©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Energetics of ATP Dissociation from the Mitochondrial ATPase during Oxidative Phosphorylation (*)

Abdul-Kader Souid (§) , Harvey S. Penefsky (¶)

From the (1) Department of Biochemistry and Molecular Biology and the Department of Pediatrics, State University of New York, Health Science Center, Syracuse, New York 13210

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The dissociation constant ( K) for ATP bound in the high affinity catalytic site of membrane-bound beef heart mitochondrial ATPase (F) was calculated from the ratio of the rate constants for the reverse dissociation step ( k) and the forward binding step ( k). kfor ATP bound to submitochondrial particles or to submitochondrial particles washed with KCl so as to activate ATPase activity was accelerated by about five orders of magnitude during respiratory chain-linked oxidations of NADH. In the presence of NADH and 0.1 m M ADP, kincreased more than six orders of magnitude. These energy-dependent dissociations of ATP were sensitive to the uncoupler carbonyl cyanide p-trifluoromethyloxyphenylhydrazone. Only small changes in kwere observed in the presence of NADH or NADH and ADP. Kat 23 °C in the absence of NADH and ADP was 10 M, in the presence of NADH, 3 µ M, and in the presence of NADH and 0.1 m M ADP, 60 µ M. Thus, the dissociation of ATP during the transition from non-energized to energized states was, under these conditions, accompanied by observed free energy changes of 8 and 9.7 kcal/mol, respectively.


INTRODUCTION

The FF() -ATPase catalyzes the terminal transphosphorylation reaction of oxidative phosphorylation, that is, the formation of ATP from ADP and P. The required energy for the reaction, stored as a proton motive force (Mitchell, 1961), is supplied by oxidations in the respiratory chain. Although the coupling reaction, that is, the manner in which proton flux is utilized to drive ATP formation, is only poorly understood, progress has been made in elucidating the mechanism of action of the mitochondrial ATPase in catalyzing ATP synthesis (for reviews see Penefsky and Cross, 1991; Hatefi, 1993). The elementary steps in the hydrolysis of ATP by both the soluble (Grubmeyer et al., 1982; Cross et al., 1982) and membrane-bound forms of beef heart mitochondrial F(Penefsky, 1985a, 1985b) supported a model for ATP synthesis in oxidative phosphorylation in which ATP could be formed from bound ADP and Pwith little or no change in free energy (Kfor the catalytic step was near unity). The free energy of binding of product ATP in high affinity catalytic sites of Fwas viewed as the driving force for ATP formation (Kfor the binding step was 10 M). Thus, the major requirement for energy in oxidative phosphorylation was for the dissociation of product ATP from high affinity catalytic sites (Penefsky and Cross, 1991). Insight into the nature of the coupling device was provided by further observations suggesting long range interactions, apparently involving the transmission of conformational changes, between proton-binding sites in Fand the catalytic sites of F(Penefsky, 1985c; Matsuno-Yagi et al., 1985). These observations support and extend the original proposals of Boyer (1993) in the binding change mechanism of oxidative phosphorylation.

The dissociation constant of 10 M for dissociation of ATP from high affinity catalytic sites of soluble (Grubmeyer et al., 1982) or non-energized membrane-bound F(Penefsky, 1985a) suggested that during oxidative phosphorylation the catalytic sites would have to undergo a decrease in affinity for ATP of many orders of magnitude. Experimental precedent for cyclic changes in the affinity of catalytic sites was established in studies of the hydrolysis of trinitrophenyl-ATP (TNP-ATP) by soluble F. TNP-ATP and TNP-ADP both were bound at site 1 of the enzyme with an affinity too high to be measured. Nevertheless, the enzyme catalyzed the hydrolysis of TNP-ATP (Grubmeyer and Penefsky, 1981a, 1981b).

However, a dissociation constant of 10 M is equivalent to a standard free energy change of about 16 kcal/mol, whereas the standard free energy change accompanying ATP hydrolysis was reported to be about -8.5 kcal/mol (Rosing and Slater, 1972) and the phosphorylation potential generated by respiring submitochondrial particles ( G) was reported to be 10.6 kcal/mol (Ferguson and Sorgato, 1977). This apparent discrepancy is resolved by observations presented in this paper that the dissociation constant for ATP ( K) from submitochondrial particles under conditions of oxidative phosphorylation used in these experiments was about 60 µ M. Thus, the observed free energy change relevant to the dissociation of ATP during the transition from non-energized to energized states was 9.7 kcal/mol. The changes in affinity for ATP were expressed largely as an increase in the rate of ATP dissociation under energized conditions. It was found that the rate of dissociation of ATP was accelerated five orders of magnitude in the presence of NADH and about six orders of magnitude in the presence of NADH and 0.1 m M ADP. The observed energized dissociation constant of 3 µ M (in the presence of NADH alone) is in the same range as values reported for the chloroplast thylakoid of 7 µ M (Magnusson and McCarty, 1976) and for Paracoccus denitrificans of 16 µ M (Perez and Ferguson, 1990a, b).


EXPERIMENTAL PROCEDURES

Materials P(enzyme grade) was purchased from ICN and used without further purification. [-P]ATP was prepared as described (Glynn and Chappel, 1964). The specific radioactivity of most preparations was 5 10counts/min/nmol. Hexokinase, ATP, ADP, and bovine serum albumin were purchased from Sigma. NADH (``100% pure'') was purchased from Boehringer Mannheim. Methods Submitochondrial particles, ETPH(Mg), were prepared from beef heart mitochondria (Beyer, 1967) and activated by washing with buffered solutions of KCl (Penefsky, 1985a). The specific ATPase activity of preparations of submitochondrial particles was about 0.8-1.0 units/mg of protein and that of KCl-washed particles 10 units/mg of protein. The amounts of Pand [-P]ATP in reaction mixtures were determined by scintillation counting of organic and aqueous phases after extraction of perchloric acid-quenched samples with isobutanol/benzene (Lindberg and Ernster, 1956). Aliquots of each of the phases were added to a scintillation mixture described earlier (Penefsky, 1977) and counted in a Beckman LS-6500 liquid scintillation counter. F(Penefsky, 1977) and OSCP (Senior, 1979) were prepared as described. The preparation of OSCP was carried out through the step of extraction with ammonia. The resulting supernatant solution was treated with 42% solid ammonium sulfate, and the precipitate was collected and dissolved with 20 ml of a solution containing 50 m M Tris-SO, pH 8, 0.1 m M dithiothreitol, and 0.1 m M EDTA. The solution was stored at -70 °C. After thawing, the solution was centrifuged at 165,000 g to remove the precipitate that formed. The supernatant was stored at -70 °C and dialyzed before use versus a solution containing 50 m M Tris-SO, pH 8, 0.1 m M dithiothreitol, and 0.1 m M EDTA.

Reconstitution of KCl-washed Particles with Soluble F

F(3.7 mg), OSCP (2.8 mg), and albumin (35 mg) were incubated with 44 mg of KCl-washed ETPH(Mg) in a buffer containing 0.25 M sucrose, 50 m M Tris acetate, pH 7.4, 12 m M P, pH 7.4, 5 m M ATP, 1 m M dithiothreitol, 0.5 m M EDTA, and 20 m M succinate for 5 min at room temperature with rapid stirring. The final volume was 17.5 ml. Succinate was essential for maximal and stable reconstitution. The reconstituted particles were separated from the soluble ATPase by centrifugation for 20 min at 40,000 revolutions/min in a Beckman Ti 50.2 rotor (4 °C). The pellets were washed once by resuspension in 15 ml of a buffer containing 0.25 M sucrose, 5 m M MgSO, 40 mg of albumin, and 1 m M dithiotheitol, followed by centrifugation under the same conditions, and resuspended at 15 mg/ml in the same buffer. Energy-dependent Dissociation of [-P]ATP Bound in High Affinity Catalytic Sites of Submitochondrial Particles-The reaction mixture contained 20 m M Tris-SO, pH 8, 5 m M P, 5 m M MgSO, 0.25 M sucrose, 50 m M glucose, 0.1 µ M [-P]ATP, 200 units of hexokinase, 1 m M NADH, and 1-2 mg of the particles. The final volume was 1.0 ml. The particles were incubated for 1 min at room temperature with all additions except NADH, hexokinase, and radioactive ATP. After incubation for 5-10 s with [-P]ATP (in order to form the enzyme-substrate complex), hexokinase was added and an additional 5 s of incubation was allowed to convert any free [-P]ATP to glucose-6-P. In a separate experiment, 5 s of incubation in the presence of 200 units of hexokinase was sufficient to convert all added [-P]ATP to glucose-6-P. Respiration was initiated with NADH. The reaction was terminated at the times indicated in the figures by adding 1.0 ml of 2 N HCl. [-P]ATP and Pwere determined by scintillation counting of aqueous and organic phases after extraction of samples with isobutanol/benzene as described by Penefsky (1977). Glucose-6-P was determined by the same methods after hydrolysis of the samples in 1 N HCl at 100 °C for 7 min. The amount of [-P]ATP bound in catalytic sites at each time point was determined by carrying out a cold chase measurement as described under Measurement of Unisite Catalysis.

Measurement of Unisite Catalysis

Submitochondrial particles (the type and amounts indicated in the figure legends) were mixed with substoichiometric amounts of [-P]ATP in a reaction mixture containing 20 m M Tris-SO, pH 8, 5 m M P, 5 m M MgSO, and 0.25 M sucrose. The final volume was 1.0 ml. The reaction was carried out at room temperature in a flat bottom, glass scintillation vial with rapid stirring via a magnetic stirring bar and was started by adding the [-P]ATP. At the times indicated in the figures, the reaction was stopped by adding 1 ml of 2 N HCl (acid quench), or a cold chase was carried out by adding 40 µl of 150 m M MgATP, permitting the reaction to proceed for an additional 5 s and then quenching with HCl as before. The amounts of [-P]ATP and Pin reaction mixtures were determined by scintillation counting of aqueous and organic phases after extraction of deproteinized reaction mixtures with isobutanol/benzene. In separate experiments, it was determined that the extent of hydrolysis of the cold chase in these experiments was less than 2% of the added nonradioactive ATP. Thus, no correction was made for cold chase hydrolysis. Corrections were made for the 1-2% of free Ppresent in the [-P]ATP.

Measurement of ATP Binding in High Affinity Catalytic Sites of the Membrane-bound Enzyme

Binding was determined using acid quench and cold chase techniques as described earlier (Penefsky, 1985a). Reconstituted, KCl-washed ETPH(Mg), 0.25 mg, or 2 mg of ETPH(Mg), were mixed with 0.01 nmol or 0.1 nmol, respectively, of [-P]ATP, and the enzyme-substrate complex was allowed to form for periods of time indicated on the abscissae before the reactions were quenched with acid (lower curves) or chased by an excess of cold ATP (upper curves). The difference between hydrolysis in the acid quench and hydrolysis in the cold chase is set equal to the amount of radioactive ATP bound in catalytic sites. The amount of Pformed in cold chase experiments (upper curves) was set equal to the amount of enzyme-substrate complex formed (F P). This latter value was used to calculate the bimolecular rate constants for ATP binding. The results are plotted in the form of a second order rate equation, as explained in the legends to Fig. 1and 2.


Figure 1: Rate of binding of [-P]ATP in catalytic sites of reconstituted, KCl-washed ETPH(Mg). A, 0.125 mg of the particles were incubated for 1 min at room temperature in a buffer containing 20 m M Tris-SO, pH 8, 5 m M KHPO, 5 m M MgSO, and 0.25 M sucrose. The final volume was 1.0 ml. The reaction was started by adding 0.005 nmol of [-P]ATP. The acid quench ( lower curves) represents the amount of Pformed after the indicated seconds of incubation. The cold chase ( upper curves) represents the amount of Pformed plus the amount of [-P]ATP hydrolyzed following addition of 6 m M MgATP. After addition of the cold chase, the enzyme was allowed to turn over for 5 s before the reaction was stopped by addition of 1.0 ml of 2 N HCl. In the acid quench experiment, 1.0 ml of 2 N HCl was added before the nonradioactive ATP. Pformed was separated from [-P]ATP as described under ``Experimental Procedures.'' Hydrolysis is expressed as the percent of the total [-P]ATP added. Low concentrations of enzyme and [-P]ATP were used in panel A to permit a better resolution of the initial portion of the on reaction. For the experiments in panels B-D, the reaction mixtures contained 0.25 mg of particles and 0.01 nmol of [-P]ATP. B, the reaction mixture was the same as that described for panel A, except that 0.25 mg of the particles and 0.01 nmol of [-P]ATP were used, and 0.1 m M ADP was present in the incubation mixture. C, the reaction mixture was the same as that described for B except that ADP was not added, and respiration was started by adding 1 m M NADH, followed 10 s later by addition of 0.01 nmol of [-P]ATP. D, the reaction mixture was the same as that described for C except that 0.1 m M ADP also was present in the reaction mixture. E, graphical determination of k, the bimolecular rate constants for [-P]ATP binding shown in panels B-E. The constants were calculated from the slopes by using the equation: k = slope/F-ATP. Fand ATPrepresent the initial concentrations of Fand ATP, respectively. FP is the concentration of FP complex at time t. The concentration of Fin reaction mixtures was calculated on the assumption that each milligram of submitochondrial particles contained 0.4 nmol of F(Harris et al., 1977; Beltran et al., 1986; Matsuno-Yagi and Hatefi, 1988). The designations of each of the slopes refer to panels A-D. The calculated rate constant in the absence of ADP and NADH was 3.5 10 Ms; in the presence of 0.1 m M ADP, 8 10 Ms; in the presence of 1 m M NADH, 8.6 10 Ms, and in the presence of both ADP and NADH, 4.1 10 Ms.



Measurement of Oxidative Phosphorylation

The reaction mixture contained 50 m M Tris acetate, pH 7.5, 0.25 M sucrose, 50 m M glucose, 5 m M magnesium acetate, 0.4 m M EDTA, 20 m M potassium phosphate, P(100-300 counts/min/nmol), 1 m M ADP, 50 units of hexokinase, and 60-70 µg of submitochondrial particles. After 5 min of incubation at 30 °C, the reaction was initiated by adding 1 m M NADH. The final volume was 0.65 ml. The reaction was stopped by adding 0.05 ml of 70% perchloric acid. Glucose-6-P formed was determined after extraction with isobutanol/benzene as described (Penefsky, 1977). Negligibly small amounts of glucose-6-P were formed during the 5-min preincubation period. The rate of oxidation of NADH was measured polarographically using a Yellow Springs Instruments model 53 device under exactly the same conditions used for the ATP synthesis measurement except that Pwas omitted and the final volume of the reaction mixture was 1.35 ml.

Assay of ATPase Activity

ATPase was determined using a regenerating system for ATP as described by Pullman et al. (1960). One unit of ATPase activity is defined as 1 µmol of ATP hydrolyzed per min. Specific activity is defined as units/milligram of protein.

Protein Concentration

The concentration of mitochondrial proteins was determined by a modified Biuret procedure (Pullman et al., 1960) using bovine serum albumin as standard.


RESULTS

A summary of the specific activities of the submitochondrial particles used in these experiments is shown in Table I. The ATPase activity of ETPH(Mg), 0.8-1 µmol of ATP/min/mg, line 1, was in the range commonly observed with these particles and a substantial rate of ATP synthesis with NADH as substrate also was found (1.06 µmol of ATP formed/min/mg protein). Washing with KCl increased the ATPase activity of the particles to a value of 11 units/mg, line 2, but reduced the rate of ATP synthesis as well as the P/O ratio. Reconstitution of KCl-washed particles with F, line 3, was without effect on ATPase activity but substantially enhanced the rate of ATP synthesis. It should be noted that ATPase activity of the particles in lines 1-3 of the table was inhibited more than 90% by oligomycin.

Rate of Binding of ATP in High Affinity Catalytic Sites of Reconstituted, KCl-washed ETPH(Mg)

Binding measurements were carried out using the techniques of acid quench and cold chase as described previously (Penefsky, 1985a, 1985b). Fig. 1 A, lower curve, shows that relatively little hydrolysis of 0.005 µ M [-P]ATP (8%) occurred during 4 s of incubation with 0.125 mg of KCl-washed ETPH(Mg). However, the cold chase revealed that substantial binding of [-P]ATP (50%) as such occurred during the same time period, Fig. 1 A, upper curve. The apparent rate of binding was similar to that previously reported (Penefsky, 1985a), but reflected instead the lower ligand concentrations used. Throughout this paper it was assumed that each milligram of submitochondrial particles contained 0.4 nmol of F(Harris et al., 1977; Ferguson et al., 1976; Beltran et al., 1986; Matsuno-Yagi and Hatefi, 1988). Thus, the molar ratio of Fto ATP in the experiment was calculated to be 10. Fig. 1 B shows that hydrolysis of 0.01 µ M [-P]ATP in the acid quench measurement, lower curve, was considerably enhanced in the presence of 0.1 m M ADP and that the acceleration of hydrolysis in the cold chase, upper curve, was correspondingly reduced. This observation is reminiscent of earlier findings that ADP promoted the hydrolysis of ATP bound in high affinity catalytic sites (Penefsky, 1985a). The rate of hydrolysis in the acid quench of 0.01 µ M[-P]ATP in the presence of 1 m M NADH, Fig. 1 C, lower curve, was similar to that found in the absence of NADH, Fig. 1 A, lower curve. The cold chase experiment, Fig. 1 C, upper curve, indicates that a substantial amount of [-P]ATP (23% of that which was added) was bound in catalytic sites after 5 s of incubation in the presence of NADH. However, in the presence of both ADP and NADH (Fig. 1 D), that is, under the conditions of oxidative phosphorylation, almost no ATP as such was found in the high affinity catalytic site. Hydrolysis in the acid quench was 14% after 5 s while hydrolysis in the cold chase was only 19%. The lower concentrations of enzyme and [-P]ATP used in Fig. 1 A permitted better resolution of the initial portion of the ``on'' reaction. The results for Fig. 1, A- D, were plotted in the form of a second-order rate equation in Fig. 1 E. The calculated bimolecular rate constants ( k), that is, the on rates, are summarized in Table II. kin the absence of any additions, Fig. 1 A, 3.5 10 Ms, compares well with the value previously reported for KCl-washed submitochondrial particles, Penefsky (1985a), of 4.0 10 Ms. The small differences in the values of kfor the various experimental conditions of Fig. 1were reproducible ().

Rate of Binding of ATP in High Affinity Catalytic Sites of ETPH(Mg)

The measurements with ETPH(Mg) were carried out under exactly the same conditions used for the study of KCl-washed and reconstituted submitochondrial particles except that the protein and [-P]ATP concentrations were somewhat higher in the former. The experiments of Fig. 2, ETPH(Mg), panel A, were done using an 80-fold excess of enzyme over [-P]ATP, 2 mg of particles (0.8 nmol of F) and 0.01 nmol of [-32P]ATP, may be compared with those of Fig. 1 A, KCl-washed and reconstituted particles. When the concentration of [-P]ATP was increased 10-fold, the rate of hydrolysis in the acid quench, panel B of Fig. 2, lower curve, was considerably higher than that in panel A of Fig. 2, lower curve. Nevertheless, the on rate constants were virtually identical in both experiments (Fig. 2, legend). In addition, the cold chase showed that 37% of added [-]ATP was bound as such (calculated from the difference between cold chase and acid quench hydrolysis) in catalytic sites of ETPH(Mg) and activated particles (Fig. 2, panel A and Fig. 1, panel A, respectively) but only 24% in ETPH(Mg) in panel B of Fig. 2. The presence of 100 µ M ADP was without effect on acid quench hydrolysis but reduced the level of bound [-P]ATP as such to only 10% of that added, Fig. 2 C. Similar low levels of [-P]ATP binding were seen in the presence of NADH, Fig. 2 D, and even less binding in the presence of NADH and ADP, Fig. 2 E. Pwas present in all of these experiments but had little effect on either the on rates or the ``off'' rates. Calculations of kfrom the slopes of second-order plots, Fig. 2 F, were carried out on the assumption that the particles contained 0.4 nmol of F/mg of protein and that the observed binding took place in a single catalytic site. The results are shown in . Similar to experiments with KCl-washed particles, small but reproducible decreases in kwere observed when the particles were energized (). Measurement of the Rate of Binding of [-P]ATP in High Affinity Catalytic Sites of ETPH(Mg) Using Hexokinase-The rate of binding of [-P]ATP was determined as hexokinase-inaccessible ATP (see legend to Fig. 3) in order to allow for the possibility that the presence of ADP, which is known to inhibit ATPase activity (Pullman et al., 1960; Vasilyeva et al., 1982; Feldman et al., 1985) would interfere with measurements made via the cold chase technique.()The on rate in the absence of ADP by the hexokinase method was 8 10 Msand may be compared with a value of 2.3 10 Msobtained by the cold chase technique, Fig. 2 A. The higher value of the on rate by the hexokinase method reflects the fact that 20-30% of the bound radioactive ATP that is inaccessible to hexokinase, cannot be chased into P. The on rate by the hexokinase technique in the presence of 0.1 m M ADP was 1.6 10 Msand was the same in the presence of 1 m M NADH.


Figure 2: Rates of binding of [-P]ATP in catalytic sites of ETPH(Mg). The composition of the reaction mixtures was exactly the same as described in Fig. 1 except that the concentration of ETPH(Mg) was 2 mg/ml. The concentration of [-P]ATP was 0.01 µ M for the experiments in panel A and 0.1 µ M for the experiments in panels B-D. Experiments in panels A and B were done in the absence of NADH and ADP, in panel C, in the presence of 0.1 m M ADP, in panel D, in the presence of 1 m M NADH, and in panel E, in the presence of both NADH and ADP. The designations in panel F on each of the slopes refer to panels B-E. The calculated rate constant in the absence of ADP and NADH was 2.3 10 Ms; in the presence of 0.1 m M ADP, 1.6 10 Ms; in the presence of 1 m M NADH, 1.0 10 Msand in the presence of both ADP and NADH, 3.8 10 Ms. The calculated kin panel A was 1.53 10 Ms. The calculated rate constant in a different preparation in the absence of ADP and NADH was 2.0 10 Ms; in the presence of 0.1 m M ADP, 0.23 10 Ms; in the presence of 1 m M NADH, 0.45 10 Ms, and in the presence of both ADP and NADH, 2.1 10 Ms.




Figure 3: Rate of binding of [-P]ATP in catalytic sites of ETPH(Mg) in the presence of ADP and NADH and in the absence of P in the buffer. Panel A, ATP binding was measured as hexokinase-inaccessible ATP as described earlier (Penefsky, 1985c). The reaction mixtures contained, in a final volume of 1.0 ml, 2 mg of ETPH(Mg), 20 m M Tris-SO, pH 8, 5 m M MgSO0.1 m M ADP, and 0.25 M sucrose. The temperature was 23 °C. After a 1-min preincubation, the reaction was started by adding 1 m M NADH followed 10 s later by 0.1 nmol of [-P]ATP (specific activity 1.7 10counts/min/nmol). Hexokinase (200 units) was then added at the time points indicated on the abscissa, and the reaction was continued for an additional 5 s before termination with 1.0 ml of 2 N HCl. The solutions were heated for 7 min in a boiling water bath, and glucose-6-P was separated and quantitated as described under ``Experimental Procedures.'' Hexokinase-inaccessible ATP was calculated as the difference between the amount of glucose-6-P formed in reaction mixtures and the amount formed in the absence of submitochondrial particles. Panel B, the data of panel A are plotted in the form of a second-order rate equation. kwas 5 10 MsPanel C, ATP binding was measured by cold chase as described in Fig. 2. The composition of the reaction mixtures was exactly as described for panel D of Fig. 2 except that Pwas not present in the buffer. Hydrolysis during the acid quench ( lower curve) and cold chase ( upper curve) was determined at points indicated on the abscissa. Panel D, the data of panel C are plotted in the form of a second-order rate equation. kwas 2.1 10 Ms.



The on rate constants in ETPH(Mg) in the presence of NADH and 0.1 m M ADP were also measured under the exact conditions used in but without P(Fig. 3). This was done in order to eliminate the possibility that ATP synthesis might interfere if it occurred during the on rate measurements. kin the absence of Pwas 5.0 10 Msby the hexokinase method and 2.1 10 Msby cold chase (Fig. 3, panels B and D, respectively). In reconstituted, KCl-washed ETPH(Mg), the on rate in the presence of NADH and 0.1 m M ADP and in the absence of Pwas 1.0 10 Ms. The Effect of ADP on the Energy-dependent Dissociation of [-P]ATP from High Affinity Catalytic Sites on ETPH(Mg)-Incubation of submitochondrial particles, containing [-P]ATP bound in high affinity catalytic sites, under the conditions of oxidative phosphorylation, that is, in the presence of NADH, ADP, and P, resulted in a kinetically competent, energy-dependent dissociation of the bound radioactive nucleotide. That is, the rate of dissociation of [-P]ATP was at least as fast as the rate of ATP synthesis catalyzed by the same particles, Penefsky (1985b). Fig. 4 B, upper curve shows that 10 µ M ADP was sufficient to provide maximal dissociation of bound [-P]ATP during a 2-s incubation with NADH. In the presence of NADH and FCCP, Fig. 4 A, lower curve, only about 4% of added [-P]ATP was dissociated in the presence of 300 µ M ADP. Two points of importance emerged from this experiment. First, proton flux was itself sufficient to cause an energy-dependent dissociation of bound ATP (Fig. 4 A, upper curve (no added ADP) and Penefsky (1985b)). Second, ADP served, in a cooperative manner, to enhance the energy-dependent dissociation of bound [-P]ATP. In contrast to earlier observations (Penefsky, 1985b), addition of NADH to submitochondrial particles containing [-P]ATP bound in high affinity catalytic sites did not result in an increase in hydrolysis of [-P]ATP.NADH used in the earlier studies apparently contained a contaminant responsible for the stimulation of hydrolysis. The Rate of Dissociation of [-P]ATP Bound in High Affinity Catalytic Sites-The rate of dissociation of bound [-P]ATP in the presence of NADH or NADH and ADP was too fast to be measured by manual methods. Fig. 5 shows the results of a series of experiments with a rapid mixing device described earlier (Grubmeyer et al., 1982). The enzyme-substrate complex was formed in the first section of aging hose and was then mixed in a second section of aging hose with NADH and hexokinase or NADH, 0.1 m M ADP, and hexokinase. The time of incubation in the energized state is shown on the abscissa and was determined by a delay programmed into the instrument. It was necessary to use very large amounts of hexokinase (800 units/ml) in order to compensate for the relatively slow rate of the hexokinase reaction. Nevertheless, it was not possible to use higher concentrations of ADP or temperatures greater than 23 °C because the resulting higher rates of dissociation would not have been resolved by the rate-limiting hexokinase reaction. The Rate of Dissociation of [-P]ATP Bound in High Affinity Catalytic Sites of F-reconstituted, KCl-washed ETPH(Mg)- shows that the rate of dissociation of bound ATP in the absence of any additions was 10s, in agreement with earlier values for the soluble, Grubmeyer, et al. (1982) and the membrane-bound, Penefsky (1985a), forms of the ATPase. The presence of 100 µ M ADP in the reaction mixture was without effect on the rate of dissociation. However, in the presence of NADH or NADH and ADP, the rate of dissociation was 0.083 and 0.24 s, respectively. Thus, under the conditions of oxidative phosphorylation, that is, in the presence of NADH, ADP, and P, the rate of dissociation of [-P]ATP bound in high affinity catalytic sites was increased about 10times above the non-energized rate.


Figure 4: Effect of ADP on the dissociation of ATP bound in high affinity catalytic sites of energized and nonenergized ETPH(Mg). A, 2 mg of ETPH(Mg) was preincubated for 1 min at room temperature in a buffer containing 20 m M Tris-SO, pH 8, 5 m M KHPO, pH 8, 5 m M MgSO, 50 m M glucose, and 0.25 M sucrose. The final volume was 1.0 ml. After incubation with 0.1 nmol of [-P]ATP for 10 s, 200 units of hexokinase were added to convert any free [-P]ATP to glucose-6-P. Five s later, the indicated amounts of NADH or NADH and ADP were added. Where indicated (+ FCCP), 20 µ M FCCP dissolved in ethanol was added to a reaction mixture identical with that described above. All incubations were continued for 2 s after the addition of NADH or NADH and ADP, and the reactions were terminated by adding 1 ml of 2 N HCl. The amounts of glucose-6-P formed were determined as described under ``Experimental Procedures.'' In a separate experiment, 200 units of hexokinase converted 0.1 nmol of [-P]ATP to glucose-6-P in less than 5 s. B, the reactions were carried out exactly as described for panel A. ADP was added to a final concentration indicated on the abscissa with 1 m M NADH. The amounts of glucose-6-P formed after 2 s were determined as described under ``Experimental Procedures.'' The total amounts of [-P]ATP free (set equal to the amount of glucose-6-P formed) are shown on the ordinate.



The rate of the energy-dependent dissociation of [-P]ATP ( k) as well as the rate of binding of [-P]ATP ( k) was the same in the presence and absence of added P.It is interesting that energization of Escherichia coli membrane vesicles decreased the dissociation constant for P( KP) and blocked rebinding of ATP (Al-Shawi et al., 1990; Weber et al., 1994).

The dissociation constants for ATP, K, were calculated from the ratios of the individual rate constants, k/ k(). Energization decreased the affinity for [-P]ATP in high affinity catalytic sites by five to six orders of magnitude. Calculation of Gfrom the expression G= - RT ln Kprovided the change in standard free energy, in kcal/mol, equivalent to the individual dissociation constants. The observed free energy change ( G) was calculated from the expression G = -RT ln K. It may be seen that energization with NADH, 0.1 m M ADP, and Pgave G of 8.5 kcal/mol (). The Rate of Dissociation of [-P]ATP Bound in High Affinity Catalytic Sites of ETPH(Mg)-Similar to KCl-washed particles, the off rates for [-P]ATP were accelerated by about five orders of magnitude in the presence of NADH and by about six orders of magnitude in the presence of NADH and 0.1 m M ADP (). Calculation of Kfrom the ratio of the reverse and forward rate constants indicated that the affinity for ATP was reduced six orders of magnitude in the presence of NADH and seven orders of magnitude in the presence of NADH and ADP. Thus, the observed free energy change ( G) in the transition from non-energized to energized states was 8 kcal/mol in the presence of NADH and 9.7 kcal/mol in the presence of NADH and ADP ().

Effect of ADP Concentration and Temperature on the Rate of ATP Synthesis by ETPH(Mg) and Reconstituted, KCl-washed ETPH(Mg)

The turnover number for ATP synthesis in both types of particles was sensitive to ADP concentration and temperature, I. Maximal rates of ATP synthesis were observed in both types of particles at 1 m M ADP and 30 °C. The off rate was commensurate with the turnover number of the enzyme under the same conditions described for the off rate measurement, that is, 0.1 m M ADP and 23 °C (Tables II and III).


DISCUSSION

This study describes changes in the affinity of the membrane-bound FF-ATPase of beef heart mitochondria for product ATP during oxidative phosphorylation. It is shown that the affinity of the non-energized enzyme for ATP is high in both ATPase-activated and non-activated submitochondrial particles. The dissociation constant for ATP ( K) was calculated from the ratio of reverse off ( k) and forward on ( k) rate constants. In non-energized membranes, Kwas 10 M ( and Penefsky, 1985a). However, in the presence of NADH, 0.1 m M ADP and P, Kwas about 60 µ M (), ETPH(Mg). Thus, the affinity of the enzyme for ATP must decrease seven orders of magnitude when the proton pumps are activated by initiating respiration. The observed change in Kis expressed almost entirely as an increase in the rate of ATP dissociation ( kincreased by five orders of magnitude over the rate observed under non-energized conditions ()) with only small changes in the rate of ATP binding to catalytic sites under energized conditions ().

The rate of the energy-dependent dissociation of ATP bound in high affinity catalytic sites of Fwas enhanced in the presence of ADP. The enhancement was dependent on the ADP concentration and was seven times greater in the presence of ADP than in its absence (Table II). The ADP-dependent rate enhancement did moreover reflect an energy-dependent cooperativity between catalytic subunits since FCCP prevented the ADP-promoted dissociation of bound ATP, Fig. 4. Thus, the energy of respiration brought about cooperativity between subunits of the enzyme with respect to release of product ATP. These observations are compatible with earlier findings of cooperativity with regard to both ATP hydrolysis (Grubmeyer and Penefsky, 1981a, 1981b) and synthesis (Matsuno-Yagi and Hatefi, 1985). The observed free energy change associated with the cooperative effects of ADP was calculated to be 1.7 kcal/mol (). This cooperative contribution to net ATP synthesis is of considerable importance since the rate of ATP synthesis at low concentrations of ADP is very low (Matsuno-Yagi and Hatefi, 1985; Perez and Ferguson, 1990a, 1990b). Further support for the importance of ADP concentration is shown in Fig. 2and , ETPH(Mg). In the presence of NADH (but absence of added ADP), Kwas about 3 µ M. Thus the affinity for product ATP in the presence of low ADP levels was too high to permit efficient net ATP synthesis. These observations are consistent with conditions observed during respiratory control. Low rates of ATP synthesis and of respiration occur at low concentrations of ADP.

Among the most interesting observations reported in this paper are the changes in standard free energy accompanying ATP dissociation from high affinity catalytic sites of membrane-bound F. Thus, the standard free energy associated with a Kabout 10 M was 15-17 kcal/mol (). The observed free energy change ( G) on the transition from non-energized membranes to membranes energized by NADH alone was 7.5 kcal/mol (reconstituted, KCl-washed particles) and 8 kcal/mol (ETPH(Mg)) (Table II). It is significant that proton flux was itself sufficient to bring about a change in the affinity of the enzyme for ATP of six orders of magnitude. These values of 7.5-8 kcal/mol are the most reasonable values to compare with reported values for the standard free energy of hydrolysis of ATP under physiological conditions of -8.4 kcal/mol (Rosing and Slater, 1971). In the presence of NADH, 0.1 m M ADP and P(that is, under the conditions of oxidative phosphorylation) a further increase in G was observed, 8.5 and 9.7 kcal/mol for KCl-washed ETPH(Mg) and ETPH(Mg) particles, respectively (). It should be emphasized that these values of G were obtained from experiments with suboptimal concentrations of ADP. Maximal rates of ATP synthesis (1 µmol of ATP/min/mg) were observed with ETPH(Mg) particles at 1 m M ADP (I). The calculated turnover number for this rate is 44 mol of ATP/mol of Fper s (I). If the turnover number was set equal to the off rate, k, the calculated Kat 30 °C in the presence of 1 m M ADP would be in the m M range (consistent with the m M range of ATP concentration in the mitochondrial matrix) and G would be correspondingly larger. It was not possible to measure the rate of ATP dissociation directly, as described in Fig. 4, in the presence of 1 m M ADP or at higher temperatures, because the hexokinase-catalyzed conversion of ATP to glucose-6-P was too slow to resolve the kinetics. On the other hand, measurement of the rate of dissociation in the presence of 10 µ M ADP and ETPH(Mg) yielded a value intermediate between 0.32 and 2.3 s. The temperature dependence of ATP synthesis in I can be explained as an effect of temperature on the rate of respiration.

These values for Kand their implications for G may be compared with earlier reports of Kin light-energized spinach chloroplasts of 7 µ M (Magnusson and McCarty, 1976) and of 16 µ M with phosphorylating vesicles from P. denitrificans by Perez and Ferguson (1990a, 1990b). However, the chloroplast values were obtained by measuring the exchange of ATP between the enzyme and the medium in the presumed absence of ADP. Consequently, the 7 µ M Kof Magnusson and McCarty (1976) might best be compared with a Kof 0.1-3 µ M observed with submitochondrial particles in the presence of NADH alone (). Perez and Ferguson (1990a, 1990b) determined K , which they could not measure directly in their system, by equating it with a measurable product inhibition constant, K. It is more difficult to compare this latter value with the numbers reported in this paper, but if product inhibition by ATP may be presumed to reflect ATP binding to an enzyme which does not contain ADP in a second catalytic site, then the 16 µ M value of Perez and Ferguson may also be comparable with numbers observed in this paper in the absence of added ADP, that is 0.1 to 3 µ M. It is important to point out that in the case of maximally phosphorylating submitochondrial particles exhibiting a Kin the millimolar range, it is not necessary to postulate product inhibition by ATP (Perez and Ferguson, 1990a, 1990b) in order to understand how net formation of ATP could occur in the presence of m M concentrations of intramitochondrial ATP.

The rate of ATP hydrolysis by KCl-washed ETPH(Mg) under unisite conditions was slow (10s) and due to the slow rate of dissociation of Pfrom membrane-bound F(Souid and Penefsky; Penefsky, 1985a). Matsuno-Yagi and Hatefi (1993) found a Poff rate of 0.12 susing conditions apparently the same as those of Penefsky (1985a). An explanation of the discrepancy is not apparent. The fact that more [-P]ATP was available for energy-dependent dissociation from KCl-washed particles than from ETPH(Mg) was explained by the slower rate of unisite hydrolysis of ATP by the former particles. A slow rate of hydrolysis of ATP, consistent with unisite catalysis, was observed in ETPH(Mg) when the ratio of Fto [-P]ATP was increased to 80-fold ( panel A of Fig. 2). Thus, it is possible that the hydrolysis in panel B of Fig. 2( lower curve), that is, at a ratio of F/[-P]ATP of 8-old, reflects catalysis at more than one site. Nevertheless, the on rate was the same in both experiments (legend of Fig. 2). This is due to the fact that the rate of ATP binding at a second catalytic site is the same as the rate of binding at the first site (Cross et al., 1982).

It is noteworthy that the kinetics of ATP binding and release by the two types of particles under energized and non-energized conditions were essentially the same (). It should be emphasized, however, that under the experimental conditions described in this paper 1 mg of KCl-washed ETPH(Mg) or 2 mg of ETPH(Mg) was fully capable of binding 97% or more of 0.1 nmol of [-P]ATP added to reaction mixtures (measured as hexokinase-inaccessible [-P]ATP).

It is clear from the experiments of that energization brought about a reduction of almost 10-fold in the rate of binding of ATP in high affinity catalytic sites. This would seem intuitively reasonable since under energized conditions the system is poised to dissociate ATP rather than bind it. It should be pointed out, however, that the on rates shown in for ETPH(Mg) are approximately 10-fold lower than for reconstituted, KCl-washed ETPH(Mg). There is an ambiguity in the calculation of the on rate since it requires a value for the concentration of membrane-bound F. Throughout this study, we have used a value of 0.4 nmol of F/mg of particle protein (Harris et al., 1977; Ferguson et al., 1976; Beltran et al., 1986; Matsuno-Yagi and Hatefi, 1988). This value seems reasonable for ATPase-activated particles, particularly reconstituted, KCl-washed ETPH(Mg). However, if one assumes that the concentration of available catalytic sites on ETPH(Mg) is about one-tenth that of KCl-washed particles (commensurate with the difference in ATPase activities ()), then the calculated on rates for ETPH(Mg) would rise about 10-fold.

The question may arise whether heterogeneity in catalytic sites might influence the kinetic constants determined in this paper. shows that the ATPase activity of the submitochondrial particles used in these experiments was inhibited at least 90% by oligomycin. If 10% of the membrane-bound enzyme was non-energizable but participated in ATP binding, that binding would be rapid (10 Ms). If it did occur, such binding did not seem to have a noticeable effect on the reduced rate of ATP binding under energized conditions (). In addition, it may be noted that in experiments with ETPH(Mg) all of the [-P]ATP bound to the membranes (determined as the difference between cold chase and acid quench hydrolysis) was dissociated within 1 s after adding NADH and 0.1 m M ADP.This experiment argues strongly for homogeneity in the high affinity catalytic sites that bind [-P]ATP. In non-energized ETPH(Mg), 2 mg of the particles bind about 97% of the added 0.1 nmol [-P]ATP in 5 s. About two-thirds of that [-P]ATP is bound in the catalytic sites of the enzyme and one-third is not accessible to hexokinase and cannot be promoted to hydrolyses by cold chase or to dissociation by NADH or NADH and ADP.In 5 s, one-third of the total [-P]ATP is hydrolyzed (Fig. 2, panel B, lower curve), and one-third can be promoted to hydrolysis by cold chase (Fig. 2, panel B, upper curve), ADP, or NADH and ADP in the presence of FCCP.In the absence of FCCP, NADH or NADH and ADP promoted the release of [-P]ATP that was bound as such rather than its hydrolysis.There was no detectable cold chase [-P]ATP following energy-dependent dissociation.These results support the conclusions that the cold chase [-P]ATP is the same as NADH-dissociable [-P]ATP and the same catalytic sites participate in the on rate and off rate measurements.

A variety of observations suggest that the ATP on rates reported in this study are a reasonable measure of the rate of ATP binding in catalytic sites of membrane-bound F. For example, the on rate in non-energized ETPH(Mg) was only 8-10-fold lower than the rates reported for ATPase-activated particles (Penefsky, 1985a) and soluble F(Grubmeyer et al., 1982). In addition, the decrease in the on rate values observed in the presence of NADH, ADP, or NADH and ADP was very similar in both types of particles. The on rate values measured with cold chase or hexokinase trap were similar. Somewhat higher numbers obtained with hexokinase reflect the fact that 20-30% of bound ATP, that is, ATP inaccessible to hexokinase, could not be chased into Por dissociated by NADH oxidation. The amount of chaseable [-P]ATP bound to ETPH(Mg) was equal to the amount of [-P]ATP dissociated by NADH. This observation supports the conclusion that [-P]ATP was bound to catalytic sites of coupled FFcomplexes during on rate measurements. Two further indications that the reported values for the ATP on rate are reasonable is found in the fact that, first, the Kvalues calculated from the ratio of the rate constants are in good agreement with values reported by Magnuson and McCarty (1976) and by Perez and Ferguson (1990) and second, that the decrease in the on rate for ATP in the presence of NADH and ADP is consistent with the observed increase in the on rate for ADP during ATP synthesis (Matsuno-Yagi and Hatefi, 1986). Finally, in spite of the insensitivity of the thermodynamic calculations to differences in K, G should not exceed the phosphorylation potential, G, that is, 10.6 kcal/mol (Ferguson and Sorgato, 1977). Thus, during steady state ATP synthesis, G in ETPH(Mg) was about 4.8 kcal/mol (15.4 kcal/mol minus 10.6 kcal/mol, ). If the off rate under these conditions is taken to be 44/s (I), Kwould be 0.3 m M and k1.5 10 Ms. These considerations provide insight into observations that ATP synthesis can occur readily even at high ATP/ADP ratios (Ferguson and Sorgato, 1977). This is so because under energized conditions the affinity of catalytic sites for ATP was reduced more than seven orders of magnitude (), while the affinity for ADP increased during ATP synthesis (Matsuno-Yagi and Hatefi, 1986).

In summary, this paper shows that the energy-dependent dissociation of ATP bound in high affinity catalytic sites of submitochondrial particles received contributions from two important variables. First, proton flux which brought about a 10-fold decrease in affinity. Second, the cooperative effects of ADP which, at 0.1 m M concentrations, decreased the affinity an additional 10-fold. The implications of these calculations is that under the conditions of oxidative phosphorylation the dissociation constant for ATP is in the m M range.

  
Table: ATP hydrolysis and synthesis by submitochondrial particles

ETPH(Mg), reconstituted, KCl-washed ETPH(Mg) and methods for measurement of ATPase, respiration, and phosphorylation are described under ``Experimental Procedures.'' The numbers in parentheses refer to rates of ATP hydrolysis in the presence of 5 µg of oligomycin. All of these experiments were carried out at 30 °C.


  
Table: Rate constants, equilibrium constants and free energy changes during ATP synthesis catalyzed by ETPH(Mg) and KCl-washed ETPH(Mg)

Preparation of submitochondrial particles and the conditions used for each of the measurements are described under ``Experimental Procedures.'' G (the observed free energy change) was calculated from the expression G = - RT ln K.


  
Table: Effect of ADP concentration and temperature on the rate of ATP synthesis by ETPH(Mg) and reconstituted, KCl-washed ETPH(Mg)

ATP synthesis was measured at the temperatures shown using the protocol described under ``Experimental Procedures.''



FOOTNOTES

*
This work was supported in part by National Institutes of Health Research Grant GM21737 (to H. S. P.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked `` advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
This work was submitted in partial fulfillment of the requirements for a Ph.D. degree in the Department of Biochemistry and Molecular Biology.

To whom correspondence should be addressed. Tel.: 315-464-8736; Fax: 315-464-8750.

The abbreviations used are: F, membrane-imbedded portion, and F, extrinsic, easily solubilized portion of the FF-ATPase; FCCP, carbonyl cyanide p-trifluoromethyoxyphenylhydrazone; TNP, trinitrophenyl; OSCP, oligomycin sensitivity-conferring protein.

A.-K. Souid and H. S. Penefsky, unpublished observations.


ACKNOWLEDGEMENTS

We thank R. L. Cross and J. D. Robinson for useful discussions during the preparation of the manuscript. Marcus Hutcheon provided excellent technical assistance.


REFERENCES
  1. Al-Shawi, M. K., Parsonage, D., and Senior, A. E. (1990) J. Biol. Chem. 265, 4402-4410 [Abstract/Free Full Text]
  2. Beltran, C., Tuena de Gomez-Puyou, M. Darszon, A., and Gomez-Puyou, A. (1986) Eur. J. Biochem. 160, 163-168 [Abstract]
  3. Beyer, R. E. (1967) Methods Enzymol. 10, 186-194
  4. Boyer, P. D. (1993) Biochim. Biophys. Acta 1140, 215-250 [Medline] [Order article via Infotrieve]
  5. Cross, R. L., Grubmeyer, C., and Penefsky, H. S. (1982) J. Biol. Chem. 257, 12101-12105 [Free Full Text]
  6. Feldman, R. I., and Boyer, P. D. (1985) J. Biol. Chem. 260, 13088-13094 [Abstract/Free Full Text]
  7. Ferguson, S. J., and Sorgato, M. C. (1977) Biochem. J. 168, 299-303 [Medline] [Order article via Infotrieve]
  8. Ferguson, S. J., Lloyd, W. J., and Radda, G. K. (1976) Biochem. J. 159, 347-353 [Medline] [Order article via Infotrieve]
  9. Glynn, I. M., and Chappell, J. B. (1964) Biochem. J. 90, 147-149 [Medline] [Order article via Infotrieve]
  10. Gresser, M. J., Myers, J. A., and Boyer, P. D. (1982) J. Biol. Chem. 257, 12030-12038 [Free Full Text]
  11. Grubmeyer, C., and Penefsky, H. S. (1981a) J. Biol. Chem. 256, 3718-3727 [Abstract/Free Full Text]
  12. Grubmeyer, C., and Penefsky, H. S. (1981b) J. Biol. Chem. 256, 3728-3734 [Abstract/Free Full Text]
  13. Grubmeyer, C., Cross, R. L., and Penefsky, H. S. (1982) J. Biol. Chem. 257, 12092-12100 [Free Full Text]
  14. Harris, D. A., Radda, G. K., and Slater, E. C. (1977) Biochim. Biophys. Acta 459, 560-572 [Medline] [Order article via Infotrieve]
  15. Hatefi, Y. (1993) Eur. J. Biochem. 218, 759-767 [Medline] [Order article via Infotrieve]
  16. Heldt, H. W., Klingenberg, M., and Milovancev, M. (1972) Eur. J. Biochem. 30, 434-440 [Medline] [Order article via Infotrieve]
  17. Lindberg, O., and Ernster, L. (1956) in Methods of Biochemical Analysis (Glick, D., ed) Vol. 3,pp. 1-23, John Wiley & Sons, New York [Medline] [Order article via Infotrieve]
  18. Magnusson, R. P., and McCarty, R. E. (1976) J. Biol. Chem. 251, 7417-7422 [Abstract]
  19. Matsuno-Yagi, A., and Hatefi, Y. (1985) J. Biol. Chem. 260, 14424-14427 [Abstract/Free Full Text]
  20. Matsuno-Yagi, A., and Hatefi, Y. (1988) Biochemistry 27, 335-340 [Medline] [Order article via Infotrieve]
  21. Matsuno-Yagi, A., and Hatefi, Y. (1986) J. Biol. Chem. 261, 14031-14038 [Abstract/Free Full Text]
  22. Matsuno-Yagi, A., and Hatefi, Y. (1993) J. Biol. Chem. 268, 1539-1545 [Abstract/Free Full Text]
  23. Matsuno-Yagi, A., Yagi, T., and Hatefi, Y. (1985) Proc. Natl. Acad. Sci. U. S. A. 82, 7550-7554 [Abstract]
  24. Mitchell, P. (1961) Nature 191, 144-148
  25. Penefsky, H. S. (1977) J. Biol. Chem. 252, 2891-2899 [Abstract]
  26. Penefsky, H. S. (1985a) J. Biol. Chem. 260, 13728-13734 [Abstract/Free Full Text]
  27. Penefsky, H. S. (1985b) J. Biol. Chem. 260, 13735-13741 [Abstract/Free Full Text]
  28. Penefsky, H. S. (1985c) Proc. Natl. Acad. Sci. U. S. A. 82, 1589-1593 [Abstract]
  29. Penefsky, H. S., and Cross, R. L. (1991) Adv. Enzymol. Rel. Areas Mol. Biol. 64, 173-214 [Medline] [Order article via Infotrieve]
  30. Perez, J. A., and Ferguson, S. J. (1990a) Biochemistry 29, 10503-10518 [Medline] [Order article via Infotrieve]
  31. Perez, J. A., and Ferguson, S. J. (1990b) Biochemistry 29, 10518-10526 [Medline] [Order article via Infotrieve]
  32. Pullman, M. E., and Monroy, G. C. (1963) J. Biol. Chem. 238, 3762-3769 [Free Full Text]
  33. Pullman, M. E., Penefsky, H. S., Data, A., and Racker, E. (1960) J. Biol. Chem. 235, 3322-3329 [Medline] [Order article via Infotrieve]
  34. Rosing, J., and Slater, E. C. (1972) Biochim. Biophys. Acta 267, 275-290 [Medline] [Order article via Infotrieve]
  35. Senior, A. E. (1979) Methods Enzymol. 55, 391-397 [Medline] [Order article via Infotrieve]
  36. Vasilyeva, E. A., Minkov, I. B., Fitin, A. F., and Vinogradov, A. D. (1982) Biochem. J. 202, 9-14 [Medline] [Order article via Infotrieve]
  37. Weber, J., Wilke-Mounts, S., and Senior, A. E. (1994) J. Biol. Chem. 269, 20462-20467 [Abstract/Free Full Text]

©1995 by The American Society for Biochemistry and Molecular Biology, Inc.