Site-directed spin-labeling of the catalytic sites yields insight into structural changes within the F0F1-ATP synthase of Escherichia coli

Electron spin resonance (ESR) spectroscopy using site-specific cysteine spin-labeling of the catalytic nucleotide binding sites of F(1)-ATPase was employed to investigate conformational changes within the nucleotide binding sites of the enzyme. Mutant Escherichia coli F(1) that had been modified at position beta-Y331C with a spin label showed almost normal catalytic activity and enabled us to study the effects of binding of different nucleotides and of the F(o) subunit b on the conformation of the catalytic binding sites. The ESR spectra of the spin-labeled, nucleotide-depleted F(1) indicate asymmetry within the sites as is expected from the structural models of the enzyme. Nucleotide binding to the enzyme clearly affects the conformation of the sites; the most pronounced feature upon nucleotide binding is the formation of catalytic site(s) in a very open conformation. Using the same beta-331 spin-labeled F(1) and a truncated form of F(o) subunit b, b(24)(-)(156), we found that binding of b(24)(-)(156) to spin-labeled F(1) significantly changes the conformation of the catalytic sites. In this paper we present data that for the first time directly show that a conformational binding change takes place upon binding of nucleotides to the nucleotide binding sites and that also show that binding of b(24)(-)(156) strongly affects the conformation of the catalytic sites, most likely by increasing the population of binding sites that are in the open conformation.     

Second stalk of ATP synthase. Cross-linking of gamma subunit in F1 to truncated Fob subunit prevents ATP hydrolysis

ATP synthase consists of two portions, F(1) and F(o), connected by two stalks: a central rotor stalk containing gamma and epsilon subunits and a peripheral, second stalk formed by delta and two copies of F(o)b subunits. The second stalk is expected to keep the stator subunits from spinning along with the rotor. We isolated a TF(1)-b'(2) complex (alpha(3)beta(3)gammadeltaepsilonb'(2)) of a thermophilic Bacillus PS3, in which b' was a truncated cytoplasmic fragment of F(o)b subunit, and introduced a cysteine at its N terminus (bc'). Association of b'(2) or bc'(2) with TF(1) did not have significant effect on ATPase activity. A disulfide bond between the introduced cysteine of bc' and cysteine 109 of gamma subunit was readily formed, and this cross-link caused inactivation of ATPase. This implies that F(o)b subunit bound to stator subunits of F(1) with enough strength to resist rotation of gamma subunit and to prevent catalysis. Contrary to this apparent tight binding, some detergents such as lauryldodecylamine oxide tend to cause release of b'(2) from TF(1).     

The structure of the central stalk in bovine F(1)-ATPase at 2.4 A resolution

The central stalk in ATP synthase, made of gamma, delta and epsilon subunits in the mitochondrial enzyme, is the key rotary element in the enzyme's catalytic mechanism. The gamma subunit penetrates the catalytic (alpha beta)(3) domain and protrudes beneath it, interacting with a ring of c subunits in the membrane that drives rotation of the stalk during ATP synthesis. In other crystals of F(1)-ATPase, the protrusion was disordered, but with crystals of F(1)-ATPase inhibited with dicyclohexylcarbodiimide, the complete structure was revealed. The delta and epsilon subunits interact with a Rossmann fold in the gamma subunit, forming a foot. In ATP synthase, this foot interacts with the c-ring and couples the transmembrane proton motive force to catalysis in the (alpha beta)(3) domain.     

Both rotor and stator subunits are necessary for efficient binding of F1 to F0 in functionally assembled Escherichia coli ATP synthase

In F1F0-ATP synthase, the subunit b2delta complex comprises the peripheral stator bound to subunit a in F0 and to the alpha3beta3 hexamer of F1. During catalysis, ATP turnover is coupled via an elastic rotary mechanism to proton translocation. Thus, the stator has to withstand the generated rotor torque, which implies tight interactions of the stator and rotor subunits. To quantitatively characterize the contribution of the F0 subunits to the binding of F1 within the assembled holoenzyme, the isolated subunit b dimer, ab2 subcomplex, and fully assembled F0 complex were specifically labeled with tetramethylrhodamine-5-maleimide at bCys64 and functionally reconstituted into liposomes. Proteoliposomes were then titrated with increasing amounts of Cy5-maleimide-labeled F1 (at gammaCys106 and analyzed by single-molecule fluorescence resonance energy transfer. The data revealed F1 dissociation constants of 2.7 nm for the binding of F0 and 9-10 nm for both the ab2 subcomplex and subunit b dimer. This indicates that both rotor and stator components of F0 contribute to F1 binding affinity in the assembled holoenzyme. The subunit c ring plays a crucial role in the binding of F1 to F0, whereas subunit a does not contribute significantly.     

Nucleotide binding sites on mitochondrial F1-ATPase. Electron spin resonance spectroscopy and photolabeling by azido-spin-labeled adenine nucleotides support an adenylate kinase-like orientation.

A spin-labeled photoaffinity ATP analog, 2-N3-2',3'-SL-ATP (2-N3-SL-ATP) was specifically loaded at catalytic (exchangeable) or noncatalytic (nonexchangeable) nucleotide-binding sites on nucleotide-depleted mitochondrial F1-ATPase. Photolysis of the enzyme complexes resulted in the specific modification of beta-Tyr-345 when the catalytic sites were occupied and beta-Tyr-368 when noncatalytic sites were filled. These are the same amino acid assignments that were made previously using 2-N3ATP. The results demonstrate that the attachment of a spin label moiety to the ribose ring does not prevent proper binding of the analog at both types of nucleotide sites on F1-ATPase and suggest that the probe can be used for investigations of the nucleotide-binding sites using ESR spectroscopy. Enzyme that is in complex with the 2-N3-SL-ATP exhibits an ESR spectrum that is typical for highly immobilized nitroxyl radicals both in the dark or after photolysis. Additional peaks in the high- and low-field regions arise due to dipolar spin interactions most likely involving a pair of catalytic and noncatalytic sites. The two sites are calculated to be approximately 15 A apart. This distance, obtained through ESR spectroscopy, combined with the finding that the 2 labeled amino acids are only 23 residues apart from each other, further supports an adenylate kinase-like arrangement of nucleotide binding sites on F1-ATPase where catalytic and noncatalytic sites are in close proximity (Vogel, P. D., and Cross, R. L. (1991) J. Biol. Chem. 266, 6101-6105).     

Conformation of the gamma subunit at the gamma-epsilon-c interface in the complete Escherichia coli F(1)-ATPase complex by site-directed spin labeling

Structure-function relationships of the gamma-epsilon-c subunit interface of F(O)F(1) ATP synthase, a region of subunit interactions important in coupling between catalysis and transport, were investigated by site-directed spin labeling and electron paramagnetic resonance (EPR) spectroscopy. The EPR line widths and collision accessibilities of 18 spin-labeled, unique cysteine F(1) mutants from gammaLeu198 to gammaLeu215 indicate an alternating pattern in the mobility and accessibility parameters for positions gamma201-209, which is reminiscent of a beta-strand. Labels at positions gamma204 and gamma210 show tertiary contact upon F(1) binding to F(O) and gammaD210C has reduced coupling efficiency. gammaE208C could not be spin labeled, but the uncoupling effects of gammaE208K are suppressed by second-site mutations in the polar loop of subunit c [Ketchum, C. J. and Nakamoto, R. K. (1998) J. Biol. Chem. 273, 22292-22297]. The restricted mobility and accessibility of spin labels in the odd-numbered positions between gamma201 and gamma207 plus the 2-4-fold higher values in k(cat) for ATP hydrolysis of these same mutant F(1) indicate that the interactions of these residues with the epsilon subunit mediate its inhibitory activity. Disrupted interactions with epsilon subunit also cause reduced coupling efficiency. We propose a model for the gamma-epsilon-c interface of Escherichia coli F(O)F(1) ATP synthase in which side chains from the odd-numbered residues of the gammaLys201-gammaTyr207 beta-strand directly and functionally interact with the epsilon subunit, while the even-numbered, acidic residues gammaAsp204, gammaGlu208, and gammaAsp210 interact with the F(O) sector, probably with subunit c. gamma Subunit interactions with both subunits in this region are important for coupling efficiency.     

The molecular neighborhood of subunit 8 of yeast mitochondrial F1F0-ATP synthase probed by cysteine scanning mutagenesis and chemical modification

The detailed membrane topography and neighboring polypeptides of subunit 8 in yeast mitochondrial ATP synthase have been determined using a combination of cysteine scanning mutagenesis and chemical modification. 46 single cysteine substitution mutants encompassing the length of the subunit 8 protein were constructed by site-directed mutagenesis. Expression of each cysteine variant in yeast lacking endogenous subunit 8 restored respiratory phenotype to cells and had little measurable effect on ATP hydrolase function. The exposure of each introduced cysteine residue to the aqueous environment was assessed in isolated mitochondria using the fluorescent thiol-modifying probe fluorescein 5-maleimide. The first 14 and last 13 amino acids of subunit 8 were accessible to fluorescein 5-maleimide in osmotically lysed mitochondria and are thus extrinsic to the lipid bilayer, indicating a 21-amino acid transmembrane span. The C-terminal region of subunit 8 was partially occluded by other ATP synthase subunits, especially in a small region surrounding Val-40 that was demonstrated to play an important role in maintaining the stability of the F(1)-F(0) interaction. Cross-linking using heterobifunctional reagents revealed the proximity of subunit 8 to subunits b, d, and f in the matrix and to subunits b, f, and 6 in the intermembrane space. A disulfide bridge was also formed between subunit 8(F7C) or (M10C) and residue Cys-23 of subunit 6, demonstrating a close interaction between these two hydrophobic membrane subunits and confirming the location of the N termini of each in the intermembrane space. We conclude that subunit 8 is an integral component of the stator stalk of yeast mitochondrial F(1)F(0)-ATP synthase.     

Isolated epsilon subunit of thermophilic F1-ATPase binds ATP

F1-ATPase, a soluble part of the F0F1-ATP synthase, has subunit structure alpha3beta3gammadeltaepsilon in which nucleotide-binding sites are located in the alpha and beta subunits and, as believed, in none of the other subunits. However, we report here that the isolated epsilon subunit of F1-ATPase from thermophilic Bacillus strain PS3 can bind ATP. The binding was directly demonstrated by isolating the epsilon subunit-ATP complex with gel filtration chromatography. The binding was not dependent on Mg2+ but was highly specific for ATP; however, ADP, GTP, UTP, and CTP failed to bind. The epsilon subunit lacking the C-terminal helical hairpin was unable to bind ATP. Although ATP binding to the isolated epsilon subunits from other organisms has not been detected under the same conditions, a possibility emerges that the epsilon subunit acts as a built in cellular ATP level sensor of F0F1-ATP synthase.     

Site-directed cross-linking of b to the alpha, beta, and a subunits of the Escherichia coli ATP synthase

The b subunit dimer of the Escherichia coli ATP synthase, along with the delta subunit, is thought to act as a stator to hold the alpha(3)beta(3) hexamer stationary relative to the a subunit as the gammaepsilonc(9-12) complex rotates. Despite their essential nature, the contacts between b and the alpha, beta, and a subunits remain largely undefined. We have introduced cysteine residues individually at various positions within the wild type membrane-bound b subunit, or within b(24-156), a truncated, soluble version consisting only of the hydrophilic C-terminal domain. The introduced cysteine residues were modified with a photoactivatable cross-linking agent, and cross-linking to subunits of the F(1) sector or to complete F(1)F(0) was attempted. Cross-linking in both the full-length and truncated forms of b was obtained at positions 92 (to alpha and beta), and 109 and 110 (to alpha only). Mass spectrometric analysis of peptide fragments derived from the b(24-156)A92C cross-link revealed that cross-linking took place within the region of alpha between Ile-464 and Met-483. This result indicates that the b dimer interacts with the alpha subunit near a non-catalytic alpha/beta interface. A cysteine residue introduced in place of the highly conserved arginine at position 36 of the b subunit could be cross-linked to the a subunit of F(0) in membrane-bound ATP synthase, implying that at least 10 residues of the polar domain of b are adjacent to residues of a. Sites of cross-linking between b(24-156)A92C and beta as well as b(24-156)I109C and alpha are proposed based on the mass spectrometric data, and these sites are discussed in terms of the structure of b and its interactions with the rest of the complex.     

Assembly of the stator in Escherichia coli ATP synthase. Complexation of alpha subunit with other F1 subunits is prerequisite for delta subunit binding to the N-terminal region of alpha

Alpha subunit of Escherichia coli ATP synthase was expressed with a C-terminal 6-His tag and purified. Pure alpha was monomeric, was competent in nucleotide binding, and had normal N-terminal sequence. In F1 subunit dissociation/reassociation experiments it supported full reconstitution of ATPase, and reassociated complexes were able to bind to F1-depleted membranes with restoration of ATP-driven proton pumping. Therefore interaction between the stator delta subunit and the N-terminal residue 1-22 region of alpha occurred normally when pure alpha was complexed with other F1 subunits. On the other hand, three different types of experiments showed that no interaction occurred between pure delta and isolated alpha subunit. Unlike in F1, the N-terminal region of isolated alpha was not susceptible to trypsin cleavage. Therefore, during assembly of ATP synthase, complexation of alpha subunit with other F1 subunits is prerequisite for delta subunit binding to the N-terminal region of alpha. We suggest that the N-terminal 1-22 residues of alpha are sequestered in isolated alpha until released by binding of beta to alpha subunit. This prevents 1/1 delta/alpha complexes from forming and provides a satisfactory explanation of the stoichiometry of one delta per three alpha seen in the F1 sector of ATP synthase, assuming that steric hindrance prevents binding of more than one delta to the alpha3/beta3 hexagon. The cytoplasmic fragment of the b subunit (bsol) did not bind to isolated alpha. It might also be that complexation of alpha with beta subunits is prerequisite for direct binding of stator b subunit to the F1-sector.     

The second stalk of Escherichia coli ATP synthase

Two stalks link the F(1) and F(0) sectors of ATP synthase. The central stalk contains the gamma and epsilon subunits and is thought to function in rotational catalysis as a rotor driving conformational changes in the catalytic alpha(3)beta(3) complex. The two b subunits and the delta subunit associate to form b(2)delta, a second, peripheral stalk extending from the membrane up the side of alpha(3)beta(3) and binding to the N-terminal regions of the alpha subunits, which are approx. 125 A from the membrane. This second stalk is essential for binding F(1) to F(0) and is believed to function as a stator during rotational catalysis. In vitro, b(2)delta is a highly extended complex held together by weak interactions. Recent work has identified the domains of b which are essential for dimerization and for interaction with delta. Disulphide cross-linking studies imply that the second stalk is a permanent structure which remains associated with one alpha subunit or alphabeta pair. However, the weak interactions between the polypeptides in b(2)delta pose a challenge for the proposed stator function.     

Membrane assembly of the 16-kDa proteolipid channel from Nephrops norvegicus studied by relaxation enhancements in spin-label ESR

The 16-kDa proteolipid from the hepatopancreas of Nephrops norvegicus belongs to the class of channel proteins that includes the proton-translocation subunit of the vacuolar ATPases. The membranous 16-kDa protein from Nephrops was covalently spin-labeled on the unique cysteine Cys54, with a nitroxyl maleimide, or on the functionally essential glutamate Glu140, with a nitroxyl analogue of dicyclohexylcarbodiimide (DCCD). The intensities of the saturation transfer ESR spectra are a sensitive indicator of spin-spin interactions that were used to probe the intramembranous structure and assembly of the spin-labeled 16-kDa protein. Spin-lattice relaxation enhancements by aqueous Ni(2+) ions revealed that the spin label on Glu140 is located deeper within the membrane (around C9-C10 of the lipid chains) than is that on Cys54 (located around C5-C6). In double labeling experiments, alleviation of saturation by spin-spin interactions with spin-labeled lipids indicates that spin labels both on Cys54 and on Glu140 are at least partially exposed to the lipid chains. The decrease in saturation transfer ESR intensity observed with increasing spin-labeling level is evidence of oligomeric assembly of the 16-kDa monomers and is consistent with a protein hexamer. These results determine the locations and orientations of transmembrane segments 2 and 4 of the 16-kDa putative 4-helix bundle and put constraints on molecular models for the hexameric assembly in the membrane. In particular, the crucial DCCD-binding site that is essential for proton translocation appears to contact lipid.     

Study of the binding of substrates and paramagnetic Mn2+ ions with phosphoglycerate kinase by saturating ESR spectra of a spin-labelled protein]

A single SH-group of phosphoglycerate kinase from yeast was modified by mercury-containing spin label. The saturation curves of ESR spectra of the spin-labeled enzyme were studied. The paramagnetic ions of Mn2+ bound to the centre of ion nonspecific binding or active centre in the complex with ATP can influence the saturation of the spin-labeled enzyme. The saturation curves of the ESR signal of the spin-labeled enzyme in the presence of paramagnetic complex of CrATP were studied. It has been demonstrated that the second nonspecific centre of ATP binding is located at the active site of the enzyme (3-phosphoglycerate binding centre).     

Chloroplast ATP synthase contains one single copy of subunit delta that is indispensable for photophosphorylation

F0F1 ATP synthases synthesize ATP in their F1 portion at the expense of free energy supplied by proton flow which enters the enzyme through their channel portion F0. The smaller subunits of F1, especially subunit delta, may act as energy transducers between these rather distant functional units. We have previously shown that chloroplast delta, when added to thylakoids partially depleted of the coupling factor CF1, can reconstitute photophosphorylation by inhibiting proton leakage through exposed coupling factor CF0. In view of controversies in the literature, we reinvestigated two further aspects related to subunit delta, namely (a) its stoichiometry in CF0CF1 and (b) whether or not delta is required for photophosphorylation. By rocket immunoelectrophoresis of thylakoid membranes and calibration against purified delta, we confirmed a stoichiometry of one delta per CF0CF1. In CF1-depleted thylakoids photophosphorylation could be reconstituted not only by adding CF1 and subunit delta but, surprisingly, also by CF1 (-delta). We found that the latter was attributable to a contamination of CF1 (-delta) preparations with integral CF1. To lesser extent CF1 (-delta) acted by complementary rebinding to CF0 channels that were closed because they contained delta [CF0(+delta)]. This added catalytic capacity to proton-tight thylakoid vesicles. The ability of subunit delta to control proton flow through CF0 and the absolute requirement for delta in restoration of photophosphorylation suggest an essential role of this small subunit at the interface between the large portions of ATP synthase: delta may be part of the coupling site between electrochemical, conformational and chemical events in this enzyme.     

Interaction between delta and epsilon subunits of F1-ATPase from pig heart mitochondria. Circular dichroism and intrinsic fluorescence of purified and reconstituted delta epsilon complex

A delta epsilon complex has been purified as a molecular entity from pig heart mitochondrial F1-ATPase. This delta epsilon complex has also been reconstituted from purified delta and epsilon subunits. Both isolated and reconstituted delta epsilon complexes have delta 1 epsilon 1 stoichiometry and are indistinguishable by their chromatographic behavior, their circular dichroism spectra (CD spectra), and their intrinsic fluorescence features. The content of secondary structures deduced from CD spectra of the delta epsilon complex appears to be the sum of the respective contributions of purified delta and epsilon subunits. All intrinsic fluorescence studies carried out on isolated epsilon subunit and delta epsilon complex show that the single tryptophan residue located on epsilon is involved in the interaction between delta and epsilon subunits. Results obtained with F1-ATPase are in favor of the same delta epsilon interaction in the entire enzyme.     

Nucleotide binding to the multidrug resistance P-glycoprotein as studied by ESR spectroscopy

Electron spin resonance (ESR) spectroscopy using spin-labeled ATP was used to study nucleotide binding to and structural transitions within the multidrug resistance P-glycoprotein, P-gp. Spin-labeled ATP (SL-ATP) with the spin label attached to the ribose, was observed to be an excellent substrate analogue for P-gp. SL-ATP was hydrolyzed in a drug-stimulated fashion at about 14% of the rate for normal ATP and allowed reversible trapping of the enzyme in transition and ground states. Equilibrium binding of a total of two nucleotides per P-gp was observed with a binding affinity of 366 microM in the presence of Mg2+ but in the absence of transport substrates such as verapamil. Binding of SL-ATP to wild-type P-gp in the presence of verapamil resulted in reduction of the protein-bound spin-label moiety, most likely due to a conformational transition within P-gp that positioned cysteines in close proximity to the spin label to allow chemical reduction of the radical. We circumvented this problem by using a mutant of P-gp in which all naturally occurring cysteines were substituted for alanines. Equilibrium binding of SL-ATP to this mutant P-gp resulted in maximum binding of two nucleotides; the binding affinity was 223 microM in the absence and 180 microM in the presence of verapamil. The corresponding ESR spectra of wild-type and Cys-less P-gp in the presence of SL-ATP indicate that a cysteine side chain of P-gp is located close to the ribose of the bound nucleotide. Trapping SL-ATP as an AlF(x)-adduct resulted in ESR spectra that showed strong immobilization of the radical, supporting the formation of a closed conformation of P-gp in its transition state. This study is the first to employ ESR spectroscopy with the use of spin-labeled nucleotide analogues to study P-glycoprotein. The study shows that SL-ATP is an excellent substrate analogue that will allow further exploration of structure and dynamics within the nucleotide binding domains of this important enzyme.     

FoF1-ATPase activity regulated by external links on β subunits

F_oF₁-ATPase activity is regulated by external links on β subunits with different molecular weight. It is inhibited when anti-β subunit antibody, streptavidin and H9 antibody link on the β subunits successively, but is activated when virus was binded. Western blotting indicated that the employed anti-β antibody target was on the non-catalytic site of the β subunit. Furthermore, an ESR study of spin-labeled ATP (SL-ATP) showed that the affinity of ATP to the holoenzyme increases with increasing external links on the β subunits. This simple regulation method may have great potential in the design of rapid, free labeled, sensitive and selective biosensors.     

Isolated epsilon subunit of Bacillus subtilis F1-ATPase binds ATP

Previously, we demonstrated ATP binding to the isolated epsilon subunit of F1-ATPase from thermophilic Bacillus PS3 [Kato-Yamada Y., Yoshida M. (2003) J. Biol. Chem. 278, 36013]. However, whether it is a general feature of the epsilon subunit from other sources is yet unclear. Here, using a sensitive method to detect weak interactions between fluorescently labeled epsilon subunit and nucleotide, it was shown that the epsilon subunit of F1-ATPase from Bacillus subtilis also bound ATP. The dissociation constant for ATP binding at room temperature was calculated to be 2 mM, which may be suitable for sensing cellular ATP concentration in vivo.     

Role of the asymmetry of the homodimeric b2 stator stalk in the interaction with the F1 sector of Escherichia coli ATP synthase

The b subunit dimer in the peripheral stator stalk of Escherichia coli ATP synthase is essential for enzyme assembly and the rotational catalytic mechanism. Recent protein chemical evidence revealed the dimerization domain of b to contain a novel two-stranded right-handed coiled coil with offset helices. Here, the existence of this structure in more complete constructs of b containing the C-terminal domain, and therefore capable of binding to the peripheral F1-ATPase, was supported by the more efficient formation of intersubunit disulfide bonds between cysteine residues that are proximal only in the offset arrangement and by the greater thermal stabilities of cross-linked heterodimers trapped in the offset configuration as opposed to homodimers with the helices trapped in-register. F1-ATPase binding analyses revealed the offset heterodimers to bind F1 more tightly than in-register homodimers. Mutations near the C terminus of b were incorporated specifically into either the N-terminally or the C-terminally shifted polypeptide, bN or bC, respectively, to determine the contribution of each position to F1 binding. Deletion of the last four residues of bN substantially weakened F1 binding, whereas the effect of the deletion in bC was modest. Similarly, benzophenone maleimide introduced at the C terminus of bN, but not bC, mediated cross-linking to the delta subunit of F1. These results imply that the polypeptide in the bN position is more important for F1 binding than the one in the bC position and illustrate the significance of the asymmetry of the b dimer in the enzyme.     

The membrane ATPase of alkalophilic Bacillus firmus RAB is an F1-type ATPase

At the optimal pH for growth (pH 10.5), alkalophilic Bacillus firmus RAB, an obligate aerobe, exhibits normal rates of oxidative phosphorylation despite the low transmembrane proton electrochemical gradient, about -60 mV (delta psi = -180 mV and delta pH = +120 mV). This bioenergetic problem might be resolved by use of an Na+ coupled ATP synthase; otherwise an F1F0-ATPase must be able to utilize low driving forces in this organism. The ATPase activity was extracted from everted membrane vesicles by low ionic strength treatment and purified to homogeneity by hydrophobic interaction chromatography and sucrose density gradient centrifugation. The ATPase preparation had the characteristic F1-ATPase subunit structure, with Mr values of 51,500 (alpha), 48,900 (beta), 34,400 (gamma), 23,300 (delta), and 14,500 (epsilon); the identity of the alpha and beta subunits was confirmed by immunoblotting with anti-beta of Escherichia coli and anti-B. firmus RAB F1. Methanol and octyl glucoside, agents that stimulated the low basal membrane ATPase activity 10- to 12-fold, dramatically elevated the MgATPase activity of the purified F1, more than 150-fold, to 50 mumol min-1 mg protein-1. Anti-F1 inhibited membrane ATPase activity greater than or equal to 80%. The membranes exhibited no Na+-stimulated or vanadate-sensitive ATPase activity when prepared in the absence or presence of Na+ or ATP. These findings, which are consistent with previous studies, establish that in alkalophilic bacteria, ATP hydrolysis, and presumably ATP synthesis is catalyzed by an F1F0-ATPase rather than a Na+ ATPase.