Characteristics of protection by MgADP and MgATP of α3ß3γ subcomplex of thermophilic Bacillus PS3 ßY341W-mutant F1-ATPase from inhibition by 7-chloro-4-nitrobenz-2-oxa-1,3-diazole support a bi-site mechanism of catalysis.

MgADP and MgATP binding to catalytic sites of ßY341W-α(3)ß(3)γ subcomplex of F(1)-ATPase from thermophilic Bacillus PS3 has been assessed using their effect on the enzyme inhibition by 7-chloro-4-nitrobenz-2-oxa-1,3-diazole (NBD-Cl). It was assumed that NBD-Cl can inhibit only when catalytic sites are empty, and inhibition is prevented if a catalytic site is occupied with a nucleotide. In the absence of an activator, MgADP and MgATP protect ßY341W-α(3)ß(3)γ subcomplex from inhibition by NBD-Cl by binding to two catalytic sites with an affinity of 37 µM and 12 mM, and 46 µM and 15 mM, respectively. In the presence of an activator lauryldimethylamine-N-oxide (LDAO), MgADP protects ßY341W-α(3)ß(3)γ subcomplex from inhibition by NBD-Cl by binding to a catalytic site with a K(d) of 12 mM. Nucleotide binding to a catalytic site with affinity in the millimolar range has not been previously revealed in the fluorescence quenching experiments with ßY341W-α(3)ß(3)γ subcomplex. In the presence of activators LDAO or selenite, MgATP protects ßY341W-α(3)ß(3)γ subcomplex from inhibition by NBD-Cl only partially, and the enzyme remains sensitive to inhibition by NBD-Cl even at MgATP concentrations that are saturating for ATPase activity. The results support a bi-site mechanism of catalysis by F(1)-ATPases.

Probes of inhibition of Escherichia coli F(1)-ATPase by 7-chloro-4-nitrobenz-2-oxa-1,3-diazole in the presence of MgADP and MgATP support a bi-site mechanism of ATP hydrolysis by the enzyme.

Binding of MgADP and MgATP to Escherichia coli F(1)-ATPase (EcF(1)) has been assessed by their effects on extent of the enzyme inhibition by 7-chloro-4-nitrobenz-2-oxa-1,3-diazole (NBD-Cl). MgADP at low concentrations (K(d) 1.3 microM) promotes the inhibition, whereas at higher concentrations (K(d) 0.7 mM) EcF(1) is protected from inhibition. The mutant betaY331W-EcF(1) requires much higher MgADP, K(d) of about 10 mM, for protection. Such MgADP binding was not revealed by fluorescence quenching measurements. MgATP partially protects EcF(1) from inactivation by NBD-Cl, but the enzyme remains sensitive to NBD-Cl in the presence of MgATP at concentrations as high as 10 mM. The activating anion selenite in the absence of MgATP partially protects EcF(1) from inhibition by NBD-Cl. A complete protection of EcF(1) from inhibition by NBD-Cl has been observed in the presence of both MgATP and selenite. The results support a bi-site catalytic mechanism for MgATP hydrolysis by F(1)-ATPases and suggest that stimulation of the enzyme activity by activating anions is due to the anion binding to a catalytic site that remains unoccupied at saturating substrate concentration.

Bi-site activation occurs with the native and nucleotide-depleted mitochondrial F1-ATPase.

Experiments are reported on the uni-site catalysis and the transition from uni-site to multi-site catalysis with bovine heart mitochondrial F1-ATPase. The very slow uni-site ATP hydrolysis is shown to occur without tightly bound nucleotides present and with or without Pi in the buffer. Measurements of the transition to higher rates and the amount of bound ATP committed to hydrolysis as the ATP concentration is increased at different fixed enzyme concentrations give evidence that the filling of a second site can initiate near maximal turnover rates. They provide rate constant information, and show that an apparent Km for a second site of about 2 microM and Vmax of 10 s-1, as suggested by others, is not operative. Careful initial velocity measurements also eliminate other suggested Km values and are consistent with bi-site activation to near maximal hydrolysis rates, with a Km of about 130 microM and Vmax of about 700 s-1. However, the results do not eliminate the possibility of additional 'hidden' Km values with similar Vmax:Km ratios. Recent data on competition between TNP-ATP and ATP revealed a third catalytic site for ATP in the millimolar concentration range. This result, and those reported in the present paper, allow the conclusion that the mitochondrial F1-ATPase can attain near maximal activity in bi-site catalysis. Our data also add to the evidence that a recent claim, that the mitochondrial F1-ATPase does not show catalytic site cooperativity, is invalid.

Nucleotide-depleted beef heart F1-ATPase exhibits strong positive catalytic cooperativity.

Catalytic cooperativity is a central feature of the binding change mechanism for F0F1-ATP synthases. However, in a recent publication (Reynafarje, B. D., and Pedersen, P. L. (1996) J. Biol. Chem. 271, 32546-32550), Reynafarje and Pedersen claim that cooperative effects are an artifact caused by endogenous nucleotides and that when such nucleotides are removed, the multiple catalytic sites on MF1 behave independently during ATP hydrolysis. In contrast to this conclusion, we show here that when ATP is loaded at a single catalytic site on nucleotide-depleted MF1, the rate of product release is accelerated by up to 5 x 10(4)-fold by the binding of ATP at adjacent catalytic sites. Hence, nucleotide-depleted MF1 is not an exception but does in fact show strong cooperative interactions. In addition, evidence is presented supporting a random order for product release during ATP hydrolysis.

Studies on reconstitution of the Rhodospirillum rubrum nicotinamide nucleotide transhydrogenase.

The energy-transducing nicotinamide nucleotide transhydrogenase of Rhodospirillum rubrum is composed of 3 subunits alpha 1, alpha 2 and beta, with M(r) values, respectively, of 40.3, 14.9 and 47.8 kDa. Subunit alpha 1 is water-soluble, loosely bound to chromatophores, and can be easily and reversibly removed. Subunits alpha 2 and beta are integral membrane proteins, and their removal from chromatophores requires the use of detergents. Treatment of chromatophores with various detergents inhibited reconstitution of transhydrogenase activity when alpha 1 was added to the detergent-treated chromatophores. This apparent inhibition could be reversed by addition of a divalent metal ion. The best condition for extraction of alpha 2/beta from chromatophores was the use of 1% deoxycholate in the presence of 0.34 M KCl. Under these conditions, the extracted alpha 2/beta mixed with purified alpha 1 was completely inactive, but gained full activity when the assay medium was supplemented with 2-3 mM MgCl2 or CaCl2. It was shown that metal ions had little effect on the apparent Km of substrates, but greatly increased the affinity between purified alpha 1 and the detergent-treated or detergent-solubilized alpha 2/beta. It seems possible that the R. rubrum transhydrogenase contains a detergent-extractable metal ion, which is required for proper binding of the soluble alpha 1 subunit to the chromatophore-bound alpha 2/beta subunits.

Nucleotide-binding sites on Escherichia coli F1-ATPase. Specificity of noncatalytic sites and inhibition at catalytic sites by MgADP.

Nucleotide-depleted EcF1 binds a maximum of two GTP, ATP, or ADP at noncatalytic sites, whereas all three sites can only be filled by a combination of nucleoside di- and triphosphates. MgPPi prevents binding of GTP and significantly slows ATP binding, suggesting that non-catalytic sites also bind PPi. No binding of GDP at non-catalytic sites could be detected. The slow rate of GTP dissociation from noncatalytic sites (t1/2 = 175 min) is increased 2-8-fold by EDTA, MgPPi, MgADP, or EDTA/ATP, but 23-fold by conditions for ATP hydrolysis. ATP hydrolysis by EcF1, depleted of both its inhibitory epsilon-subunit and endogenous nucleotides, shows a burst of activity. However, it shows a lag if preincubated with MgADP but not when preincubated with Mg2+ alone. For epsilon-depleted EcF1 containing endogenous inhibitory ADP, preincubation with an ATP-regenerating system results in a burst of activity, whereas the control shows a lag. This same enzyme form shows significant inhibition with increasing concentrations of Mg2+ during ATP hydrolysis but lesser levels of inhibition when other NTP substrates are used. With the five-subunit enzyme, increasing amounts of azide cause an increase in the level of inhibition with a corresponding increase in amount of bound nucleotide resistant to rapid chase. Azide-trappable nucleotide is bound at catalytic sites as shown by covalent incorporation of 2-azido-ADP. The results suggest that ligand specificity may not be a reliable means of distinguishing between catalytic and noncatalytic sites and that MgADP inhibition should be taken into account in the kinetic analysis of EcF1 mutants.

Structure of cucumarioside G2, a novel nonholostane glycoside from the sea cucumber Eupentacta fraudatrix.

The new triterpene glycoside cucumarioside G2 [1] has been isolated from the sea cucumber Eupentacta fraudatrix. Glycoside 1 is the first triterpene glycoside with the 23,24,25,26,27-pentanorlanostane type of aglycone. Its structure has been established by chemical transformations as well as 13C- and 1H-nmr, eims, and liquid sims studies.

Nucleotide binding sites on beef heart mitochondrial F1-ATPase. Cooperative interactions between sites and specificity of noncatalytic sites.

We have studied the properties of beef heart mitochondrial F1 having inhibitory MgADP bound at one of the three catalytic sites and various levels of occupancy of the three noncatalytic nucleotide sites including zero, two, or three ADP/ATPs or two ADP/ATP plus one GTP. The properties examined include the rate of MgATP-dependent reactivation and the rate of increase in the fraction of F1 containing transiently bound intermediates. For each form of the enzyme tested, the rate of reactivation closely paralleled the rate of increase in the level of bound intermediates, indicating that when one catalytic site on F1 is blocked by inhibitory MgADP, the remaining two sites are incapable of residual uni- or bi-site activity. It was also found that the stability of the MgADP-inhibited complex decreases with full occupancy of the noncatalytic sites. This demonstrates that the noncatalytic sites modulate the properties of catalytic sites. Finally, it was found that the noncatalytic sites on mitochondrial F1 do not, as has long been believed, bind adenine nucleotides exclusively. Evidence is presented that both GTP and PPi bind tightly at noncatalytic sites.

The permeability transition in heart mitochondria is regulated synergistically by ADP and cyclosporin A.

Heart mitochondria respiring in a sucrose medium containing P(i) show a permeability transition when challenged with Ca2+ and an oxidant such as cumene hydroperoxide. The transition results from the opening of a Ca(2+)-dependent pore and is evidenced by loss of membrane potential (delta psi) and osmotic swelling due to uptake of sucrose and other solutes. In the absence of oxidant, high concentrations of Ca2+ (100-150 microM) are necessary to induce loss of delta psi and initiate swelling. Cyclosporin A delays the loss of delta psi but enhances swelling under these conditions, apparently by promoting better retention of accumulated Ca2+. Cyclosporin A and ADP together restore delta psi in respiring mitochondria that have undergone the permeability transition at levels that are not effective when either is added alone. When the state of the Ca(2+)-dependent pore is assessed using passive osmotic contraction in response to polyethylene glycol (Haworth, R. A., and Hunter, D. R. (1979) Arch. Biochem. Biophys. 195, 460-467), cyclosporin A is found to be a partial inhibitor of solute flow through the open pore. Cyclosporin A decreases the Vmax of passive contraction and increases the Km for Ca2+ without affecting the Hill slope. ADP in the presence of carboxyatractyloside closes the pore almost completely even in the presence of 40 microM Ca2+. ADP shows mixed type inhibition of the Ca(2+)-dependent pore, and cyclosporin A increases the affinity of the pore for ADP. It is concluded that cyclosporin A and ADP act synergistically to close the Ca(2+)-dependent pore of the mitochondrion and that the pore is probably not formed directly from the adenine nucleotide transporter.

When beef-heart mitochondrial F1-ATPase is inhibited by inhibitor protein a nucleotide is trapped in one of the catalytic sites.

Inactivation of the isolated ATPase portion of ATP synthase from beef-heart mitochondria (F1) by its natural inhibitor protein (IP) during steady-state ATP hydrolysis is accompanied by a trapping of 1 mol nucleotide/mol F1 in one of the catalytic sites. The trapped nucleotide is not released during incubation of IP-inhibited F1 in the presence of MgATP at pH 8.0 for at least 20 min, indicating a very low turnover rate of the IP.F1 complex. The ATP/ADP ratio of the trapped nucleotides is higher than that found for transitorily bound nucleotides under the same conditions but in the absence of IP. The IP impairs the acceleration of ATP hydrolysis and product release steps that results from the binding of ATP to an alternate catalytic site. It also inhibits ATP hydrolysis by a single catalytic site or shifts the equilibrium toward ATP formation from bound ADP and Pi. At high pH, an active acidic form of the free IP is transformed to the inactive basic one with a half-time of 3-4 s. This process seems to be prevented by IP binding to F1. The inactive basic form of IP does not compete with the active acidic IP for the binding to F1. The data do not favor the existence of a long-lived catalytically active IP.F1 intermediate during IP action on F1. The reactivation of IP-inhibited membrane-bound F1 by energization may be due to a conformational change in the IP.F1 complex allowing the transformation of IP into an inactive basic state that rapidly dissociates.

Characteristics of the combination of inhibitory Mg2+ and azide with the F1 ATPase from chloroplasts.

The interactions between ADP, Mg2+, and azide that result in the inhibition of the chloroplast F1 ATPase (CF1) have been explored further. The binding of the inhibitory Mg2+ with low Kd is shown to occur only when tightly bound ADP is present at a catalytic site. Either the tightly bound ADP forms part of the Mg(2+)-binding site or it induces conformational changes creating the high-affinity site for inhibitory Mg2+. Kinetic studies show that CF1 forms two catalytically inactive complexes with Mg2+. The first complex results from Mg2+ binding with a Kd for Mg2+ dissociation of about 10-15 microM, followed by a slow conversion to a complex with a Kd of about 4 microM. The rate-limiting step of the CF1 inactivation by Mg2+ is the initial Mg2+ binding. When medium Mg2+ is chelated with EDTA, the two complexes dissociate with half-times of about 1 and 7 min, respectively. Azide enhances the extent of Mg(2+)-dependent inactivation by increasing the affinity of the enzyme for Mg2+ 3-4 times and prevents the reactivation of both complexes of CF1 with ADP and Mg2+. This results from decreasing the rate of Mg2+ release; neither the rate of Mg2+ binding to CF1 nor the rate of isomerization of the first inactive complex to the more stable form is affected by azide. This suggests that the tight-binding site for the inhibitory azide requires prior binding of both ADP and Mg2+.

The characteristics and effect on catalysis of nucleotide binding to noncatalytic sites of chloroplast F1-ATPase.

The recent finding that the presence of ATP at non-catalytic sites of chloroplast F1-ATPase (CF1) is necessary for ATPase activity (Milgrom, Y. M., Ehler, L. L., and Boyer, P. D. (1990) J. Biol. Chem. 265,18725-18728) prompted more detailed studies of the effect of noncatalytic site nucleotides on catalysis. CF1 containing at noncatalytic sites less than one ADP or about two ATP was prepared by heat activation in the absence of Mg2+ and in the presence of ADP or ATP, respectively. After removal of medium nucleotides, the CF1 preparations were used for measurement of the time course of nucleotide binding from 10 to 100 microM concentrations of 3H-labeled ADP, ATP, or GTP. The presence of Mg2+ strongly promotes the tight binding of ADP and ATP at noncatalytic sites. For example, the ADP-heat-activated enzyme in presence of 1 mM Mg2+ binds ADP with a rate constant of 0.5 x 10(6) M-1 min-1 to give an enzyme with two ADP at noncatalytic sites with a Kd of about 0.1 microM. Upon exposure to Mg2+ and ATP the vacant noncatalytic site binds an ATP rapidly and, as an ADP slowly dissociates, a second ATP binds. The binding correlates with an increase in the ATPase activity. In contrast the tight binding of [3H]GTP to noncatalytic sites gives an enzyme with no ATPase activity. The three noncatalytic sites differ in their binding properties. The noncatalytic site that remains vacant after the ADP-heat-activated CF1 is exposed to Mg2+ and ADP and that can bind ATP rapidly is designated as site A; the site that fills with ATP as ADP dissociates when this enzyme is exposed to Mg2+ and ATP is called site B, and the site to which ADP remains bound is called site C. Procedures are given for attaining CF1 with ADP at sites B and C, with GTP at sites A and/or B, and with ATP at sites A, B, and/or C, and catalytic activities of such preparations are measured. For example, little or no ATPase activity is found unless ATP is at site A, but ADP can remain at site C with no effect on ATPase. Maximal GTPase activity requires ATP at site A but about one-fifth of maximal GTPase is attained when GTP is at sites A and B and ATP at site C. Noncatalytic site occupancy can thus have profound effects on the ATPase and GTPase activities of CF1.

ATP binding at noncatalytic sites of soluble chloroplast F1-ATPase is required for expression of the enzyme activity.

The F1-ATPase from chloroplasts (CF1) lacks catalytic capacity for ATP hydrolysis if ATP is not bound at noncatalytic sites. CF1 heat activated in the presence of ADP, with less than one ADP and no ATP at non-catalytic sites, shows a pronounced lag in the onset of ATP hydrolysis after exposure to 5-20 microM ATP. The onset of activity correlates well with the binding of ATP at the last two of the three noncatalytic sites. The dependence of activity on the presence of ATP at non-catalytic sites is shown at relatively low or high free Mg2+ concentrations, with or without bicarbonate as an activating anion, and when the binding of ATP at noncatalytic sites is slowed 3-4-fold by sulfate. The latent CF1 activated by dithiothreitol also requires ATP at noncatalytic sites for ATPase activity. A similar requirement by other F1-ATPases and by ATP synthases seems plausible.

The ADP that binds tightly to nucleotide-depleted mitochondrial F1-ATPase and inhibits catalysis is bound at a catalytic site.

Previous studies have shown that the initial complex formed when ADP binds to nucleotide-depleted F1-ATPase is transformed with a half time of 2 to 3 min to form with a much lower rate of ADP release. The ADP binding results in a strong inhibition of ATPase activity. The present paper reports appraisal of where the inhibitory ADP binds by use of the photoreactive ADP analog, 2-N3-ADP. In presence of Mg2+ the 2-N3-ADP like ADP induces reversible inhibition of nucleotide-depleted F1 (ndF1) with a Kd of about 10 nM. Photoirradiation of the inactive 2-N3-[beta-32P]ADP-ndF1 complex results in labeling of only the beta-subunit. The major labeled peptide isolated from a trypic digest consists of residues from Ala-338 to Arg-356, with Tyr-345 as the site of labeling. This identifies the site of the inhibitory ADP binding as one of the catalytic sites of the enzyme.

The F1-type ATPase in anaerobic Lactobacillus casei.

An ATPase from anaerobic Lactobacillus casei has been isolated and 100-times purified. The 400 kDa enzyme molecule was found to have a hexagonal structure 10 nm in diameter composed of at least six protein masses. SDS-electrophoresis reveals four or, under certain conditions, five types of subunit, of apparent molecular masses 57 (alpha), 55 (beta), 40 (gamma), 22 (delta) and 14 (epsilon) kDa with stoichiometry of 3 alpha, 3 beta, gamma, delta, epsilon. The following features resembling F1-ATPases from other sources were found to be inherent in the solubilized L. casei ATPase. (i) Detachment from the membrane desensitizes ATPase to low DCCD concentrations and sensitizes it to water-soluble carbodiimide. (ii) Soluble ATPase is inhibited by Nbf chloride and azide, is resistant to SH-modifiers and is activated by sulfite and octyl glucoside, the activating effect being much stronger than in the case of the membrane-bound ATPase. Substrate specificity of the enzyme is also similar to that of other factors F1. Divalent cations strongly activate the soluble enzyme when added at a concentration equal to that of ATP. An excess of Mn2+, Mg2+ or Co2+ inhibits ATPase activity of F1, whereas that of Ca2+ induces its further activation. No other F1-like ATPases are found in L. casei. It is concluded that this anaerobic bacterium possesses a typical F1-ATPase similar to those in mitochondria, chloroplasts, aerobic and photosynthetic eubacteria.

The effect of inorganic pyrophosphate on the activity and Pi-binding properties of mitochondrial F1-ATPase.

Interaction of F1-ATPase from beef heart mitochondria with PPi has been investigated. The presence of PPi in the ATPase assay medium does not affect the initial rate of ATP hydrolysis by F1-ATPase, but slows down the decrease of enzyme activity in the course of ATP hydrolysis and increases the steady-state rate of ATP hydrolysis. Being present in the ATPase assay medium, PPi accelerates the ATP-dependent reactivation of an inactive complex formed by F1-ATPase and ADP. This inactive complex is also reactivated after preincubation with PPi. F1-ATPase, preincubated with PPi, is inactivated by azide much more slowly than is the non-preincubated enzyme. PPi stimulates the binding of Pi to F1-ATPase by decreasing mainly the Kd for Pi and only slightly raising the stoichiometry of high-affinity Pi binding. It follows from the results obtained that PPi interacts with the non-catalytic site(s) of F1-ATPase.

Electron-microscopic studies on location of SH-groups in mitochondrial F1-ATPase using a ferritin label.

A new approach has been suggested for electron-microscopic study of the structure of mitochondrial F1-ATPase based on ferritin labeling. By means of sequential treatment with 2-iminothiolane and Nbs2 we obtained a modified ferritin (NbsSPrCNH-Ft) able to react with SH-groups of proteins and to form conjugates in which the protein and ferritin are bound by disulfide bonds. An electron-microscopic investigation of the negatively stained preparations of mitochondrial F1-ATPase, preincubated with modified ferritin, revealed such enzyme-ferritin conjugates. In case of modified ferritin, containing 360 mol SH-groups per mol protein, and F1-ATPase, pretreated with N-ethylmaleimide and then with dithiothreitol, conjugates were obtained in which ferritin molecules are bound to several (as many as four) of the six protein masses, comprising a bilayer molecule of the enzyme. Taking into consideration the biochemical data on the location of accessible SH-groups (only in alpha, gamma or epsilon subunits), it is inferred from the results obtained that one of the protein masses is a complex between beta subunit and at least one of the minor subunits located partially on the molecule's external side. This indicates the nonequivalence of different copies of the major subunits. Averaged images of the particles of the F1-F0 complex from bovine heart mitochondria and bacteria Micrococcus lysodeicticus were obtained. It was found that F0 component is bound to two adjacent protein masses of the F1-ATPase molecule. It is suggested that this binding may be due the nonequivalency of single-type major subunits.

The interaction of mitochondrial transhydrogenase with derivatives of coenzyme A.

The 2,4-dinitrophenyl derivative of dephospho-CoA and the 7-nitrobenzofurazan-4-yl derivative of CoA are competitive inhibitors (Ki 3 microM and 2.6 microM respectively) of mitochondrial transhydrogenase with regard to NAD+ and NADPH respectively. The 7-nitrobenzofurazan-4-yl derivative of dephospho-CoA is a competitive inhibitor with regard to both transhydrogenase substrates with the same Ki equal to 0.3 microM. The pattern of transhydrogenase inhibition with the 7-nitrobenzofurazan-4-yl derivative of dephospho-CoA indicates that one molecule of the inhibitor binds simultaneously to both the NADP(H) and the NAD(H) binding sites of the enzyme. This result is evidence of the short distance between the NADP(H) and the NAD(H) binding sites.

Using 2'(3')-O-trinitrophenyl derivatives of adenine nucleotides to study the structure and mechanism of functioning of soluble mitochondrial ATPase.

The 2'(3')-O-trinitrophenyl (N3ph) derivatives of the adenine nucleotides are strong competitive inhibitors of isolated mitochondrial ATPase (factor F1). Ki decreases in the order N3phAdo greater than N3phAdo greater than N3phAMP greater than N3phADP and is equal to 8 nM for N3phADP. Picric acid, which activates the ATPase reaction of factor F1 without changing the Km(app), prevents the inhibiting action of N3phADP. At pH 7.6 the inhibiton of factor F1 is accompanied by the binding of one molecule of N3phADP to a molecule of the enzyme. This binding leads to changes in the absorption spectrum, but not in the intensity of the fluorescence of the N3phADP. At pH 6.7 one or two molecules of N3phADP bind with the tight binding sites of factor F1. This binding is accompanied by the manifold enhancement of the fluorescence of N3phADP. The results obtained indicate that the sites of factor F1 that tightly bind nucleotides are immersed in the hydrophobic pocket of the protein molecule.

Isolation of alpha-subunits of factor F1 from submitochondrial particles and the reconstitution of active ATPase from isolated alpha-subunits and beta-subunits bound to the mitochondrial membrane.

The alpha-subunits of factor-F1 ATPase are removed by extraction of submitochondrial particles with 1.75 M-LiCl, with the consequent loss of ATPase activity. ATPase activity is reconstituted by incubation of LiCl-extracted particles with purified alpha-subunits, and the reconstituted ATPase activity is oligomycin-sensitive. Reconstitution is enhanced by maintenance of the alpha-subunits in reduced form by dithiothreitol or NaBH4 and by modification of the alpha-subunits by p-chloromercuribenzoate, iodoacetic acid or N-ethylmaleimide. Experiments with the mixed anhydride of ATP and mesitylene-carboxylic acid, which was previously shown to interact with the F1 active site, localized on the beta-subunits, indicate that the active site of ATPase is shielded by the alpha-subunits.