunit in the presence o f Mg 2÷. The a m o u n t o f firmly b o u n d ATP and ADP is unaltered b y these treatments. (Wagenvoord, R.J., van der Kraan, I. and Kemp, A., unpublished). Thus, it is very likely that there exist at least t w o and possibly four weak binding sites for ADP under the conditions of t h e experiments described in this paper.

No u n c e r t a i n t y exists concerning the n u m b e r of tight ADP-binding sites. The binding data could be fitted only to models with a single tight site.

Although the u n c e r t a i n t y in the n u m b e r of weak sites precludes an un- ambiguous calculation of the dissociation constant o f ligand b o u n d to these sites, calculations were made in order to obtain an idea of the order of magni- t u d e of this constant. This is o f interest in c o n n e c t i o n with t h e study of inhibition b y ADP o f the ATPase activity of F~ [23]. The following assump- tions were made: the F1 preparation is h o m o g e n e o u s , t h e F~ concentration has b e e n calculated correctly, t h e binding sites are fully accessible and are mutually i n d e p e n d e n t , and the b o u n d and added ADP function as a single pool. Cal- culations have been carried o u t for a model (A) with 1 strong and 2 weak sites and for a second (B) with 1 strong and 4 weak sites. In b o t h models the weak sites are considered equivalent. The results are given in Table II. It must be m e n t i o n e d here that in these models a relatively small error in the F1 concentration will lead to a large error in the affinity constants. For example, b y using a molecular weight of 347 000 instead of 319 000, the Kd values are decreased b y 52% and 27% for the strong and the weak sites, respectively, in m o d e l A (Expt. 2).

The dissociation constant o f ADP b o u n d to the strong sites is rather higher than had been believed, b u t it is consistent with the finding that repeated washing and dialysis o f FI removes a considerable a m o u n t of this 'tightly b o u n d ' ADP. Which subunit bears this single strong site is n o t known.

Experiments with p h o t o a f f i n i t y labels have shown that weak ADP-binding sites are present on t h e ~ and ~ subunits. Azido-ADP-, and presumably ADP-, binding sites are present on b o t h a and ~ subunits, b u t it i s n o t k n o w n if ADP, in the absence o f Mg 2÷ and EDTA, binds to b o t h a and ~ subunits or to only one o f each. In the f o r m e r case (model B) the calculated dissociation constants (assuming equal affinities o f the sites) are 114 and 162/~M in t h e t w o experiments: If only t w o sites can be occupied (model A) t h e dissociation constants are 41 and 65 pM, respectively. Whereas the p h o t o a f f i n i t y labelling favours t h e former possibility, a b e t t e r fit to t h e binding data judged f r o m the residual sums of squares was obtained with m o d e l A. It does seem reasonable to con-

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clude from t h e data presented in Table II that there is a single binding site with a dissociation c o n s t a n t o f a b o u t 0.5/~M and additional binding sites with dissociation c o n s t a n t of minimally a b o u t 50 #M.

Since no ATP was d e t e c t e d in the ultrafiltrates, exchange o f ADP for tightly b o u n d ATP, observed b y Harris et al. [24] did n o t take place in our experiments. This m a y be d u e to b o t h the high pH and the relatively low concentrations of ADP used in our experiments.

Tight and weak sites of ADP binding were previously observed b y Hilborn and Hammes [3], w h o reported one strong site and one weak. The dissociation constants were 0.28 and 47 pM, respectively, in the presence o f Mg 2÷ and 11 and 43 #M, respectively, in its absence. Strangely, our values obtained in the absence o f Mg 2+ agree fairly closely with theirs obtained in its presence, except for the n u m b e r of weak sites, b u t n o t in its absence. Catterall and Pedersen [2] also f o u n d a single firmly b o u n d ADP (Kd 0.9/~M) in liver FI, in the absence o f Mg 2+. Since ADP already b o u n d to the F1 was n o t taken into

a c c o u n t in these studies, the K d values may be under-estimated, especially for

the strong sites.

Acknowledgements

The contributions o f Dr. O.A. Roveri to the experimental part, and of It. H.N. Linssen and Mr. R. Kool, who did the c o m p u t e r calculations, are gratefully acknowledged. This w o r k was supported b y grants from t h e Nether- lands Organization for the Advancement o f Pure Research (Z.W.O.), o n e o f which under auspices of the Netherlands F o u n d a t i o n for Chemical Research (S.O.N.).

References

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