Immunological properties of membrane-bound adenosine triphosphatase: immunological identification of rutamycin-sensitive F0.F1ATPase from Micrococcus luteus ATCC 4698 established by crossed immunoelectrophoresis.

(1) F0.F1ATPase (EC 3.6.1.3) from Micrococcus luteus ATCC 4698 was solubilized from plasma membranes by the non-ionic detergent Triton X-100 in the presence of 0.05 M MgCl2. (2) The antibiotics rutamycin, Dio-9, quercetin, oligomycin, botrycidin, efrapeptin, leucinostatin, valinomycin, and venturicidin as well as N,N'-dicyclohexylcarbodiimide and dinitrophenol are potent inhibitors of F0.F1ATPase activity.(3) F0.F1ATPase activity is completely inhibited by anti-F1ATPase antibodies. The inhibition is non-competitive. (4) Crossed immunoelectrophoresis reveals a reaction of immunological identity of F0.F1ATPase and F1ATPase indicating that both enzymes have in common antigenic sites.     

Mitochondrial adenosine triphosphatase of the fission yeast Schizosaccharomyces pombe 972h-. Changes in inhibitor sensitivities during the cell cycle indicate similarities and differences in binding sites.

1. We used 11 different inhibitors of energy conservation as inhibitors of ATPase (adenosine triphosphatase) in extracts of Schizosaccharomyces pombe obtained from cells at different stages of the cell cycle. 2. All the inhibitors showed cell-cycle-dependent variations in their I50 values (microng of inhibitor/mg of protein giving 50% inhibition of inhibitor-sensitive ATPase at pH 8.6). 3. From the sensitivity profiles through the cell cycle it was concluded that: (a) oligomycin, venturicidin, triethyltin sulphate and dibutylchloromethyltin chloride all act at closely associated site(s); (b) NN'-dicyclohexylcarbodi-imide and leucinostatin both act at a similar site, which is, however, distinct from that at which other inhibitors of the membrane factor (Fo) act. 4. The variations in I50 values for efrapeptin closely followed changes in specific activity of ATPase, as would be expected for an inhibitor acting at catalytic sites; these fluctuations were different from those for aurovertin, Dio-9, 4-chloro-7-nitrobenzofurazan, quercetin and spegazzinine, all of which show different sensitivity profiles from one another. 5. Anomalous stepwise inhibitor-titration curves were obtained for spegazzinine, NN'-dicyclohexylcarbodiimide, dibutylchloromethyltin chloride and leucinostatin. 6. Possible explanations are proposed for the discontinuous expression of inhibitor-binding sites during the cell cycle.     

The mother-cell-membrane adenosine triphosphatase of sporulating Clostridium pasteurianum

1. Sporulation of Clostridium pasteurianum effects several changes in its proton-translocating cell-membrane H(+)-ATPase. Notable among these are the acquisition of susceptibility to activation by trypsin and a changed protein subunit composition. 2. A protein was isolated from the mother-cell membrane that inhibited the ATP phosphohydrolase activity of purified vegetative-cell-membrane H(+)-ATPase [BF(0)F(1) complex, which consists of soluble ATPase (BF(1)) and the proton-channel component (BF(0))] and rendered it susceptible to trypsin activation. 3. This trypsin-sensitive inhibitor protein had a molecular weight of 10000 and on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis was indistinguishable from the novel protein subunit e of the mother-cell-membrane ATPase 4. In bacteriorhodopsin-containing everted membrane vesicles, the specific ATP synthetase activity of the mother-cell-membrane ATPase was significantly greater than that of the vegetative-cell-membrane ATPase. 5. Treatment with trypsin-sensitive inhibitor protein of artificial proteoliposomes containing bacteriorhodopsin and vegetative-cell-membrane H(+)-ATPase (BF(0)F(1)) significantly increased the specific ATP synthetase activity of this enzyme. 6. The ATP synthetase activity of crude cell-membrane preparations from cultures of Clostridium pasteurianum increased during that period in the course of sporulation when the membrane ATP phosphohydrolase was both most rapidly decreasing in specific activity and acquiring its susceptibility to activation by trypsin.     

Adenosine triphosphatase activity of Tritrichomonas foetus.

Homogenates of Tritrichomonas foetus exhibited a Mg2+-dependent adenosine triphosphatase (ATPase) activity, with a pH optimum in Tris buffers of 8.2 to 8.3. The activity was not sensitive to oxygen. At high concentrations, quercetin and 4-chloro-7-nitrobenzofurazan inhibited ATPase activity in the cytoplasmic extract by 20 and 70%, respectively, whereas oligomycin, venturicidin, triethyltin, leucinostatin, dibutylchloromethyltin chloride, spegazzinine, efrapeptin, citreoviridin and sodium azide had no effect and N,N'-dicyclohexylcarbodi-imide stimulated the activity somewhat. The activity was localized in a population of small cytoplasmic particles which also contained an acid phosphatase. There was no indication of an association of ATPase with hydrogenosomes. The ATPase activity (or activities) in this aerotolerant anaerobe is different from the ATPases characteristic of mitochondria or of anaerobic bacteria.     

Diethylstilbestrol. A novel F0-directed probe of the mitochondrial proton ATPase

At low concentrations, diethylstilbestrol (DES) is shown to be a potent F0-directed inhibitor of the F0F1-ATPase of rat liver mitochondria. In analogy to other F0-directed inhibitors, DES inhibits both the ATPase and ATP-dependent proton-translocation activities of the purified and membrane bound enzyme. When added at low concentrations with dicyclohexylcarbodiimide (DCCD), a covalent inhibitor, DES acts synergistically to inhibit ATPase activity of the complex. At higher concentrations, DES restores DCCD-inhibited ATPase activity. However, there is no restoration of ATP-dependent proton translocation. Under these conditions DCCD remains covalently bound to the F0F1-ATPase complex and F1 remains bound to Fo. Significantly, when the F0F1-ATPase is inhibited by the Fo-directed inhibitor venturicidin rather than DCCD, DES is also able to restore ATPase activity. In contrast, DES is unable to restore ATPase activity to F0F1 preparations inhibited by the Fo-directed inhibitors oligomycin or tricyclohexyltin. However, combinations of [DES + DCCD] or [DES + venturicidin] can restore ATPase activity to F0F1 preparations inhibited by either oligomycin or tricyclohexyltin. Results presented here indicate that the F0 moiety of the rat liver mitochondrial proton ATPase contains a distinct binding site for DES. In addition, they suggest that at saturating concentrations simultaneous occupancy of the DES binding site and sites for either DCCD or venturicidin promote "uncoupled" ATP hydrolysis.     

Butyricin 7423 and the membrane H+-ATPase of Clostridium pasteurianum

The bacteriocin butyricin 7423 inhibited the activity of the membrane H⁺-ATPase (BF₀, F₁) of vegetative cells of Clostridium pasteurianum but not that of its soluble BF₁ component. In vitro studies with the H⁺-ATPases of mutant strains selected for diminished sensitivity (a) to butyricin 7423 and (b) to dicyclohexylcarbodi-imide, confirmed that butyricin 7423 interacts with the BF₀ component of this enzyme complex. Even so, certain other mutant strains displaying decreased sensitivity to butyricin 7423 possessed H⁺-ATPases which in vitro showed undiminished sensitivity to inhibition by the bacteriocin. Furthermore, from the changes in intracellular ATP concentration and in the rates and net extent of efflux of intracellular ⁸⁶Rb⁺ ions that were provoked by exposure of the parent and several of the mutant strains to butyricin 7423, it was concluded that its primary bactericidal action was not attributable to stoichiometric inhibition of the membrane H⁺-ATPase. High extracellular concentrations of K⁺ ions enabled Cl. pasteurianum to survive exposure to low concentrations of this membrane-active bacteriocin.Non-standard abbreviations H⁺-ATPase proton translocating adenosine 5-triphosphatase (EC 3.6.1.3) - DCCD dicyclohexylcarbodiimide     

Specific labelling of the (Ca2+ + Mg2+)-ATPase of Escherichia coli with 8-azido-ATP and 4-chloro-7-nitrobenzofurazan.

1. 8-Azido-ATP is a substrate for Escherichia coli (Ca2+ + Mg2+)-ATPase (E. coli F1). 2. Illumination of E. coli F1 in the presence of 8-azido-ATP causes inhibition of ATPase activity. The presence of ATP during illumination prevents inhibition. 3. 8-Azido-ATP and 4-chloro-7-nitrobenzofurazan (NbfCl) bind predominantly to the alpha subunit of the enzyme, but also significantly to the beta subunit. 4. The alpha subunit of E. coli F1 seems to have some properties that in other F1-ATPases are associated with the beta subunit.     

Conformational interactions between alpha and beta subunits in the F1 ATPase of Escherichia coli as shown by chemical modification of uncA401 and uncD412 mutant enzymes

In contrast to wild-type F1 adenosine triphosphatase, the beta subunits of soluble ATPase from Escherichia coli mutant strains AN120 (uncA401) and AN939 (uncD412) were not labeled by the fluorescent thiol-specific reagents 5-iodoacetamidofluorescein, 2-(4'-iodoacetamidoanilino)naphthalene-6-sulfonic acid or 4-[N-(iodoacetoxy)ethyl-N-methyl]amino-7-nitrobenzo-2-oxa-1,3-diazole. The mutation in the alpha subunit (uncA401) of F1 ATPase thus influences the accessibility of the single cysteinyl residue in the beta subunit. Following reaction of ATPase with 4-chloro-7-nitrobenzo-2-oxa-1,3-diazole or N,N'-dicyclohexylcarbodiimide, the alpha and beta subunits of the uncA401, but not of the uncD412 mutant F1 ATPase were intensely labeled by a fluorescent thiol reagent. The mutation in the beta subunit (uncD412) thus influences the accessibility of the cysteinyl residues in the alpha subunit. In other work [Stan-Lotter, H. and Bragg, P.D. (1986) Arch. Biochem. Biophys. 248] we have shown that 4-chloro-7-nitrobenzo-2-oxa-1,3-diazole and 2-(4'-iodoacetamidoanilino)naphthalene-6-sulfonic acid react with a different beta subunit from that labeled by N,N'-dicyclohexylcarbodiimide. This asymmetry with respect to modification by 4-chloro-7-nitrobenzo-2-oxa-1,3-diazole and N,N'-dicyclohexylcarbodiimide was seen in both mutant enzymes. In addition, the modification of one beta subunit of the uncA401 F1 ATPase induced the previously unreactive sulfhydryl group of another beta subunit to react with 2-(4'-iodoacetamidoanilino-naphthalene-6-sulfonic acid. These results provide evidence for at least three types of conformational interactions of the major subunits of F1 ATPase: from alpha to beta, from beta to alpha, and from beta to beta. As in wild-type ATPase, labeling of membrane-bound unc mutant ATPase by a fluorescent thiol reagent modified the alpha subunits. This suggests that a conformational change of yet a different type occurs when the enzyme binds to the membrane.     

Membrane ATPase of Vibrio alginolyticus. Ion transport activity and homology with F0F1-ATPase from E. coli]

F0F1-ATPase has been isolated from the marine alkali-resistant bacterium Vibrio alginolyticus. The enzyme subunits cross-reacted with antibodies against subunits alpha, beta, gamma, epsilon, and b of E. coli ATPase. The purified ATPase was reconstituted into liposomes effecting an ATP-dependent uptake of H+. Proton transport was inhibited by the ATPase blockers DCCD, triphenyltin, and venturicidin. Na+ ions had no effect on ATP-dependent proton transport. No ATP-dependent transport of Na+ was detected in proteoliposomes.     

Reaction of membrane-bound F1-adenosine triphosphatase of Escherichia coli with chemical ligands and the asymmetry of beta subunits

The three beta subunits of the isolated Escherichia coli F1-ATPase react independently with chemical reagents (Stan-Lotter, H. and Bragg, P.D. (1986) Arch. Biochem. Biophys. 284, 116-120). Thus, one beta subunit is readily cross-linked to the epsilon subunit, Another reacts with N,N'-dicyclohexylcarbodiimide (DCCD), and the third one is modified on a lysine residue by 4-chloro-7-nitrobenzofurazan (NbfCl). The binding site for the ATP analog, 2-azido-ATP, was not associated with a specific type of beta subunit (Bragg, P.D. and Hou, C. (1989) Biochim. Biophys. Acta 974, 24-29). We now show that this binding site is a catalytic site as opposed to a noncatalytic nucleotide-binding site. NbfCl reacted with a tyrosine residue on the DCCD-reacting beta subunit in contrast to the different subunit location of the lysine residue labeled by the reagent. Thus, O to N transfer of the Nbf group in the free F1-ATPase involves transfer between subunits. The chemical labelling pattern of membrane-bound F1-ATPase differed from that of free F1. The strict asymmetry of labeling of the free F1-ATPase was not observed. Thus, double labeling of beta subunits by several reagents was found. This suggests that the asymmetry was not induced by chemical modification, but is inherent in the structure of the ATPase.     

Immunological and reconstitution studies on the adenosine triphosphatase complex from Rhodospirillum rubrum.

Studies on restoration of membrane-bound adenosinetriphosphatase (ATP phosphohydrolase, EC 3.6.1.3) from Rhodospirillum rubrum show that the delta-subunit is capable of binding to the F1 factor or to the F0 moiety of the F0-F1 ATPase complex. This subunit is thus likely involved in linking the F0 and F1 factor. During solubilization of the oligomycin-sensitive F0-F1 ATPase complex with Triton X-100 the detergent becomes specifically associated with the lipophilic F0 part of the enzyme complex. Crossed immunoelectrophoresis, agglutination tests, and kinetic studies with anti-F1 ATPase antibodies reveal a reaction of immunological identity of membrane-bound ATPase, F0-F1 ATPase, and F1 ATPase.     

Membrane integration and function of the three F0 subunits of the ATP synthase of Escherichia coli K12

Integration into the cytoplasmic membrane and function of the three F0 subunits, a, b and c, of the membrane-bound ATP synthase of Escherichia coli K12 were analysed in situations where synthesis of only one or two types of subunits was possible. This was achieved by combined use of atp mutations and plasmids carrying and expressing one or two of the atp genes coding for ATP synthase subunits. AU three F0 subunits were found to be required for the establishment of efficient H+ conduction. Subunits a and b individually as well as together were found to bind F1 ATPase to the membrane while subunit c did not. The ATPase activity bound to either of these single subunits, or in pairwise combinations, was not inhibited by N,N'-dicyclohexylcarbodiimide. Also ATP-dependent H+ translocation was not catalysed unless all three F0 subunits were present in the membrane. The integration into the membrane of the subunits a and b was independent of the presence of other ATP synthase subunits.     

Mitochondrial adenosine triphosphatase of the fission yeast, Schizosaccharomyces pombe 972h-. Changes in activity and inhibitor-sensitivity in response to catabolite repression.

1. The specific activity of mitochondrial ATPase (adenosine triphosphatase) in extracts of Schizosaccharomyces pombe decreased 2.5-fold as the glucose concentration in the growth medium decreased from 50mM to 15mM. 2. During the late exponential phase of growth, ATPase activity doubled. 3. Sensitivity to oligomycin and Dio-9 as measured by values for I50(mug of inhibitor/mg of protein giving 50% inhibition) at pH 6.8 increased sixfold and ninefold respectively during the initial decrease in ATPase activity, and this degree of sensitivity was maintained for the remainder of the growth cycle. 4. Increased sensitivity to NN'-dicyclohexylcarbodi-imide, triethyltin and venturicidin was also observed during the early stage of glucose de-repression. 5. Smaller increases in sensitivity to efrapeptin, aurovertin, 7-chloro-4-nitrobenzo-2-oxa-1,3-diaz-le, quercetin and spegazzinine also occurred. 6. The ATPase of glycerol-grown cells was less sensitive to inhibitors than that of glucose-repressed cells; change in values for I50 were not so marked during the growth cycle of cells growing with glycerol. 7. When submitochondrial particles from glycerol-grown cells were tested by passage through Sephadex G-50, a fourfold increase in activity was accompanied by increased inhibitor resistance. 8. Gel filtration of submitochondrial particles from glucose-de-repressed cells gave similar results, whereas loss of ATPase occurred in submitochondrial particles from glucose-repressed cells. 9. It is proposed that alterations in sensitivity to inhibitors at different stages of glucose derepression may be partly controlled by a naturally occuring inhibitor of ATPase. 10. The inhibitors tested may be classififed into two groups on the basis of alterations of sensitivity of the ATPase during physiological modification: (a) oligomycin, Dio-9, NN'-dicyclohexylcarbodi-imide, venturicidin and triethyltin, and (b) efrapeptin, aurovertin, 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole, quercetin and spegazzinine.     

Ultrafast purification and reconstitution of His-tagged cysteine-less Escherichia coli F1Fo ATP synthase

His-tagged cysteine-less F1Fo ATP synthase from Escherichia coli was purified using Ni-NTA affinity chromatography. During the purification procedure the loss of total ATPase activity did not exceed 50%, and the extent of purification was about 80-fold. The purified enzyme was essentially free of other proteins, was highly active in ATP hydrolysis (75 units/mg at pH 8 and 37 degrees C), and was sensitive to N,N'-dicyclohexylcarbodiimide (70%). Incorporation of F1Fo into soybean liposomes yielded well-coupled and highly active proteoliposomes. The entire procedure, from the disruption of cells by French press to the preparation of proteoliposomes, took only about 8 h. Some improvements in procedures for the estimation of rates of both ATP hydrolysis and ATP-dependent 9-amino-6-chloro-2-methoxyacridine (ACMA) fluorescence quenching are described.     

The mitochondrial ATPase. Evidence for a single essential tyrosine residue.

1. Evidence is presented which indicates that inactivation of the mitochondrial ATPase from bovine heart by the reagent 4-chloro-7-nitrobenzofurazan results from modification of one tyrosine residue per enzyme molecule. Activity can be restored by a variety of sulphydryl reagents. 2. In sodium dodecyl sulphate, the nitrogenzofurazan group on tyrosine is transfered to newly exposed sulphydryl groups on the enzyme. 3. The rate of transfer of the nitrobenzofurazan moiety from theenzyme to sulphydryl compounds is compared with that for transfer from the model compound N-acetyl-tyrosine-0(7-nitrobenzo-furazan) ethyl ester, the synthesis and properties of which are also described. 4. The ligands ATP and ADP exert a protective effect on the rate of reaction between the mitochondrial ATPase and 4-chloro-7-nitrobenzofurazan. The variation in rate of this reaction with change in pH has also been examined and a pKa of 9.5 estimated for the tyrosine residue. 5. The modification does not prevent substrate binding as judged by changes in the fluorescence of aurovertin, an antibiotic with specific affinity for mitochondiral ATPases. 6. When the ATPase activity of submitochondrial particles is inhibited by 4-chloro-7-nitrobenzo-furazan, there is a parallel decrease in the extent of the energy-linked fluorescence enhancement of 1-anilino-naphthalene-8-sulphonate induced by ATP hydrolysis. Both ATPase activity and the fluorescence enhancement are restored by sluphydryl reagents.     

Ligand-induced conformational changes in the Escherichia coli F1 adenosine triphosphatase probed by trypsin digestion

Digestion of the F1-ATPase of Escherichia coli with trypsin stimulated ATP hydrolytic activity and removed the delta and epsilon subunits of the enzyme. A species represented by the formula alpha 1(3) beta 1(3) gamma 1, where alpha 1, beta 1 and gamma 1 are forms of the native alpha, beta and gamma subunits which have been attacked by trypsin, was formed by trypsin digestion in the presence of ATP. In the presence of ATP and MgCl2, conversion of gamma to gamma 1 was retarded and the enzyme retained the epsilon subunit. These results imply that binding of ATP to the beta subunits alters the conformation of ECF1 to increase the accessibility of the gamma subunit to trypsin. The likely trypsin cleavage sites in the alpha, beta and gamma subunits are discussed. ECF1 from the alpha subunit-defective mutant uncA401, or after treatment with N,N'-dicyclohexylcarbodiimide or 4-chloro-7-nitrobenzofurazan, was present in a conformation in which the gamma subunit was readily accessible to trypsin and could not be protected by the presence of ATP and MgCl2. In a similar manner to native E. coli F1-ATPase, the hydrolytic activity of the trypsin-digested enzyme was stimulated by the detergent lauryldimethylamine N-oxide. Since the digested enzyme lacked the epsilon subunit, a putative inhibitor of hydrolytic activity, a mechanism for the stimulation which involves loss or movement of this subunit is untenable.     

N,N'-dicyclohexylcarbodiimide and 4-chloro-7-nitrobenzofurazan bind to different beta subunits of the F1 ATPase of Escherichia coli

The fluorescent thiol reagent 2-(4'-iodoacetamidoanilino)naphthalene-6-sulfonic acid (IAANS) labels the gamma, delta, and one of the three beta subunits of the F1 ATPase from Escherichia coli (ECF1). This is the same beta subunit which incorporates 4-chloro-7-nitrobenzofurazan (Nbf) [H. Stan-Lotter and P. D. Bragg (1986) Eur. J. Biochem. 154, 321-327]. After inactivation of ECF1 with N,N'-dicyclohexylcarbodiimide (DCCD), IAANS labels in addition to the beta, gamma, and delta subunits also the alpha subunit. This suggests a conformational change of ECF1 upon binding of DCCD. The beta subunit which incorporates DCCD does does not bind IAANS. Likewise, IAANS-modified ECF1 does not incorporate DCCD into the same beta subunit. It is concluded that DCCD and Nbf bind to different beta subunits. Since neither of these reagents binds to that beta subunit which can be crosslinked to to the epsilon subunit by 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide, these data show that there is a difference in the chemical reactivity of each of the three beta subunits of ECF1, despite their identical primary structures. This suggests that there is an asymmetry in the F1 molecule.     

Purification and reconstitution of the F1F0-ATP synthase from alkaliphilic Bacillus firmus OF4. Evidence that the enzyme translocates H+ but not Na+

The F1F0-ATP synthase from the alkaliphilic Bacillus firmus OF4 was purified in a reconstitutively active form, in good yield and with a high specific ATPase activity when appropriately activated. The purification procedure involved octyl glucoside extraction of washed membrane vesicles in the presence of 20% glycerol and asolectin followed by ammonium sulfate fractionation and sucrose density gradient centrifugation. The purified preparation was resolved into seven bands by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, corresponding to the five F1 subunits, alpha, beta, gamma, delta, and epsilon, and to the b and c subunits of the F0. Two-dimensional sodium dodecyl sulfate-poly-acrylamide gel analysis revealed a candidate for the alpha subunit of F0. The MgATPase activity of B. firmus OF4 F1F0 was barely detectable but could be stimulated, optimally more than 100-fold, by sulfite, methanol, and octyl thioglucoside. The enzyme was inhibited by N,N'-dicyclohexylcarbodiimide and sodium azide, but not by aurovertin, an inhibitor of the F1 from Escherichia coli. The F1F0 reconstituted into proteoliposomes catalyzed ATPase activity, ATP-Pi exchange, and ATP-dependent delta pH and delta psi formation. ATP hydrolysis was stimulated by protonophores while the other activities were abolished by protonophores. These activities were neither dependent on added sodium ions nor significantly affected by them. F1F0 proteoliposomes made from crude octyl glucoside extracts that also contained the Na+/H+ antiporter were shown to catalyze ATP-dependent Na+ uptake that was completely sensitive to carbonyl cyanide m-chlorophenyl-hydrazone; Na+ uptake activity was absent in proteoliposomes containing more purified F1F0 but lacking the Na+/H+ antiporter. These data show that the F1F0 translocates protons and does not substitute Na+ for H+ in energy coupling.     

Correlation of energy-dependent cell cohesion with social motility in Myxococcus xanthus.

Membrane-bound ATPase was found in membranes of the archaebacterium Methanosarcina barkeri. The ATPase activity required divalent cations, Mg2+ or Mn2+, and maximum activity was obtained at pH 5.2. The activity was specifically stimulated by HSO3- with a shift of optimal pH to 5.8, and N,N'-dicyclohexylcarbodiimide inhibited ATP hydrolysis. The enzyme could be solubilized from membranes by incubation in 1 mM Tris-maleate buffer (pH 6.9) containing 0.5 mM EDTA. The solubilized ATPase was purified by DEAE-Sepharose and Sephacryl S-300 chromatography. The molecular weight of the purified enzyme was estimated to be 420,000 by gel filtration through Sephacryl S-300. Polyacrylamide gel electrophoresis in sodium dodecyl sulfate revealed two classes of subunit, Mr 62,000 (alpha) and 49,000 (beta) associated in the molar ratio 1:1. These results suggest that the ATPase of M. barkeri is similar to the F0F1 type ATPase found in many eubacteria.     

Recent developments on structural and functional aspects of the F1 sector of H+-linked ATPases

This review concerns the catalytic sector of F1 factor of the H+-dependent ATPases in mitochondria (MF1), bacteria (BF1) and chloroplasts (CF1). The three types of F1 have many similarities with respect to the structural parameters, subunit composition and catalytic mechanism. An alpha 3 beta 3 gamma delta epsilon stoichiometry is now accepted for MF1 and BF1; the alpha 2 beta 2 gamma 2 delta 2 epsilon 2 stoichiometry for CF1 remains as matter of debate. The major subunits alpha, beta and gamma are equivalent in MF1, BF1 and CF1; this is not the case for the minor subunits delta and epsilon. The delta subunit of MF1 corresponds to the epsilon subunit of BF1 and CF1, whereas the mitochondrial subunit equivalent to the delta subunit of BF1 and CF1 is probably the oligomycin sensitivity conferring protein (OSCP). The alpha beta gamma assembly is endowed with ATPase activity, beta being considered as the catalytic subunit and gamma as a proton gate. On the other hand, the delta and epsilon subunits of BF1 and CF1 most probably act as links between the F1 and F0 sectors of the ATPase complex. The natural mitochondrial ATPase inhibitor, which is a separate protein loosely attached to MF1, could have its counterpart in the epsilon subunit of BF1 and CF1. The generally accepted view that the catalytic subunit in the different F1 species is beta comes from a number of approaches, including chemical modification, specific photolabeling and, in the case of BF1, use of mutants. The alpha subunit also plays a central role in catalysis, since structural alteration of alpha by chemical modification or mutation results in loss of activity of the whole molecule of F1. The notion that the proton motive force generated by respiration is required for conformational changes of the F1 sector of the H+-ATPase complex has gained acceptance. During the course of ATP synthesis, conversion of bound ADP and Pi into bound ATP probably requires little energy input; only the release of the F1-bound ATP would consume energy. ADP and Pi most likely bind at one catalytic site of F1, while ATP is released at another site. This mechanism, which underlines the alternating cooperativity of subunits in F1, is supported by kinetic data and also by the demonstration of partial site reactivity in inactivation experiments performed with selective chemical modifiers. One obvious advantage of the alternating site mechanism is that the released ATP cannot bind to its original site.(ABSTRACT TRUNCATED AT 400 WORDS)