Biochimica et Bioph),sica Acta, 463 (1978) 245-273 © E l s e v i e r / N o r t h - H o l l a n d Biomedical Press B B A 86044

THE

INTERACTIONS

OF COUPLING

ATPases

WITH

NUCLEOTIDES

D A V I D A. H A R R I S

Department of Biochemistry, University of Oxford, South Parks Road, Oxford ( U.K.) (Received M a y 17th, 1977)

CONTENTS I.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. T h e n a t u r e of coupling A T P a s e s . . . . . . . . . . . . . . . . . . . . . . . B. Nucleotide binding to coupling A T P a s e s . . . . . . . . . . . . . . . . . . .

II. 'Tightly b o u n d ' nucleotides on coupling A T P a s e s A. Detection a n d estimation . . . . . . . . . B. Isolated A T P a s e s . . . . . . . . . . . . C. M e m b r a n e - b o u n d A T P a s e s . . . . . . . . III.

IV.

B e h a v i o u r o f b o u n d nucleotides . . . . . A. Isolated A T P a s e s . . . . . . . . . . . 1. Studies on nucleotide binding . . . 2. Reactions o f nucleotides in isolated

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245 245 246 247 247 248 250 252 252 252 254

B. M e m b r a n e - b o u n d A T P a s e s . . . . . . . . . . . . . . . . . . . . . . . . . 1. I n t r o d u c t i o n . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. Non-energised m e m b r a n e s . . . . . . . . . . . . . . . . . . . . . . . . 3. Chloroplast m e m b r a n e s . . . . . . . . . . . . . . . . . . . . . . . . . 4. O t h e r systems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5. Inhibition studies . . . . . . . . . . . . . . . . . . . . . . . . . . . .

254 255 255 255 258 258

Mechanism of phosphorylation . . . . . . . . . . . . . . . . . . . . . . . . . A. Initial p h o s p h a t e acceptor . . . . . . . . . . . . . . . . . . . . . . . . . . B. E n e r g y i n p u t during A T P synthesis . . . . . . . . . . . . . . . . . . . . . .

259 259 261

V.

Models for A T P synthesis . . . . . . . . . . . . . . . . . . . . . . . . . . .

262

VI.

Nucleotide binding in regulation o f coupling A T P a s e s . . . . . . . . . . . . . . .

268

VII. C o n c l u d i n g r e m a r k s Acknowledgements References

. . . . . . . . . . . . . . . . . . . . . . . . . . . . .

268

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269

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269

I. I N T R O D U C T I O N

1.4. The nature o f coupling ,4TPases Coupling plast

thylakoid

membranes, membrane

such as the mitochondrial

inner membrane,

and

of

specialised

regions

all

bacterial

the chloromembranes,

Abbreviations: A T P a s e : a d e n o s i n e - 5 ' - t r i p h o s p h a t a s e ( A T P p h o s p h o h y d r o l a s e , E C 3.6.1.3); A M P - P N P : adenylyl fl-~ i m i d o d i p h o s p h a t e ; S~ 3 : 5-chloro-3-t-butyl-2'-chloro-4'-nitrosalicylanilide; F C C P : carbonyl cyanide p - t r i f l u o r o m e t h o x y p h e n y l h y d r a z o n e ; A M P - C - P : a d e n o s i n e 5"-methylene d i p h o s p h o n a t e ; F T P : formycin 5'-triphosphate; N u T P : nucleoside 5'-triphosphate.

246 contain a protein complex capable of utilising energy derived from electron transfer in the synthesis of ATP from ADP and P~, and/or of utilising energy derived from ATP hydrolysis for various processes such as ion transport. This complex can be isolated more or less intact from the membrane in the presence of detergent [1-4]. A subfraction of this complex can be obtained in a watersoluble form which contains the nucleotide binding sites of the 'ATP-synthase'. This fraction is in general termed the "coupling ATPase", or F1 [5]. The relative ease of isolation of the coupling ATPase fraction directly from a variety of membranes has led to its wide study. For recent reviews, dealing largely with structural aspects of this ATPase see refs. [6-11]. Although the molecular weights of many coupling ATPases lie in the region 300 000-400 000, and there appears to be a close relation between ATPases from different sources, no consensus has been reached as to their overall subunit composition. In the following discussion, a stoicheiometry C12fl271 (66)? iS assumed [12-15] (the subunits being designated a-e in order of decreasing molecular weight). IB. Nucleotide binding to coupling ATPases The membrane-bound coupling ATPase is responsible for the formation of ATP from ADP and Pi, in most organisms, and it therefore bears one (or more) binding sites for ADP. The ATPase (membrane-bound or isolated) is also able to hydrolyse ATP to ADP and Pi, although this activity may be latent in some preparations. One ATP-binding site must therefore also be present. The relationship between these putative sites is not unequivocally established: it is generally held that ATP hydrolysis and synthesis involve the same site on the enzyme but it has been suggested that entirely separate sites may be involved [9,16,17]. The role of the coupling ATPase is thus to interact with nucleotides, and considerable efforts have been made to elucidate the nature of these interactions. They appear to be particularly complex. Notably, in addition to the ATPase/ phosphorylation site(s) mentioned above - where binding must be rapidly reversible to account for the high rates observed - there are also present sites where nucleotide binding is slow and not rapidly reversible. As a result, it is difficult to interpret binding data obtained on this ATPase by classical methods, which assume equilibrium to be attained in the system [18-20]. The object of this review is: (a) to explain nucleotide binding and kinetic data obtained on various ATPases in terms of a common model based on two classes of site: rapidly reversible, and slowly reversible (or 'tight') ; (b) to examine the roles of these sites in ATP hydrolysis, ATP synthesis, and in regulation of both activities.

247 II. 'TIGHTLY BOUND' NUCLEOTIDES ON COUPLING ATPases

IIA. Detection and estimation 'Tightly bound' nucleotides are defined operationally as those nucleotides which remain stably bound to the coupling ATPases when all detectable free nucelotides have been removed from the enzyme solution. Rate of dissociation from the 'tight' binding sites is so slow that tightly bound ATP is not removed by ATPutilising enzymes such as hexokinase or luci ferase [21-24]. Removal of free nucleotides from systems containing a coupling ATPase may be achieved in several ways. For membrane-bound coupling ATPases, the membranes can generally be centrifuged repeatedly through solutions lacking in ATP and ADP until no nucleotides are detectable in the supernatant. Magnesium ions should be included (or low ionic strengths avoided)during this centrifugation or loss of ATPase from the membrane may occur (cf. refs. 23 and 25). Addition of pyrophosphate to the washing medium helps to remove nucleotides bound non-specifically. In the case of chloroplast preparations, the membranes may alternatively be immobilized on glass fibre filters and washed by passing buffer throngh the filter [26,27]. In the case of chloroplasts and some bacteria [7,8], soluble coupling ATPases can be liberated from the membrane by washing at low salt concentrations. Prepared thus from prewashed membranes, the ATPases will contain only tightly bound nucleotides. Mitochondrial ATPases in general require more severe treatment, such as prolonged sonication, to remove them from the membrane [7], and ATP must normally be present to stabilise the enzyme. Removal of nucleotides from these isolated enzymes without denaturation may be a more ticklish problem. We find the most reproducible procedures for the beef heart ATPase to be repeated precipitation with (NH4)zSO, (added from a saturated solution to a final concentration of 50~o saturated) [28] or passage through a Dowex 2 anion exchange resin [29]. Gel filtration or dialysis of the enzyme in the absence of nucleotide leads to partial denaturation, at least in some preparations [28,29]. However, ATPases from several other sources (e.g. Escherichia coli, Paracoccus denitrificans) are unstable to precipitation with (NH,)2SO 4. Estimation and characterisation of bound nucleotides requires their release from the protein. Commonly the protein is denatured with perchloric or tricholoroacetic acid, heat or urea. Care must be taken to ensure complete denaturation of the protein (and thus complete release of nucleotides) and that no loss of nucleotide from the solution or alteration of the A T P : A D P : A M P ratio occurs. In a commonly used procedure, the protein is denatured by 4 ~ HCIO,, protein removed by centrifugation at 0°C, and the resulting solution rapidly neutralised with K O H [30]. When this procedure is used for solutions containing low concentrations of nucleotide, as it is here, there may be several sources of error. (i) Nucleotide may co-precipitate with K C 1 0 , after neutralisation [31]. This is avoided ( < 1 0 ~ ) if nucleotide concentrations are maintained higher than 1 /~M. (ii) Some active adenylate kinase may be present in the neutralised extract and

248 cause phosphate transfer. This is avoided by adding excess EDTA to the extract before neutralisation [32]. (iii) Acid treatment of membrane systems may cause bound Pi and ADP to form ATP, by a mechanism akin to normal phosphorylation [33]. It is not clear how general this phenomenon is (cf. ref. 34). The nucleotides released are estimated enzymaticaily [35,36] or, if prelabelled, by standard radiochemical techniques. Prelabelling is best carried out in vivo [22,37,38] for bacteria, because labelling requires energisation of the ATPase (see below, C2) and this may be incomplete for membrane fragments and impossible for the isolated ATPase. Even prolonged incubation of an isolated ATPase with ATP, labelled in the adenine moiety, may not give total exchange of the bound nucleotides [28] although considerable labelling might occur [38]. A non-destructive method of measuring the nucleotide content of proteins was described by Warburg and Christian [39]. For a nucleotide-free protein, the ratio A 2 8 0 nm/A 260 n m ~ 1.7-1.8, while nucleotide, with a strong absorption at 260 nm lowers this ratio. Beef heart mitochondrial ATPase has indeed a lowered A 280 ,m/A z6o nm ~ 1.4 [40], but for this enzyme the ratio is sensitive to solvent and to protein concentration and is not, in our hands, a reliable estimate of nucleotide content (ref. 41 and unpublished results). Attempts to visualise the bound nucleotides using 31p N M R [42] failed, probably because the phosphate groups of the bound ATP and ADP are highly immobilised.

liB. Isolated ATPases The possibility that isolated beef heart ATPase contained very tight binding sites for nucleotides emerged in 1965 when Zalkin et al [43] showed that the ATPase incubated with [14C]ADP or ATP could bind up to 0.8 nucleotide per mol ATPase in a manner stable to repeated (NH4)2SO4 precipitation. Warshaw et al. [44] subsequently showed that "Factor A", a preparation containing beef heart coupling ATPase, the ATPase inhibitor protein (and possibly some other minor components) contained tightly bound ADP (1 mol/280 000 daltons protein) although prepared from mitochondria in the absence of added nucleotide. 'Tightly bound' ATP and ADP have since been demonstrated on coupling ATPases from a variety of sources - mitochondria, chloroplasts and bacteria and results to date are summarised in Table I. Although the figures are somewhat variable it is commonly found that an isolated coupling ATPase bears 1-2 mol ATP and 1-2 moi ADP per mol enzyme, i.e. nucleotides are present in amounts stoichiometric with the enzyme. The variability in these figures appears rather high. In beef heart ATPase for example, ATP levels vary from 0 to 2.2. mol/mol ATPase, and ADP levels from 1.3 to 2.3 tool/tool ATPase. Although this may be due in part to the different methods used to prepare the ATPase, it should also be noted that various conditions can lead to a loss of some nucleotide from the ATPase. These include gel filtration in the

249 TABLE I TIGHTLY BOUND NUCLEOTIDES FOUND ON ISOLATED COUPLING ATPases Source

tool/tool ATPase ATP ADP

Method of assay*

Reference

Beef heart mitochondria Beef heart mitochondria Beef heart mitochondria Rat liver mitochondria Spinach chloroplasts Spinach chloroplasts S. faecalis A. faecalis E. coli R. rubrum

2.2 0.9 0.0 i 0.5 0.8 1 ? 1.9 1.3

E E E ? L E PL L PL E

24,38 40 46 9 38 32 37 54 22 55

1.3 2.3 1.8 1 1 0,9 1 1q0.7 0.7

* E, enzymatic; L, labelling of the isolated enzyme with radioactive nucleotide; PL, labelling in vivo followed by isolation of the ATPase. absence of nucleotide [28], exposure to alcohols [40] (used to stabilise some ATPase preparations), exposure to very high ( ~ 6 0 ~ ) ammonium sulphate concentrations (unpublished results) and cold treatment [22,45]. Such treatments generally lead to more or less inactivation of the ATPase but this may be difficult to detect, particularly where the ATPase is latent and requires activation before assay. These considerations may account for the recent findings of Lee et al. [20] that only 0.025 mol each of A T P and A D P are tightly bound per mol ATPase of Mycobacterium phlei after purification by gel electrophoresis. It is likely therefore that most preparations of isolated coupling ATPases are heterogeneous with respect to nucleotide content. It is not clear how this heterogeneity relates to the finding of polymorphism within preparations of ATPase from Alcaligenes faecalis. The various forms of the enzyme detected here were interconvertible by incubation in the presence or absence of ATP [67]. Systematic attempts to remove all bound nucleotides have been made in several laboratories. The nucleotides are not removed by charcoal treatment [28]. Active nucleotide-free preparations of the beef heart ATPase can be prepared by filtering the enzyme through Sephadex in the presence of either 50~o glycerol [40] or l l0 m M sulphate ions [46]. Titration of nucleotides into this 'stripped' enzyme can be used to investigate the functions of the tight binding sites (see below, IliA). However, large differences were reported between the two preparations: the glycerol-treated enzyme rebinds 3 tool A D P or 5 mol A M P - P N P tightly per tool enzyme [40] and the sulphate-treated enzyme only 1 mol A D P or 2 mol A M P - P N P [46]. Mg 2+ is necessary for maximum binding. The reason for these discrepancies is unknown. The two preparations also showed differences in their ability to couple phosphorylation in ATPase-deficient membranes. An active, nucleotide-free preparation of the coupling ATPase from the thermophilic bacterium, PS3, has apparently been made by Kagawa and co-workers [47], by combining the isolated subunits of

250 the enzyme in the absence of nucleotides. The properties of this species were not described. The 'tightly bound' nucleotides are bound non-covalently to the ATPases, since they are lost when the ATPase is denatured simply by incubation at 0°C [22,28]. However, their dissociation constant has not been accurately determined as it is so small. A value of 10-1° M has been estimated for the beef heart ATPase [28]. The site of tight binding is uncertain. Using the chloroplast ATPase (prelabelled with [14C]ADP), Magnusson and McCarty [48] showed that digestion of all but the a and fl subunits with trypsin left > 60 ~ of the label on the protein. Thus the a and/or/3 subunits seem to be the site of tight binding, at least for ADP. These subunits also bear the ATPase site of the enzyme [49]. However, Leimgruber and Senior [46] showed that tryptic digestion of the beef heart enzyme led to complete loss of the bound nucleotides with no detectable change in the subunit structure of the enzyme. Chymotryptic digestion of the ATPase from Streptococcusfaecalis [50] leads to modification of the a subunit without loss of tightly bound ATP or ADP. Isolated coupling ATPases do not seem to contain comparable tight binding sites for AMP and Pi i.e. preparations free of AMP and P~ are readily obtained [22,28,32,40,46]. However, bound magnesium has been reported in purified ATPases from three sources [28,47,51] and seems to be important in the assembly of the ATPase from its subunits [47,51].

IIC. Membrane-bound ATPases Both rat liver [52] and beef heart [53] mitochondria contain small amounts of ATP unavailable for hydrolysis even under de-energised conditions. This was suggested to be bound to some specific site inside the mitochondria. The results above (liB) suggest that this site may be the coupling ATPase. This has been further investigated by nucleotide analysis of washed coupling membranes. Table II shows that tightly bound ATP and ADP are present on coupling membranes from a variety of sources, while AMP is generally absent or present in low amount. The picture for membranes is, in fact, more constant than for isolated ATPases, with A T P / A D P ratios 1.5-2 for most membrane preparations. Several lines of evidence suggest that the only site of tight nucleotide binding on the membrane is the coupling ATPase. Table III shows that, in the case of beef heart mitochondria, the tight binding sites for ATP and ADP 'purify' with the coupling ATPase and that the nucleotides found tightly bound to the membrane are quantitatively recovered in the isolated ATPase. Similar results have been obtained in chloroplasts [32,48,63] and in bacteria [23]. In addition, an E.coli point mutant with a deficient coupling ATPase [64], and submitochondrial particles from a ~yeast (which lack the coupling ATPase but retain the ATP-ADP transiocase [65]) lack tightly bound nucleotides on the membrane [57]. It is therefore likely that all coupling membranes contain ATP and ADP tightly bound to the coupling ATPase and that no other 'tight binding' sites for ATP and A D P exist on coupling membranes. Where the amount of ATPase on the

251 TABLE II TIGHTLY ATPases

BOUND

NUCLEOTIDES

FOUND

ON

MEMBRANE-BOUND

COUPLING

All the results in this table are the result of enzymatic assays of nucleotide levels. Labelling, where it has been used, gives values consistent with these. Source

nmol/mg protein ATP ADP AMP

Beef heart mitochondria Mg-ATP particles* 0.85 Mg particles**0.82 Mg-Mn particles*** 2.4 Rat liver mitochondria 0.74 S. cerevisiae mitochondria 0.57 E. coli 0.22 P. denitr([icans 1.4 Spinach Chloroplasts Subehloroplast particles R. rubrum * ** *** -~ ~t

nmol/mg chlorophyll ATP ADP AMP

ATP/ ATP/ ATPase A D P

Reference

0.60 0.53

0.24 0.06

2.2 -

1.4 1.5

24 24

1.9

-

-

1.3

56

0.51

0.1

-

1.45

57

0.21 0.12 1.1

0.05 0.4 0.5

-

2.7 1.8 1.3

57 57 23

2.5

1.4

0.0

2.1

1.8

32

9.5 5.3t

2.1

-

2-3 2~'t

2.6

26 58,59

Prepared as in ref. 47. Prepared as in ref. 47, but ATP omitted from the preparation. Prepared as in ref. 48. Lundin et. al. (1977) FEBS Lett. 79, 73-76 Calculated assuming 1 coupling ATPase/tight antimycin binding site.

TABLE IlI TIGHTLY BOUND NUCLEOTIDES ON FREE A N D M E M B R A N E - B O U N D ATPase IN THE BEEF H E A R T SYSTEM

Mitochondria Mg-ATP particles ATPase-deficient particles* ATPase-deficient particles*, reconstituted with ATPase Isolated ATPase

nmol/mg protein ATP ADP

AMP

Reference

0.60 0.90 0.16

0.90 0.60 0.34

3.4 0.2 0.2

24 24 24

0.89 5.9

1.04 3.7

0.0

62 28

* 2 M urea was used to deplete the membranes of ATPase.

m e m b r a n e is k n o w n , ( A T P ÷ A D P ) c a n b e c a l c u l a t e d t o b e a b o u t 3 m o l / m o l A T P a s e . T a b l e I s h o w s t h a t v a l u e s o n t h e i s o l a t e d A T P a s e a p p r o a c h this, a n d it is c o n c l u d e d t h a t , i n g e n e r a l , t h e r e a r e 3 t i g h t b i n d i n g sites f o r n u c l e o t i d e o n c o u p l i n g A T P a s e s ,

252 although some of the bound nucleotide itself may be lost in the preparation of the soluble ATPase. Leimgruber and Senior [62] have demonstrated that ATP binds more tightly to the membrane-bound ATPase, showing that ATPase unable to bind ATP tightly when isolated can do so when the ATPase is membrane-bound. The dissociation constant for tightly bound ATP on membrane-bound coupling ATPase, as for the isolated ATPase, is less than 10-1° M [23,24]. A suggestion that tightly bound nucleotides in Rhodospirillum rubrum could occupy two distinct types of site, isolable from one in acid and from the other in acid + detergent, with different functions [59], has been withdrawn [55]. Removal of tightly bound nucleotides from membrane-bound ATPase is more difficult than removal from the isolated ATPase (above) and has not been accomplished in a well defined preparation. Chloroplast membranes when illuminated with a dithiol in the absence of Mg 2+ and rapidly washed are found to have lost bound ADP, but ATP remains [32]. Sephadex filtration of submitochondrial particles, even in the presence of 550 mM sulphate, removes only 75 ~ of the bound nucleotides [56]. Trypsin treatment removes nucleotides completely from submitochondrial particles, but it is not known how much the ATPase or membrane is damaged during this treatment [56]. On mitochondrial membranes, at least, several 'tight' binding sites for Mg 2+ occur outside the coupling ATPase [66], and thus it is difficult to establish directly the presence of tightly bound Mg 2+ on the membrane-bound ATPase.

III. BEHAVIOR OF BOUND NUCLEOTIDES

IliA. Isolated ATPases IIIA-1. Studies on nucleotide binding. Studies of nucleotide binding to the mitochondrial ATPases [18,19] were performed by standard methods (equilibrium dialysis, gel filtration) before the presence of bound ATP and ADP was established. Two types of binding site for ADP were demonstrated and the binding appeared remarkably slow [68], involving minutes rather than the expected milliseconds. It now appears that these studies measured a (fast, reversible) equilibration of nucleotide, probably at the ATPase site of the enzyme, and a slow exchange of nucleotide [28] into the tight binding sites. Fig. 1 shows the reversible and 'irreversible' components of ADP binding to the beef heart mitochondrial ATPase measured by equiqbrium dialysis [69] (Fig. la) and by the method of Catterall and Pedersen [18] (Fig. lb). Exchange of bound nucleotides with added labelled nucleotide were measured directly on a well-defined ATPase preparation (initial nucleotide content 2 mol ATP, 1 mol ADP/mol enzyme) [28]. Even after 24 h in the presence of [3H]ATP or [aH]ADP only 1 5 - 3 0 ~ of the bound nucleotide had exchanged, even in the presence of Mg z+ [28,70] (Fig. 1). The initial rate of exchange was low (tv, ~ 2 rain). Using ATPase with its bound nucleotides prelabelled, it has been shown that in the chloroplast

253 a 1.5

~

~ 0-/.

1.0

"6 E o

o

o

~

g

x~ 0 ' 5 -

°

0.2,

7 10

20 30 f r e e ADP ( ~ M )

t.0

50

lb

20

30

4b

free ADP (~IM)

Fig. 1. Total and 'irreversible' binding of [3H]ADP to isolated beef heart mitochondrial ATPase. Total binding was measured either by equilibrium dialysis [69] (a) or by determining the amount of label co-precipitating with the protein from 50% saturated (NH4)2SO4 [18] (b). Aliquots of labelled protein from each procedure were washed by repeated precipitation with (NH4)2SO4 until no free nucleotides were present. The labelled nucleotide still present on the protein ('irreversible' binding) represents binding at the 'tight' nucleotide binding sites of the protein [28]. The values are corrected for dilution of the added label with nucleotide originally bound to the protein, assuming a 1 : 1 exchange [24]. © - - @, total [aH]ADP bound; [ ] - - 2 , [aH]ADP 'irreversibly' bound.

[63] and the E. coli [22] ATPases exchange of bound with added nucleotide is also small. The tight nucleotide binding sites thus respond so slowly, and to such a limited extent under turnover conditions, that their direct participation in the ATPase activity of the isolated enzyme is ruled out. Partial reversible cold denaturation of the beef heart ATPase [45], and exposure of the enzyme to low pH [70] increases the exchange of bound with added nucleotides. Using these findings, Harris et al. [71] showed that the specificity of the tight binding sites for nucleotides was much higher than that of the rapidly reversible site. 2'-dATP, iso-GTP and FTP were able to replace bound ATP, while GTP, ITP, e-ATP and N1-O-ATP - all of which were hydrolysed at comparable rates were unable to do so. This confirms that the tight sites are not directly involved in hydrolysis. However, if bound ATP were replaced by ADP[45] or AMP-PNP[40] the enzyme lost its hydrolytic activity. This modified enzyme was not dissociated into subunits, as shown by ultracentrifugation [45]. It is therefore concluded that the tight binding sites, while not directly involved in hydrolysis on the isolated enzyme, do affect this activity - presumably via allosteric interactions (see V and VI). Attempts have been made to label the nucleotide binding sites of the mitochondrial ATPase irreversibly, using photolabile ATP analogues. 8-azidoATP labels specifically the fl-subunit of an ATPase preparation containing its full complement of bound nucleotides [72], suggesting the involvement of this subunit in the rapidly reversible site of the ATPase. However, 8-azidoADP (Wagenvoord, R. J., Kemp, Jr., A. and Slater, E. C., personal communication) and N-4-azido 2-nitrophenylamino-

254 butyryl ADP [73], which apparently bind to the same site, label the a-subunit. It is not yet clear whether these analogues can occupy the tight binding sites of the ATPase. Studies on the (latent) chloroplast coupling ATPase similarly began on a preparation undefined as to nucleotide content [38,74,75] but which must still have contained firmly bound nucleotide. Results obtained, however, were very similar to those obtained by Cantley and Hammes [76], who worked with an enzyme apparently freed of nucleotide by (NH4)2SO4 precipitation and repeated gel filtration. ADP binding to these preparations was slow (required 0.5-2 h) and led to an enzyme with about 2 tool nucleotide/mol enzyme, which was stable to gel filtration [38]. The nucleotides were therefore presumably in the tight binding sites. IDP, CDP, GDP, and U D P did not compete with ADP [38], but AMP-PNP could replace ADP [74]. As in the case of the mitochondrial ATPase, chloroplast enzyme with ADP or AMP-PNP occupying the tight binding sites was not active as an ATPase, even when 'activated' [74]. The coupling ATPase from A. faecalis also binds [3H]ADP 'tightly' in that the binding is slow and the resultant labelled enzyme is isolable by gel filtration in the absence of nucleotides [54]. Variable results as to the number of nucleotide molecules bound probably reflect variable nucleotide contents of the enzyme preparation used in the assays. IIIA-2. Reactions of nucleotides in isolated ATPase preparations. The coupling ATPases in general hydrolyse ATP in the presence of a divalent cation (Mg 2+ or Ca2+), the dominant product being ADP. Even in the absence of a divalent cation a slow hydrolysis of ATP occurs [! 8,19,42,77], and the product here is entirely ADP in the case of beef heart ATPase [78]. These changes presumably occur at the 'rapidly reversible' nucleotide binding site of the ATPase, since tightly bound ATP remains present on the enzyme even in the presence of Mg 2+ [28]. In addition, there are reports of AMP formation from ATP or ADP, by ATPase preparations from chloroplasts [38,76], mitochondria [78] and some bacteria [20, 54]. AMP formation is slow and limited in amount (0.1-4 tool per tool ATPase). Slow transphosphorylations have also been reported: the reaction 2ADP -+ ATP + AMP (Mg2+-independent) on the chloroplast ATPase, [38,79] and the reaction ATP-~ A M P - + 2ADP (MgZ+-dependent) on the mitochondrial ATPase [78], although, surprisingly, not the reverse reaction in either case. It is not clear whether these reactions occur on the coupling ATPase alone or involve another enzymic impurity in these preparations, although the participation of soluble (4.2-S) adenylate kinase is ruled out in the case of the chloroplast ATPase [79]. In the mitochondrial ATPase at least, the tight nucleotide binding sites appear not to be involved, since the reaction occurs on the ATPase with its full complement of nucleotides (as well as on the stripped enzyme) [78], while on the chloroplast ATPase, ATP formed from ADP comes to occupy a tight binding site [38]. The possible role of transphosphorylations in coupled phosphorylation is discussed in IVA. Isolated coupling ATPases are in general not capable of incorporating 3 2 p I

255 into tightly bound or added nucleotide (