Adenosine Triphosphatase from Rat Liver Mitochondria

Homogeneous mitochondrial ATPase from rat liver binds ADP in a rapidly reversible manner. The enzyme-bound ADP can be recovered quantitatively as ADP. In the absence of Mg2+, the enzyme exhibits 0.88 bindingsitesfor ADP per enzyme molecule with an intrinsic dissociation constant of 0.94 pM. In the presence of MgZ+, the enzyme loses 90% of its ATPase activity but does not lose the ability to bind ADP. Short term binding experiments detect 0.65 binding sites per enzyme molecule with an intrinsic dissociation constant of 2.1 pM. The Km (ADP) for oxidative phosphorylation catalyzed by purified inner membrane vesicles, in which mitochondrial ATPase is located on the outer surface of the membrane directly available to added ADP, was found to be 3.8 pM. Aurovertin, a potent inhibitor of oxidative phosphorylation, inhibits binding of ADP by mitochondrial ATPase in the absence of Mg 2+ in a manner similar to its inhibition of oxidative phosphorylation. In the presence of Mg2+, however, binding is enhanced by aurovertin. Taken together, the results show that the binding of ADP by soluble mitochondrial ATPase has important properties in common with the interaction of ADP with the functional oxidative phosphorylation system.

In the absence of Mg2+, the enzyme exhibits 0.88 bindingsitesfor ADP per enzyme molecule with an intrinsic dissociation constant of 0.94 pM. In the presence of MgZ+, the enzyme loses 90% of its ATPase activity but does not lose the ability to bind ADP.
Short term binding experiments detect 0.65 binding sites per enzyme molecule with an intrinsic dissociation constant of 2.1 pM.
The Km (ADP) for oxidative phosphorylation catalyzed by purified inner membrane vesicles, in which mitochondrial ATPase is located on the outer surface of the membrane directly available to added ADP, was found to be 3.8 pM. Aurovertin, a potent inhibitor of oxidative phosphorylation, inhibits binding of ADP by mitochondrial ATPase in the absence of Mg 2+ in a manner similar to its inhibition of oxidative phosphorylation.
In the presence of Mg2+, however, binding is enhanced by aurovertin.
Taken together, the results show that the binding of ADP by soluble mitochondrial ATPase has important properties in common with the interaction of ADP with the functional oxidative phosphorylation system.
Membrane ATPases have been isolated in soluble and homogeneous form from mitochondria of bovine heart (1, 2) and rat liver (3,4).
The importance of these enzymes in oxidative phosphorylation has been extensively documented (for a review, see Reference 5). However, the physical properties of the isolated enzymes have only recently been elucidated (2)(3)(4).
We have reported (3) that the rat liver enzyme has a molecular weight of 384,000 and consists of three classes of polypeptide chain sub- units with molecular weights of 62,500, 57,000, and 36,000. Results similar in many respects to those reported by us (3) have been obtained by Lambeth and Lardy (4). In this communication we report studies on the interaction of mitochondrial ATPase with ADP. Three considerations led us to undertake this study.
First, determination of the number of binding and catalytic sites is an important first step in analysis of the subunit structure in functional terms. Second, membrane ATPase from chloroplasts, a closely related enzyme in both structural and functional terms' (6), binds ADP in an unusual manner (7). Therefore, it is important to assess the possibility that mitochondrial ATPase might have similar properties. Third, comparison of the binding of ADP by soluble mitochondrial ATPase with the interaction of ADP with mitochondrial membrane vesicles catalyzing oxidative phosphorylation provides a measure of the functional similarity between purified soluble mitochondrial ATPase and membrane-bound mitochondrial ATPase functioning in oxidative phosphorylation.
The results presented below show that mitochondrial ATPase binds a single molecule of ADP in a rapidly reversible manner with a dissociation constant in the range of 1 PM and that the binding process has important properties in common with oxidative phosphorylation.

Materials
The following materials were purchased commercially and used as received: Sephadex A-50 from Pharmacia; thin layer sheets of polyethyleneimine-impregnated cellulose (Polygram Cel PEI) from Brinkmann; carrier-free 32Pi and 3H-labeled nucleotides (specific radioactivity of approximately 10 Ci per mmole) from New England Nuclear; unlabeled nucleotides from P-L Biochemicals, and special enzyme-grade ammonium sulfate from Mann.
All other chemicals were of reagent-grade purity. Oligomycin was purchased from Sigma. Atractyloside was generously donated by Professor R. Santi and Dr. A. Bruni of the University of Padova, Italy.
Aurovertin was a product of Pitman-Moore, Indianapolis, Ind. Because the sample used was quite old, the potency of the inhibitor was tested. Half-maximal inhibition of ADP-stimulated respiration was achieved at 0.24 nmole aurovertin per mg of mitochondrial protein in close agreement with the published value of 0.25 nmole per mg (8).

Isolation of Mitochondria-Rat
liver mitochondria were isolated by differential centrifugation as described by Schnaitman and Greenawalt (9).
Isolation of Mitochondrial A TPase-Mitochondrial ATPase was isolated exactly as described previously (3). The specific activities of the preparations used were between 20 and 22 pmoles of ATP hydrolyzed per min per mg under the described assay conditions (3).
Preparation of Inner Membrane Vesicles-Purified inner membrane vesicles were prepared by sequential treatments of mitochondria with digitonin and Lubrol WX exactly as described by Chan et al. (10). The isolated vesicles were washed once in isolation medium before use in P:O ratio studies.
separation of Nucleotides-Preparative separations of nucleotides were carried out by using columns (0.5 x 8 cm) of Sephadex A-50 (HC03 form) developed with a linear gradient of triethylammonium bicarbonate (40 to 400 mM, pH 8.0) (11). Analytical separations were carried out by using thin layer sheets of polyethyleneimine-substituted cellulose as described by Randerath and Randerath (12). Development was in 1 .O M LiCl for separation of ATP, ADP, and AMP + Pi, or in 1.0 M LiCl-1.0 M formic acid for separation of ATP from ADP + AMP + Pi. Nucleotide spots were located by ultraviolet absorbance, cut out, and analyzed for radioactivity.

Measurement of Binding by Ammonium Sulfate Precipitation of Enzyme-&gad
Complex-Samples (200 ~1, 3 to 4 mg per ml) of purified enzyme in Buffer l2 were treated with 500 ~1 of saturated (at 23") (NH&S04-1 mM EDTA that had been brought to pH 7.5 with solid Tris base. The precipitated enzyme was sedimented by centrifugation at 27,000 x g for 20 min at room temperature.
The supernatant was carefully removed. The sediment was dissolved in 250 ~1 of 10 mM Tris-HCl-1 mM EDTA (pH 7.5). The (NHb)gSOa concentration of the enzyme samples thus prepared was measured with Nessler reagent. Samples (0.5 ml, 0.1 to 0.4 pmole of NHd+) were treated with 0.2 ml of Nessler reagent (Koch and McMeekin type from A. H. Thomas), and A420 was measured after 2 min. The concentration of (NH&SO4 was 118 f 8 mM. Ligand binding was then determined as follows. An aliquot (10 ~1) of 10 mM Tris-HCl (pH 7.5) containing 70,000 cpm of aHlabeled adenine nucleotide, an aliquot (10 ~1) of 10 mM Tris-HCl, 100 mM (NHd)zSOd, 2 mM EDTA (pH 7.5) or 10 IIIM Tris-HCl, 100 DIM (NHJS04, 2 mM EDTA, 20 mM MgClz (pH 7.5) containing 1 .O to 80 pM adenine nucleotide, and an aliquot (20 ~1) of the enzyme sample were mixed in l-ml conical centrifuge tubes. The solutions were incubated at room temperature for the periods of time indicated in the figure legends. The incubations were terminated by addition of 0.4 ml of 3.5 M (NH&S04, 10 mM Tris-HCl, 1 mu EDTA (pH 7.5) or 0.4 ml of 3.5 M (NH&Sod, 10 mM Tris-HCl, 1 InM EDTA, 5 InM MgClz (pH 7.5). Samples were allowed to stand for 10 min at room temperature and were then subjected to centrifugation at 27,000 x g for 20 min. Supernatants were carefully removed.
The sedimented precipitates were dissolved in 100 ~1 of 10 M urea. Radioactivity and protein were determined on aliquots of the 10 M urea solutions.
Correction for unbound radioactivity in sedimented precipi-tates was made by subtracting from each sample the counts per min in the dissolved precipitate from a control incubated with a 1000.fold excess of unlabeled adenine nucleotide (10 mM). Such a control sample was included in each experiment.
This correction was less than 20% of the sample counts per min at the highest ADP concentrations and less than 2% at the low ADP concentrations.
In companion experiments, distribution of label in ATP, ADP, and AMP was determined at various time intervals by removing aliquots from samples identical to those above and separating ATP, ADP, and AMP by chromatography on polyethyleneimineimpregnated cellulose thin layer sheets. Bound nucleotide was released by the 1.0 M LiCl so that total adenine nucleotide was analyzed by this procedure.
No change of label was detected in any of the experiments.
Initial adenine nucleotide concentrations were determined by adsorbance at 259 nm by using a molar extinction coefficient of 15,400. The average number of ligand molecules bound per molecule of enzyme (r) was calculated from the corrected radioactivity and measured protein in each sedimented precipitate. The concentration of free ligand (L) was calculated from the difference between the total ligand concentration and the concentration of bound ligand.
The data were then plotted as r/L versu.s r as recommended by Scatchard (13). If the binding is to a single set of n independent binding sites of dissociation constant Kdiss, the data fall on a straight line described by the following equation (13): r/L = (n/Kdi,,) -(r/Kdiss). If the binding is to multiple sets of sites or to a set of interacting sites, the data fall on a curved plot. Measurement of Binding by Equilibrium Dialysis-Lucite dialysis cells exactly like those designed by Englund et al. (14) were used. Each cell contained two pairs of sample compartments (30 ~1 each) separated by a semipermeable dialysis membrane. Dialysis membranes were prepared from Visking dialysis tubing (A. H. Thomas, inflated diameter = 27/32 inch) which had been boiled in 2 large volumes of 5% NazCOs-2% EDTA and stretched linearly and circularly by the technique of Craig and King (15). The equilibration rate of ADP was increased approximately a-fold by the stretching procedure. A l-mm glass bead was inserted in each compartment to aid in mixing.
Enzyme samples were prepared exactly as described above. The experiment was initiated by adding 20 ~1 of enzyme solution to the "inside" cell compartment and 20 ~1 of a solution of adenine nucleotide (0.5 to 40 PM) containing about 70,000 cpm of %labeled adenine nucleotide in 50 InM (NH&SO+ 10 mrvr Tris-HCl, 1 mM EDTA (pH 7.5) to the "outside" cell compartment. Additions were made with a 25.~1 Hamilton syringe with a blunt needle point.
Cells were then sealed with tape and incubated for 3 hours at room temperature while being rotated at 4 rpm about the long axis of the cell. After dialysis, one 15+1 aliquot was removed from each "inside" compartment and pipetted into 200 ~1 of Buffer 1. Protein concentration, ATPase activity, and radioactivity were determined on aliquots of this solution.
One 15+1 aliquot was removed from each "outside" compartment and pipetted into 100 ~1 of HzO. Radioactivity and distribution of radioactivity among ATP, ADP, and AMP were determined on aliquots of this solution.
The polyethyleneimine-impregnated cellulose thin layer method was used to separate ATP, ADP, and AMP.
Recoveries of protein, total radioactivity, and radioactivity in labeled compound were quantitative.
Recovery of enzymatic activity was between 85 radioactivity in the "outside" compartment. The concentration of bound ligand was calculated from the difference in radioactivity between the two compartments.
The average number of ligand molecules bound per molecule of enzyme (r) was calculated from the concentration of bound ligand and the input protein concentration.
The data were plotted as described above. Measurement of P:O Ratios-P : 0 ratio measurements were carried out in a reaction medium consisting of 220 mM n-mannitol, 70 mM sucrose, 0.5 mM EDTA, 2.0 mM NQhydroxethyl piperazine N'%ethane sulfonic acid, 2.5 InM MgC12, 2.5 mM potassium phosphate, 0.25 PC1 of 32Pi per ml, 0.5 mg of bovine serum albumin (defatted) per ml, 5.0 mM sodium succinate, 5.4 units of hexokinase per ml, 7.1 InM glucose, 360 pg of purified inner membrane per ml, and ADP in the range of 0 to 40 PM.
The total volume was 2.8 ml at pH 7.4. Oxygen consumption was measured with a Clark oxygen electrode (Yellow Springs Instrument Co.) in a closed 3-ml vessel. Respiration was initiated by addition of inner membrane, allowed to proceed for 5 min at room temperature, and then terminated by addition of 0.1 ml of 10 N perchloric acid. 32P-labeled glucose 6-phosphate was determined by measuring the radioactivity of 1 ml of the aqueous phase remaining after extraction of excess "Pi into isobutanol-benzene as described by Nielsen and Lehninger (16). Radioactivity was measured using a low background planchet counter.
Protein Determination-Membrane protein was estimated by the biuret reaction in the presence of 0.33 % sodium cholate (17). Soluble protein was measured by the method of Lowry et al. (18) in a total volume of 0.65 ml. Crystalline bovine serum albumin was used as standard.
The protein was routinely precipitated with 5% trichloroacetic acid and redissolved in 1 N NaOH prior to the determination of protein. iMeasurement of Radioactivity-Radioactivity of 3H-and 32Plabeled compounds was routinely determined by using a liquid scintillation spectrometer of the Beckman 100 series. Aqueous samples (0.5 ml of H20) were counted in 15 ml of a scintillation cocktail consisting of 2.51 g of 2,5-diphenyloxazole, 63 mg of 1,4 bis (2-phenyloxazolyl) benzene, 625 ml of toluene, and 375 ml of absolute ethanol.
Samples adsorbed to solid supports were counted in 10 ml of the above scintillation cocktail without ethanol.
In the P:O ratio experiments, radioactivity of 32Plabeled compounds was measured in a Nuclear-Chicago low background planchet counter after drying the samples onto planchets.

RESULTS
Recovery of Bound ADP and Reversibility of Binding-Initial experiments were designed to determine whether ADP is bound reversibly by mitochondrial ATPase and whether it can be recovered from the enzyme unchanged.
It was particularly important to answer this question in these studies on mitochondrial ATPase, considering the results of recent work on a purified membrane ATPase of strikingly similar subunit structure' involved in photophosphorylation in chloroplasts. The chloroplast ATPase binds two molecules of ADP in a slow process during which the enzyme-bound ADP is transformed to an equilibrium mixture of enzyme-bound ADP, ATP, and AMP (2: 1: 1) (7). Therefore, experiments were directed toward isolation of an ADP-enzyme complex and examination of the bound radioactivity to determine whether ATP or AMP was sedimented precipitates were dissolved in 10 M urea, and the nucleotides, were separated by chromatography on DEAE-Sephadex after addition of unlabeled carrier nucleotide.
Results of experiments in the presence and absence of MgC12 (5 mM) indicated that no detectable enzyme-bound, 3H-labeled AMP or ATP is produced (Fig. I). Reaction of 5% of the enzyme-bound ADP could have been detected.
In these experiments, as in all of the experiments described below, the total nucleotide in the reaction mixtures was analyzed as described under "Methods," and no labeled ATP or AMP could be detected.
The binding of ADP by mitochondrial ATPase is complete within 1 min at both low and high nucleotide concentrations (Fig. 2), and the bound radioactivity can be removed instantaneously by addition of unlabeled ADP.
These three results demonstrate that the binding process is rapid and reversible and that enzyme-bound ADP is not transformed.
Therefore, it is feasible to determine the number of ADP-binding sites and their intrinsic dissociation constants by studies of equilibrium binding. Equilibrium Binding of ADP in the Absence of iWg2+-The results of a typical ADP titration experiment carried out in the absence of Mg2+ by using the ammonium sulfate technique previously described to detect binding are illustrated in Fig. 3 A, mitochondrial ATPase (200 ~1, 2.2 mg per ml) in Buffer 1 was precipitated with ammonium sulfate as described under "Methods." The precipitate (410 pg) was dissolved in 100 ~1 of 10 rnM Tris-HCl, 5 mM MgC12, and 100 pM 3H-labeled ADP (pH 7.5) and incubated for 1 hour at 23". The incubation was terminated by addition of 0.6 ml of 3.5 M (NH4)&!Oa, 10 mM Tris-HCl-5 rnM MgClz (pH 7.5). The resulting precipitate was sedimented at 27,000 X 9 for 20 min. The sediment was washed by resuspension and sedimentation in 0.7 ml of the same ammonium sulfate solunroduced.

Mitochondrial
ATPase was precipitated from Buffer tion. The washed sediment was dissolved in 0.5 ml of 10 M urea 1 as described under "Methods" and incubated with 100 PM and carrier nucleotide was added. Sample was applied to a 3H-labeled ADP for 1 hour. The ADP-enzyme complex was DEAE-Sephadex column, and the nucleotides were separated as isolated by precipitation with ammonium sulfate, and the pre- Binding measurements were made in the absence of MgCl, by using the ammonium sulfate precipitation technique described under "Methods." Reactions were initiated by addition of enzyme and terminated by addition of ammonium sulfate solution.
For the sample at zero time, the ammonium sulfate solution was added before the enzyme. Total ADP concentration (bound + free) was 1.0 PM. Protein concentration was 2.5 pM.
(A molecular weight of 3.8 X lo5 was used in all calculations.) ( Fig. 3). Enzyme that has been cold-inactivated does not bind ADP.
ii plot of the ADP binding data according to Scatchard (13) is presented in Fig. 4 Thus, these experiments detect a single binding site for ADP on the enzyme. ATP is also bound by mitochondrial ATPase at concentrations in the range of 0.2 to 15 PM in the absence of Mg2+. Examination of the enzyme-bound nucleotide reveals, however, that, even in short incubations in the presence of EDTA, approximately one-half of the bound ATP is hydrolyzed to ADP.
Thus, equilibrium binding of ATP cannot be measured under these conditions. This result is of interest, however, since it suggests that ATP hydrolysis can occur in the absence of Mg"+ and other divalent metals, and that ATP is bound in the absence of Mg2+ with an Data are plotted as recommended by Scatchard (13). affinity much greater than would be expected considering the K, for hydrolysis of MgATP of 0.78 mM. 3 Two results suggested that the ammonium sulfate technique used here was in fact measuring the nucleotide bound at equilibrium.
First, if ammonium sulfate was added before enzyme, no binding occurred (Figs. 2 and 8, zero time points).
Second, the ADP-enzyme complex was stable in ammonium sulfate suspension for up to 60 min. To verify this conclusion, binding of ADP was also studied by the micro-equilibrium dialysis technique of Englund et al. (14). In preliminary experiments, the half-time for equilibration of ADP across the dialysis membrane was determined and found to be 18 min. Thus, at 3 hours, the system was within 0.1% of equilibrium. The results of these two types of binding experiments indicate that mitochondrial ATPase is able to bind a single molecule of ADP in the absence of divalent metal with a dissociation constant of approximately 0.9 PM. Equilibrium Binding of ADP in the Presence of Mg2+-Because a divalent metal ion (usually Mg"+) is required for ATPase activity of both soluble and membrane-bound mitochondrial ATPase (1) and for ATP-Pi exchange activity of membranebound ATPase complex (lo), it seemed important to measure binding of ADP to this binding site in the presence of Mg2+. These measurements were complicated, however, by the instability of the enzyme in the presence of Mg*+. The loss of enzymatic activity that follows the addition of MgClz (10 mM) to mitochondrial ATPase in Buffer 1 is illustrated in Fig. 6. Greater than 90% of the enzymatic activity is lost. The initial phase of loss of activity is approximated by a single exponential of ti/z between 6 and 7 min (Fig. 6, inset). To determine whether this loss of enzymatic activity is accompanied by a loss of ADP ADP bound and ATPase activity remaining were measured at various times after mixing enzyme, MgCL, and ADP. These results are presented in Fig. 7. Although the loss of ATPase activity is rapid (50% loss in 6 to 7 min), the ADP bound per mole enzyme increases to a maximum at the earliest measurable time (0.5 min) and then decreases much more slowly than the ATPase activity (50% loss in 60 to 80 min). These results suggest that a reliable estimate of binding of ADP in the presence of M8"f can be obtained by using the ammonium sulfate technique to measure ADP bound after short incubations.
Results of ADP titration experiments in the presence of MgZf using 2-min incubation times are presented as a Scatchard plot in Fig. 8 more than a single binding site for ADP. The dissociation constant for ADP binding is increased approximately 2-fold in the presence of Mg2+.
The average values of n and Kdiss in the three series of experiments described are collected in Table I. In the absence of Mg*, the ammonium sulfate precipitation method and equilibrium dialysis method give identical results within error. The averages are n = 0.88 and Kdiss = 0.92 PM. In the presence of Mg*, Kdiss increases to 2.1 pM, and the number of binding sites observed decreases to 0.65. The reason for the decrease in the number of binding sites observed in the presence of Mg"+ is unknown at present.
K, for ADP in Oxidative Phosphorylation-In order to compare the affinity of isolated mitochondrial ATPase for ADP with the affinity of the oxidative phosphorylation system for ADP, we have measured the dependence of the phosphorylation efficiency (P:O ratio) of purified inner membrane vesicles of rat liver mito-,chondria (10) on ADP concentration.
These vesicles are ideal for such measurements because adenylate kinase activity is very low (lo), the concentration of endogenous ADP is negligible, and mitochondrial ATPase is situated on the outer surface of the vesicle membrane directly available to added ADP (10).
The results of a series of experiments in which P:O ratios were measured with succinate as respiratory substrate at ADP concentrations between 0 and 40 PM are presented in Fig. 9. Maximum phosphorylation efficiency is achieved at ADP concentrations greater than 20 to 30 PM. Half-maximum efficiency is achieved at 3 to 4 PM. At all concentrations, the observed phosphorylation is inhibited 90% or more by 2 pg of oligomycin per mg of vesicle protein.
Atractyloside at a concentration (3.6 pM) sufficient to inhibit translocation of adenine nucleotides (19) did not cause significant reduction in phosphorylation efficiency. Since the rate of succinate oxidation by these inner membrane vesicles is not dependent on the ADP concentration, the P:O ratio can be treated as a steady state rate of phosphorylation. A double reciprocal plot of [P:O ratio]-' ver.sus [ADPI-l is illustrated in Fig. 10. The data extrapolate to a K, of 3.0 pM for ADP in oxidative phosphorylation.
The average value (f standard error of the mean) derived from a series of six experiments was K, (ADP) = 3.85 f 0.45 MM. This value is similar to the dissociation constants for ADP binding by purified mitochondrial ATPase of 2.1 and 0.9 PM in the presence and absence of Mg2+, respectively. E$ect of Aurovertin on ADP Binding-Aurovertin has been shown to be a potent inhibitor of oxidative phosphorylation in mitochondria (8,20), but a relatively poor inhibitor of ATPsupported energy-linked reactions and ATPase activity (8, 21) of mitochondria and submitochondrial vesicles. The results of a recent kinetic study of ATPase activity of sonicated submitochondrial vesicles (22) indicate that aurovertin does not change V,,, or K, (ATP) of the ATPase reaction but increases Ki (ADP) from 8.6 to 180 PM, an increase of 21-fold.
In addition, aurovertin partially inhibits the ATPase activity of soluble preparations of mitochondrial ATPase3 (4,23,24). These data suggested that aurovertin might inhibit ADP binding to mitochondrial ATPase.
The results presented in Fig. 11 indicate that in absence of Mg2+ aurovertin does indeed reduce ADP binding.
However, in the presence of Mgz+ the inhibitor enhances the binding of ADP.
Both half-maximum stimulation and inhibition occur at 8 pg of aurovertin per mg of purified enzyme. Half-maximum inhibition of oxidative phosphorylation occurs at 0.24 pg of aurovertin per mg of mitochondrial protein.
3 Since mitochondrial ATPase is purified approximately 50-fold from mitochondria, the effects on ADP binding occur at concentrations of aurovertin similar to those that inhibit oxidative phosphorylation.
The inhibition of ADP binding in the absence of MgZ+ (Fig. 11) corresponds to an increase in Kd iss (ADP) from 0.9 pM to approximately 14 PM or 16-fold, assuming that the number of ADP- 11. Effect of aurovertin on binding of ADP by mitochondrial ATPase.
Aliauots of aurovertin solution (100 pg per ml in absolute ethanol) were pipetted into l-ml conical centrifuge tubes, and the ethanol was removed by evaporation under high vacuum (50 Bm). Binding measurements were then made by using the ammonium sulfate technique as described under "Methods." Incubations were for 60 mm in the absence of Mg2+ (MS++) and for 2 min in the nresence of Ma*+. Total ADP concentration (bound + free) was 1.0 ELM. Each-point represents the average of two determinations.
Protein concentrations in the four experiments presented were between 2.7 and2.9 p&r. Control experiments with absolute ethanol indicated that the residue left on the tubes after evaporation had no effect on the measured binding of ADP. With Mg*+ (a---L); without Mg"+ (O--O).
binding sites is unchanged.
The stimulation of ADP binding in the presence of Mg* corresponds to a decrease in &iss (ADP) from 2.1 PM to approximately 0.5 pM or 4.2-fold. The stimulation of binding of ADP in the presence of Mg2+ is not due to protection of ATPase activity on treatment with Mg* since the rate of activity loss is unaffected by the presence of aurovertin. Neither is the difference in response in the presence and absence of Mg* due to the different incubation times (2 min versus 60 min) since similar inhibition is observed in the absence of Mg2+ when 2-min incubations are carried out. Thus, these data seem to indicate a fundamental difference in the response of mitochondrial ATPase to aurovertin in the presence and absence of Mg2+.

DISCUSSION
The results presented indicate that mitochondrial ATPase binds no more than a single molecule of ADP per enzyme molecule in the presence and absence of Mg2+. The binding site observed has two of the properties that might be expected of a catalytic site for oxidative phosphorylation: first, Kd iss (ADP) 2 K, (ADP) and second, ADP binding is modified by aurovertin.
It seems probable, therefore, that this binding site is part of the catalytic site for oxidative phosphorylation. A number of lines of evidence indicate that this preparation of mitochondrial ATPase consists of a total of 7 subunits: 3 of 62,500,3 of 57,000, and 1 of 36,0003 (3). Since we have observed a single binding site for ADP, suggesting a single catalytic site, the simplest conclusion is that the catalytic site is located on the lone subunit of 36,000. This conclusion cannot be made uncritically, however, because a number of enzymes (listed by Frieden (25)) have been shown to have fewer binding sites than identical subunits.
Thus, the catalytic site might reside on one of the larger subunits and a mechanism, such as negative cooperativity (26), might ensure that only one site is occupied by ADP at any given time. Further work is required before the relationship between the active site and the subunits can be firmly established.
Several characteristics of the binding reaction described here distinguish it from the reaction carried out by chloroplast ATPase (7). The reaction is rapidly reversible.
Only 1 molecule of ADP is bound.
The bound ADP can be quantitatively recovered as ADP.
Thus, although the two isolated ATPases have similar physical properties and subunit compositions' (3, B), there appear to be fundamental functional differences.
The ADP-binding site we have observed binds ADP with a dissociation constant of 0.94 pM in the absence of Mg2+ and 2.1 PM in the presence of Mg*.
The affinity of our preparation of rat liver mitochondrial ATPase for ADP is many fold greater than that of bovine heart mitochondrial ATPase as prepared by Pullman et al. (1). That enzyme preparation exhibits a Ki (ADP) for competitive inhibition of ATPase activity of 1.5 mM (I) or 30 PM (27)) depending on the assay used. These differences may be due to the different parameters measured, Kdiss versus Ki. In any case, the K, (ADP) measured for oxidative phosphorylation by inner membrane vesicles of rat liver mitochondria is 3.8 PM, indicating that mitochondrial ATPase in situ must bind ADP with a dissociation constant of 3.8 PM or less. The dissociation constants we have measured for ADP binding by soluble mitochondrial ATPase are closely similar to this limit.
The K, (ADP) for ATP production by intact mitochondria has been measured previously and the values reported range from 2 to 3 PM (28) to 20 to 30 PM (29), depending upon the conditions under which the measurements are performed (28). The interpretation of this value is ambiguous, however, because either oxidative phosphorylation itself or adenine nucleotide transport could be rate limiting.
In sonicated submitochondrial vesicle preparations, mitochondrial ATPase is located on the external surface of the vesicles directly available to added ADP.
However, measurements of Km (ADP) for oxidative phosphorylation catalyzed by submitochondrial membrane vesicles have given variable results.
The high values of K, (ADP) for ATP production by submitochondrial vesicles together with the inability of mitochondrial ATPase from bovine heart to bind ADP with high afhnity (1) have been interpreted as indicating that the K, (ADP) for oxidative phosphorylation itself is high, whereas the rate-limiting step in ATP production in intact mitochondria is the translocation of adenine nucleotide (28,29). Two results presented here demonstrate that this hypothesis is unnecessary.
First, the K, (ADP) for ATP production by purified inner membrane vesicles is 3.8 PM in agreement with the lowest values of K, (ADP) for intact mitochondria (28). Under the experimental conditions used here, K, (ADP) for intact mitochondria was estimated to be less than 10 pM (10). Second, soluble mitochondrial ATPase binds ADP with a Kdiss (1 to 2 PM) lower than K, (ADP) in oxidative phosphorylation.
Thus, it seems probable that the measured K, (ADP) for ATP synthesis by intact mitochondria reflects the affinity of mitochondrial ATPase for ADP rather than the affinity of the adenine nucleotide translocation system for ADP. The higher values for K, (ADP) for ATP synthesis obtained by some investigators by using sonicated membrane preparations may reflect damage to the coupling system during sonication. Aurovertin is a potent effector of the ADP-binding reaction described here. The effects occur at concentrations of aurovertin that correlate closely on a nanomole of aurovertin per unit of ATPase basis with those that inhibit oxidative phosphorylation. The effect of aurovertin on binding of ADP by mitochondrial ATPase is modulated by Mg*.
In the absence of Mg*, binding is inhibited; whereas in its presence, binding is enhanced.
We prefer to interpret these results in terms of a direct interaction of Mg" with mitochondrial ATPase.
The results do not, however, exclude the possibility that the difference in response to aurovertin reflects the change in bound species from ADP to MgADP.
Another result presented here strongly suggests that soluble mitochondrial ATPase is capable of direct interaction with Mg*.
Addition of Mg2+ to the enzyme under conditions of optimal stability causes a rapid loss of 90% of the enzymatic activity without a concomitant loss of ability to bind ADP. This is most easily explained in terms of an enzyme-Mg* complex that has lower ATPase activity than the uncomplexed enzyme but has similar capacity for ADP binding.
Thus, these two results are best interpreted in terms of a functionally significant interaction of Mg2+ with purified mitochondrial ATPase. This interaction may play an important role in mediation of the myriad effects of Mg2f on the characteristics of oxidative phosphorylation (31, 32). The binding of ADP by soluble mitochondrial ATPase is closely similar to the interaction of ADP with the oxidative phosphorylation system. These results, therefore, describe an important partial reaction of oxidative phosphorylation carried out by a soluble homogeneous enzyme preparation.
Experiments in our laboratory at present are directed toward development of conditions under which other partial reactions of oxidative phosphorylation including formation of a "high energy" phosphorylated intermediate and catalysis of ATP-32Pi exchange and ATP-14C-ADP exchange can be observed in systems containing soluble homogeneous mitochondrial ATPase.