The effect of Mg2+ on mitochondrial F0.F1 ATPase and characteristics of the nucleotide binding sites

The interaction of Mg2+ with nucleotide-washed F0.F1 ATPase from pig heart was studied. Mg2+ had no effect on nucleotide-washed F0.F1 ATPase, but it competitively inhibited the hydrolytic activity of washed F0.F1 ATPase preincubated with ADP and slightly activated the hydrolytic activity of washed F0.F1 ATPase preincubated with ATP. In the last two cases, it revealed negative cooperativity. The effect of Mg2+ on F0.F1 ATPase is therefore closely related to the characteristics of the nucleotide binding sites on mitochondrial F0.F1 ATPase.     

Further study on the role of Mg2+ in lipid-protein interaction in reconstituted porcine heart mitochondrial H+-ATPase

Porcine heart mitochondrial H+-ATPase was reconstituted by cholate dialysis method in liposomes containing neutral (PC, PE), acidic (PG, PI, PA, PS, DPG) or neutral and acidic phospholipids. The Mg2+ effect on the ATPase activity and its sensitivity to oligomycin, ATP-induced delta psi and delta pH formation was observed for the proteoliposomes containing acidic but not neutral phospholipids. Maleimide spin labels with varying arm lengths or bromoacetamide spin probe were used to monitor the conformational difference of H+-ATPase in the Mg2+-containing and Mg2+-'free' samples. A difference in W/S ratio (weakly immobilized/strongly immobilized component in the ESR spectra) could be detected for the F0.F1-containing and F1-depleted, (F0)-containing proteoliposomes, suggesting conformational difference in the F0-F1 complex and F0 portion induced by the Mg2+ effect. A conformational change of the beta-subunits in the F1 portion was also deduced from the ATP-induced fluorescence quenching of aurovertin-complex for Mg2+-containing samples. The results obtained are in favor of our previous assumption that Mg2+ may play its role by altering the physical state of the lipid bilayer, which would induce a conformational change in F0 (buried in the lipid core), which in turn is transmitted to the catalytic F1, resulting in a higher enzyme activity.     

A kinetic study on the hydrolytic activity of mitochondrial ATPase (F0-F1 complex) from pig heart

A kinetic study of mitochondrial ATPase (F0-F1 complex) from pig heart reported in this paper shows that when it was incubated with free Mg2+ (0-2mM), the hydrolytic activity of the ATPase was competitively activated by the Mg2+ and revealed no cooperativity. In the case of incubation with free ATP the hydrolytic activity was competitively inhibited and revealed positive cooperativity. These results are quite different from those of free F1 as obtained by Gautheron and coworkers (1). This indicates that either Mg2+ or ATP produces different effects on F1 when it is in different states, i.e., free state and membrane bound state. This may be considered to mean that the conformation of F1 in membrane bound state, which is influenced by F0 and membrane lipids is different from that of F1 in free state, thus exhibiting different catalytic site cooperativity between subunits, which is the fundamental feature of the mechanism of the enzyme action.     

Effect of polyamines on mitochondrial F1-ATPase catalyzed reactions

The effect of polyamines on F1-ATPase catalyzed reactions has been studied through the use of submitochondrial particles and F1-ATPase. ATP degradation catalyzed by submitochondrial particles and F1-ATPase was inhibited by spermine and spermidine. Spermine's inhibition was much greater than spermidine's effect. In contrast, P1-ATP exchange and succinate dependent ATP synthesis catalyzed by submitochondrial particles were both stimulated by spermine. The inhibition of ATPase activity by polyamines probably occurs through polyamine's replacement of Mg2+ on ATP, for the following reasons. (a) The ATPase activity inhibited by spermine was partially recovered when Mg2+ was added. (b) Spermine bound to ATP and phospholipids but not to F1-ATPase; yet spermine inhibited the ATPase reaction catalyzed by F1-ATPase, a protein free of phospholipid. (c) The binding of spermine to ATP was inhibited by Mg2+. The ATP content in polyamine-deficient cells definitely was lower than that in normal cells. On the basis of these results, the possible role of spermine in keeping the ATP concentration at a high level is discussed.     

Cross-reconstitution of isolated F1-ATPase from potato tuber mitochondria with F1-depleted beef heart and yeast submitochondrial particles

Cross-reconstitution of isolated potato mitochondrial F1-ATPase with F1-depleted beef heart and yeast submitochondrial particles is reported. Potato F1 binds to the heterologous membrane and confers oligomycin sensitivity on the ATPase activity of the reconstituted system. Binding of F1 is promoted by the presence of Mg2+ with the maximal stimulatory effect at 20 mM. Mg2+ increase the sensitivity to oligomycin of the reconstituted system consisting of potato F1 and yeast membranes, however, they do not influence oligomycin sensitivity of potato F1 and beef heart membranes.     

Mitochondrial ATP synthase: dramatic Mg2+-induced alterations in the structure and function of the F1-ATPase moiety

The ATPase activity of the F1 moiety of rat liver ATP synthase is inactivated when incubated prior to assay at 25 degrees C in the presence of MgCl2. The concentration of MgCl2 (130 microM) required to induce half-maximal inactivation is over 30 times higher than the apparent Km (MgCl2) during catalysis. Moreover, the relative efficacy of divalent cations in inducing inactivation during prior incubation follows an order significantly different from that promoting catalysis. Inactivation of F1-ATPase activity by Mg2+ is accompanied by the dramatic dissociation from the F1 complex of alpha subunits and part of the gamma-subunit population. The latter form a precipitate while the beta, delta, and epsilon subunits, and the remaining part of the gamma-subunit population, remain soluble. Dissociation is not a sudden "all or none" event but parallels loss of ATPase activity until alpha subunits have almost completely dissociated together with about 50% of the gamma-subunit population. Mg2+-induced loss of F1-ATPase activity cannot be prevented by including either the hydrolytic substrates ATP, GTP, or ITP in the incubation medium or the product ADP. Ethylenediaminetetraacetic acid, mercaptoethanol, and dithiothreitol are also ineffective in preventing loss of ATPase activity. Significantly, KPi at high concentration (greater than or equal to 200 mM) is effective in partially protecting F1 against inactivation. However, the most effective means of preventing Mg2+-induced inactivation of F1-ATPase activity is to rebind F1 to its F0 moiety in F1-depleted particles. When bound to F0, F1 is protected completely against divalent cation induced inactivation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Coupling factor F1 ATPase with defective beta subunit from a mutant of Escherichia coli

The defective coupling factor F1 ATPase from a mutant strain (KF11) of Escherichia coli was purified to a practically homogeneous form. The final specific activity of Mg2+-ATPase was 6-9 units/mg protein, which is about 10-15 times lower than that of F1 ATPase from the wild-type strain. The mutant F1 had a ratio of Ca2+-ATPase to Mg2+-ATPase of about 3.5, whereas the wild-type F1 had ratio of about 0.8. The mutant F1 was more unstable than wild-type F1: on storage at -80 degrees C for 2 weeks, about 80% of its activity (dependent on Ca2+ or Mg2+) was lost, whereas none of the activity of the wild-type F1 was lost. The following results indicate that the mutation is in the beta subunit. (i) High Mg2+-ATPase activity (about 20 units/mg protein) was reconstituted when the beta subunit from wild type F1 was added to dissociated mutant F1 and the mixture was dialyzed against buffer containing ATP and Mg2+. (ii) Low ATPase activity having the same ratio of Ca2+-ATPase to Mg2+-ATPase as the mutant F1 was reconstituted when a mixture of the beta subunit from the mutant F1 and the alpha and gamma subunits from wild-type F1 was dialyzed against the same buffer. (iii) Tryptic peptide analysis of the beta subunit of the mutant showed a difference in a single peptide compared with the wild-type strain.     

Purification and characterization of the F1 portion of the ATP synthase (F1Fo) of Streptomyces lividans

The F1 complex of the ATP synthase of Streptomyces lividans was isolated and purified. The procedure involved the solubilization of F1 from membranes with buffer of low ionic strength in the presence of EDTA, ion-exchange chromatography and gel filtration. The purified F1 complex from S. lividans (SLF1) consists of five subunits alpha, beta, gamma, delta and epsilon with molecular masses of 58,000, 50,000, 36,000, 28,000 and 13,000, respectively and exhibits immunological cross-reactivity with the F1 portion purified from Escherichia coli (ECF1). The enzymatic properties of SLF1 were determined by the use of microtiter-plate-based assay and compared with data obtained for ECF1. ATPase activity of SLF1 (specific activity: 20-30 U/mg) was only observed in the presence of high concentrations of Ca2+ (10mM). Stimulation of the ATPase activity by Mg2+ was not detectable; quite to the contrary, Mg2+ inhibited the Ca(2+)-stimulated activity of SLF1. SLF1 was re-bound to F1-stripped membranes of S. lividans, but not to F1-stripped membrane vesicles of E. coli. In contrast, ECF1 could be cross-reconstituted with F1-stripped membranes of S. lividans; however, a structural but not a functional reconstitution of the hybrid F1Fo complex was observed.     

Purification and characterization of the F1 ATPase from Bacillus subtilis and its uncoupler-resistant mutant derivatives.

The F1 ATPase of Bacillus subtilis BD99 was extracted from everted membrane vesicles by low-ionic-strength treatment and purified by DEAE-cellulose chromatography, hydrophobic interaction chromatography, and anion-exchange high-performance liquid chromatography. The subunit structure of the enzyme was examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the absence and presence of urea. In the absence of urea, the alpha and beta subunits comigrated and the ATPase was resolved into four bands. The mobility of the beta subunit, identified by immunoblotting with anti-beta from Escherichia coli F1, was altered dramatically by the presence of urea, causing it to migrate more slowly than the alpha subunit. The catalytic activity of the ATPase was strongly metal dependent; in the absence of effectors, the Ca2+-ATPase activity was 15- to 20-fold higher than the Mg2+ -ATPase activity. On the other hand, sulfite anion, methanol, and optimally, octylglucoside stimulated the Mg2+ -ATPase activity up to twice the level of Ca2+ -ATPase activity (specific activity, about 80 mumol of Pi per min per mg of protein). The F1 ATPase was also isolated from mutants of B. subtilis that had been isolated and characterized in this laboratory by their ability to grow in the presence of protonophores. The specific activities of the ATPase preparations from the mutant and the wild type were very similar for both Mg2+- and Ca2+ -dependent activities. Kinetic parameters (Vmax and Km for Mg-ATP) for octylglucoside-stimulated Mg2+ -ATPase activity were similar in both preparations. Structural analysis by polyacrylamide gel electrophoresis and isoelectric focusing indicated that the five F1 subunits from ATPase preparations from the mutant and wild-type strains had identical apparent molecular weights and that no charge differences were detectable in the alpha and beta subunits in the two preparations. Thus, the increased ATPase activity that had been observed in the uncoupler-resistant mutants is probably not due to a mutation in the F1 moiety of the ATPase complex.     

A plasma-membrane associated ATPase from the acidophilic bacterium Acidiphilium cryptum

A membrane-bound ATPase of Acidiphilium cryptum, an acidophilic bacterium of mine origin, has been studied. The enzyme has a pH optimum of 8.4 Mg2+ is required for its activity and could be replaced by Mn2+, but not by Ca2+. The enzyme shows a strong preference for ATP as substrate, with the apparent Km of about 0.2 mM. Sulphite ion significantly stimulated the enzyme activity. N,N'-Dicyclohexylcarbodiimide, oligomycin, and azide strongly inhibited the enzyme, whereas vanadate was without effect, suggesting that the A. cryptum ATPase might be of F0F1 type.     

Chemical modification of the F0 part of the ATP synthase (F1F0) from Escherichia coli. Effects on proton conduction and F1 binding

The purified F0 part of the ATP synthase complex from Escherichia coli was incorporated into liposomes and chemically modified by various reagents. The modified F0-liposomes were assayed for H+ uptake and, after reconstitution with F1, for total and dicyclohexylcarbodiimide-sensitive ATPase activity. The water-soluble carbodiimide, 1-ethyl-3-(-3-dimethylaminopropyl)carbodiimide methiodide, (1.2 mM), inhibited H+ uptake to a great extent. Binding of F1 was almost unaffected, but the hydrolysis of ATP was uncoupled from H+ transport. This is reflected by the inhibition of dicyclohexylcarbodiimide-sensitive ATPase activity. Woodward's reagent K, N-ethyl-5-phenylisoxazolium-3'-sulfonate, inhibited both H+ uptake and total ATPase activity. Modification of arginine residues by phenylglyoxal (20 mM) was followed by inhibition of the F1 binding activity by 80% of the control. H+ translocation was reduced to 70%. Diethylpyrocarbonate (3 mM) exhibited a strong inhibiting effect on H+ uptake but not on F1 binding. Modification of tyrosine (by tetranitromethane) as well as lysine residues (by succinic anhydride) did not affect F0 functions. From the data presented we conclude that carboxyl-groups, different from the dicyclohexylcarbodiimide-binding site, are involved in H+ translocation through F0 and, in part, in the functional binding of F1. Furthermore, for the latter function, also arginine residues seem to be important. The role of histidine residues remains unclear at present.     

Energy-linked reactions catalyzed by the purified ATPase complex (F0F1) from Rhodospirillum rubrum chromatophores

1. The isolation of F0F1-ATPase complex from Rhodospirillum rubrum chromatophores by the use of taurodeoxycholate is described. 2. The enzyme preparation contains about 12 polypeptides; five are subunits of the F1 moiety. 3. The ATPase activity of the purified enzyme is dependent on the addition of phospholipids. 4. Km-vales for Mg2+-ATP and Ca2+-ATP are similar to the values obtained for the membrane-bound enzyme. 5. The F0F1-ATPase complex is more than 70% inhibited by oligomycin and N,N'-dicyclohexylcarbodiimide. 6. The F0F1-ATPase complex was integrated into liposomes. The reconstituted proteoliposomes catalyzed energy transduction as shown by ATP-dependent quenching of acridine dye fluorescence and ATP-32Pi exchange.     

Activation of Mg-ATP hydrolysis in isolated Rhodospirillum rubrum H+-ATPase

The effects of lauryl dimethylamine oxide on the Rhodospirillum rubrum H+-ATPase have been studied. This detergent activates Mg2+-dependent ATP hydrolysis in the isolated R. rubrum F0-F1 34-fold, whereas the Ca2+-ATPase activity is only slightly modified. ATPase activation by lauryl dimethylamine oxide enhances the effect on ATP hydrolysis exerted by free Mg2+ ions. Concentrations of free Mg2+ in the range of 0.025 mM favor activation while higher concentrations inhibit ATPase activity by approximately 70%. Steady-state kinetic analysis shows that lauryl dimethylamine oxide induces a complex kinetic behavior for Mg-ATP in the chromatophores, similar to the untreated F0-F1 complex. The initial rate value for Mg-ATP unisite catalysis was found to be 6.3 times higher (3.5 X 10(-3) mol Pi per mol R. rubrum F0-F1 per second) in the presence than in the absence of detergent, where the initial rate was 5.5 X 10(-4) mol Pi per mol R. rubrum F0-F1 per second. These experiments show that lauryl dimethylamine oxide shifts the cation requirement for ATP-hydrolysis of the isolated R. rubrum H+-ATPase from Ca2+ to Mg2+ and that it activates both multisite and unisite catalysis. Results are discussed in relation to the possibility of a regulatory role by Mg2+ ions on ATP hydrolysis expressed through subunit interactions.     

Characterization of the Na+-stimulated ATPase of Propionigenium modestum as an enzyme of the F1F0 type

The ATP-hydrolyzing activity of Propionigenium modestum was extracted from the membranes with Triton X-100 or by incubation with EDTA at low ionic strength. The ATPase in the Triton extract was highly sensitive to dicyclohexylcarbodiimide but not to vanadate. These properties are characteristic for enzymes of the F1 F0 type. The ATPase was specifically activated by Na+ ions yielding a 15-fold increase in catalytic activity at 5 mM Na+ concentration. The additional presence of 1% Triton X-100 caused a further 1.5-fold activation. In the absence of Na+ Triton stimulated the ATPase about 13-fold. The Triton-stimulated ATPase was further activated about 1.5-2-fold by Na+ addition. The ATPase extracted by the low-ionic-strength treatment was purified to homogeneity by fractionation with poly(ethylene glycol) and gel chromatography. The enzyme had the characteristic F1-ATPase subunit structure with Mr values of 58,000 (alpha), 56,000 (beta), 37,600 (gamma), 22,700 (delta), and 14,000 (epsilon). The F1-ATPase was not stimulated by Na+ ions. The membrane-bound ATPase was reconstituted from the purified F1 part and F1-depleted membranes, thus further indicating an F1 F0 structure for the ATPase of P. modestum. Upon reconstitution the ATPase recovered its stimulation by Na+ ions, suggesting that the binding site for Na+ is localized on the membrane-bound F0 part of the enzyme complex.     

The H+-translocating ATP synthase in Halobacterium halobium differs from F0F1-ATPase/synthase

Cell envelope vesicles of Halobacterium halobium synthesize ATP by utilizing base-acid transition (an outside acidic pH jump) under optimal conditions (1 M NaCl, 80 mM MgCl2, pH 6.8) even in the presence of azide (a specific inhibitor of F0F1-ATPase) (Mukohata & Yoshida (1987) J. Biochem. 101, 311-318). An azide-insensitive ATPase was isolated from the inner face of the vesicle membrane, and shown to hydrolyze ATP under very specific conditions (1.5 M Na2SO4, 10 mM MnCl2, pH 5.8) (Nanba & Mukohata (1987) J. Biochem. 102, 591-598). This ATPase activity could also be detected when the vesicle components were solubilized by detergent. The relationship between ATP synthesis and the membrane-bound ATPase was investigated by modification of the vesicles with 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl) or N-ethylmaleimide (NEM). The inhibition pattern of ATP synthesis in the modified vesicles and that of ATP hydrolysis of the solubilized modified vesicles were compared under the individual optimum conditions. The inhibition patterns were almost identical, suggesting that the ATP synthesis and hydrolysis are catalyzed by a single enzyme complex. The ATP synthase includes the above ATPase (300-320 kDa), which is composed of two pairs of 86 and 64 kDa subunits. This is a novel H+-translocating ATP synthase functioning in the extremely halophilic archaebacterium. This "archae-ATP-synthase" differs from F0F1-ATPase/synthase, which had been thought to be ubiquitous among all respiring organisms on our biosphere.     

Energy-dependent transformation of the catalytic activities of the mitochondrial F0 x F1-ATP synthase

The ADP(Mg2+)-deactivated, azide-trapped F0 x F1-ATPase of coupled submitochondrial particles is capable of ATP synthesis being incapable of ATP hydrolysis and ATP-dependent delta muH+ generation [FEBS Lett. (1995) 366, 29-32]. This puzzling phenomenon was studied further. No ATPase activity of the submitochondrial particles catalyzing succinate-supported oxidative phosphorylation in the presence of azide was observed when ATP was added to the assay mixture after an uncoupler. Rapid ATP hydrolysis was detected in the same system when ATP followed by an uncoupler was added. Less than 5% of the original ATPase activity was seen when the reaction (assayed with ATP-regenerating system) was initiated by the addition of ATP to the azide-trapped coupled particles oxidizing succinate either in the presence or in the absence of the uncoupler. High ATP hydrolytic activity was revealed when the reaction was started by the simultaneous addition of the ATP plus uncoupler to the particles generating delta muH+. The energy-dependent conversion of the enzyme into latent uncoupler-activated ATPase was prevented by free ADP (Ki approximately 20 microM) and was greatly enhanced after multiple turnovers in oxidative phosphorylation. The results suggest that the catalytic properties of F0 x F1 are delta muH+-dependent which is in accord with our hypothesis on different conformational states of the enzyme participating in ATP synthesis or hydrolysis.     

Purification and properties of factors in yeast mitochondria stabilizing the F1F0-ATPase-inhibitor complex

A previously found yeast-mitochondrial protein fraction stabilizing the inactivated complex between mitochondrial ATPase and intrinsic ATPase inhibitor (Hashimoto, T., et al. (1983) J. Biochem. 94, 715-720) was separated into two proteins by high performance liquid chromatography on a cation exchanger. The molecular weights of the factors were estimated to be 9,000 and 15,000 daltons by sodium dodecyl sulfate (SDS)-gel electrophoresis. Both factors were required to stabilize a complex of inhibitor and proton-translocating ATPase (F1F0-ATPase) either in its purified form or in mitochondrial membranes. On the other hand both factors together could not stabilize a complex of the inhibitor and F1-ATPase, suggesting that both factors act together with the F0-portion. The factors also facilitated very efficiently the binding of ATPase inhibitor to F1F0-ATPase in the presence of ATP and Mg2+. Both the 15,000 and 9,000 dalton stabilizing factors were hardly distinguishable from delta- and epsilon-subunit, respectively, on an SDS-gel electrophoregram, but immuno-diffusion assay showed that neither factor was present in the purified F1-ATPase containing the delta- and epsilon-subunit.     

Ligand binding studies of the F1 moiety of rat liver ATP synthase: implications about the enzyme's structure and mechanism

F1-ATPase of rat liver was examined for its capacity to interact with both metal ions and nucleotides and for the effect of covalent ATPase inhibitors on these interactions. As isolated, rat liver F1 contains about 2 mol of Mg2+/mol of F1, 1 mol of which can be removed or exchanged. The remaining mole of Mg2+ per mole of F1 remains very tightly associated with F1 and is recovered in the alpha gamma fraction after cold denaturation. Rat liver F1 also contains as isolated a nearly equivalent amount of nucleotide (approximately 1.7 mol/mol of F1) which is readily removed by incubation at room temperature followed by column centrifugation. The "2 Mg2+ enzyme" binds almost 3 mol of 5'-adenylyl imidodiphosphate (AMP-PNP)/mol of F1 in the presence or absence of added divalent cation. When divalent cation is present as Co2+, an equivalent activator to Mg2+ in the ATPase reaction, 1 mol of F1 binds 3 mol of both AMP-PNP and Co2+. under these conditions, the very tight Mg2+ site remains loaded, the exchangeable Mg2+ site is replaced with AMP-PNPCo, and two additional AMP-PNPCo sites are filled. At this point, ADP can be loaded onto the enzyme as a fourth nucleotide at a site separate and distinct from the AMP-PNP sites. Significantly, rat liver F1 contains only a single readily detectable ADP binding site in the presence or absence of divalent cation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Determination of the roles of active sites in F1-ATPase by controlled affinity labeling

The affinity reagents 3'-O-(5-fluoro-2,4-dinitrophenyl) [alpha-32P]ATP (FDNP-[alpha-32P]ATP) and 3'-O-(5-fluoro-2,4-dinitrophenyl) [8-14C]ATP (FDNP-[14C]ATP) were synthesized and used to characterize the structure and function of the three active sites in F1-ATPase. FDNP-[alpha-32P]ATP was found to bind covalently to F1 up to two DNP-[alpha-32P]ATP labels per F1 in the absence of Mg2+ without decreasing the ATPase activity. However, when MgCl2 was subsequently added to the reaction mixture, the enzyme could be further labeled with concomitant decrease in ATPase activity that is consistent with the complete inactivation of one enzyme molecule by an affinity label at the third ATP-binding site. Partial hydrolysis of the FDNP-[14C]ATP-labeled enzyme and sequencing of the isolated peptide indicated that the affinity label was attached to Lys-beta 301 at all three active sites. Samples of F1 with covalent affinity label on Lys-beta 301 were also used to reconstitute F1-deficient submitochondrial particles. The reconstituted particles were assayed for ATPase and oxidative phosphorylation activities. These results show that the catalytic hydrolysis of ATP either by F1 in solution or by F0F1 complex attached to inner mitochondrial membrane takes place essentially at only one active site, but is promoted by the binding of ATP at the other two active sites, and that ATP synthesis during oxidative phosphorylation takes place at all three active sites [corrected].     

Binding properties of an intrinsic ATPase inhibitor and occurrence in yeast mitochondria of a protein factor which stabilizes and facilitates the binding of the inhibitor to F1F0-ATPase

The content of an intrinsic ATPase inhibitor in mitochondria was determined by a radioimmunoassay procedure which showed the molar ratio of the inhibitor to ATPase to be 1:1. The ratio in submitochondrial particles, where half of the enzyme was activated, was the same as that of mitochondria, indicating that the inhibitor protein has affinity for the mitochondrial membrane as well as for F1-ATPase. The inhibitor protein could be removed from the mitochondrial membrane by incubation with 0.5 M Na2SO4 and concomitantly the enzyme was fully activated. The enzyme fully activated by the salt treatment was inactivated again by the externally added ATPase inhibitor in the presence of ATP and Mg2+. The enzyme-inhibitor complex (inactive) on the mitochondrial membrane was more stable than the solubilized enzyme-inhibitor complex but gradually dissociated in the absence of ATP and Mg2+. However, in mitochondria, the enzyme activity was inhibited even in the absence of the cofactors. A protein factor stabilizing the enzyme-inhibitor complex on the mitochondrial membrane was isolated from yeast mitochondria. This factor stabilized the inhibitor complex of membrane-bound ATPase while having no effect on that of purified F1-ATPase. It also efficiently facilitated the binding of the inhibitor to membrane-bound ATPase to form the complex, which reversibly dissociated at slightly alkaline pH.