Labeling the Ca2+-ATPase of skeletal muscle sarcoplasmic reticulum with maleimidylsalicylic acid

Maleimidylsalicylic acid reacts with the Ca(2+)-ATPase of skeletal muscle sarcoplasmic reticulum with high affinity and inhibits the ATPase activity following a pseudo-first-order kinetic with a rate constant of 8.3 m(-1) s(-1). Calcium binding remains unaffected in the maleimide-inhibited ATPase. However, the presence of ATP, ADP, and, to a lesser extent, AMP protects the enzyme against inhibition. Furthermore, ATPase inhibition is accompanied by a concomitant decrease in ATP binding. The stoichiometry of the nucleotide-dependent maleimidylsalicylic acid binding is 6-10 nmol/mg ATPase, which corresponds to the binding of up to one molecule of maleimide/molecule of ATPase. The stoichiometry of maleimide binding is decreased in the presence of nucleotides and in the ATPase previously labeled with fluorescein-5'-isothiocyanate or N-ethylmaleimide A fluorescent peptide was isolated by high performance liquid chromatography after trypsin digestion of the maleimide-labeled ATPase. Analysis of the sequence and mass spectrometry of the peptide leads us to propose Cys(344) as the target for maleimidylsalicylic acid in the inhibition reaction. The effect of Cys(344) modification on the nucleotide site is discussed.     

Binding properties of 9K protein to F1-ATPase: a counterpart ligand to the ATPase inhibitor

A regulatory subunit of yeast mitochondrial ATP synthase, 9K protein, formed an equimolar complex with F1-ATPase in the presence of ATP and Mg2+, indicating that the binding of the protein to the enzyme took place in a similar manner to that of ATPase inhibitor. The ATP-hydrolyzing activity of F1-ATPase decreased 40% on binding of the 9K protein, and the remaining activity was resistant to external ATPase inhibitor. The apparent dissociation constant of the F1-ATPase-9K complex was determined by gel permeation chromatography to be 3.7 X 10(-6) M, which was in the same order of magnitude as that of enzyme-ATPase inhibitor complex (4.2 x 10(-6) M). When added simultaneously the binding of the inhibitor and 9K protein to F1-ATPase were competitive and the sum of their bindings did not exceed 1 mol per mol of enzyme. However, the binding of each protein ligand to F1-ATPase took more than 1 min for completion, and when one of these two proteins was added 10 min after the other, it did not replace the other. These observations strongly suggest that membrane-bound F1-ATPase always binds to either the 9K protein or ATPase inhibitor in intact mitochondria and that the complexes with the two ligands are active and inactive counterparts, respectively.     

Simultaneous bindings of ATPase inhibitor and 9K protein to F1F0-ATPase in the presence of 15K protein in yeast mitochondria

Yeast mitochondrial ATP synthase has three regulatory proteins, ATPase inhibitor, 9K protein, and 15K protein. The 9K protein binds directly to purified F1-ATPase, as does the ATPase inhibitor, but the 15K protein does not [Hashimoto, T. et al. (1987) J. Biochem. 102, 685-692]. In the present study, we found that 15K protein bound to purified F1F0-ATPase, forming an equimolar complex with the enzyme. The apparent dissociation constant was calculated to be 1.4 x 10(-5) M. The ATPase inhibitor and 9K protein also bound to F1F0-ATPase in the presence of ATP and Mg2+, and the dissociation constants of their bindings were about 3 X 10(-6) M. They bound to the enzyme competitively in the absence of 15K protein, but in its presence, they bound in equimolar amounts to the enzyme. The ATP-hydrolyzing activity of the enzyme-ligand complex was greatly influenced by the order of bindings of ATPase inhibitor and 9K protein: when the ATPase inhibitor was bound first, the activity of the enzyme was inhibited completely and was not restored by 9K protein, but when 9K protein was added first, the activity was inhibited only partially even after equimolar binding of the ATPase inhibitor to the enzyme. These observations strongly suggest that the 15K protein binds to the F0 part and functions to hold the ATPase inhibitor or 9K protein on the F1 subunit.     

Studies on the active site of the Neurospora crassa plasma membrane H+-ATPase with periodate-oxidized nucleotides

The Neurospora crassa plasma membrane H+-ATPase is inactivated by the periodate-oxidized nucleotides, oATP, oADP, and oAMP, with oAMP the most effective. Inhibition of the ATPase is essentially irreversible, because Sephadex G-50 column chromatography of the oAMP-treated ATPase does not result in a reversal of the inhibition. Inhibition of the ATPase by oAMP is protected against by the H+-ATPase substrate ATP, the product ADP, and the competitive inhibitors TNP (2',3'-O-(2,4,6-trinitrocyclohexadienylidine)-ATP and TNP-ADP, suggesting that oAMP inhibition occurs at the nucleotide binding site of the enzyme. The rate of inactivation of the ATPase by oAMP is only slightly affected by EDTA, indicating that the oAMP interaction with the nucleotide binding site of the H+-ATPase occurs in the absence of a divalent cation. The protection against oAMP inhibition by ADP is likewise unaffected by EDTA. The inhibition of the ATPase by oAMP is absolutely dependent on the presence of acidic phospholipids or acidic lysophospholipids known to be required for H+-ATPase activity, suggesting that these lipids either aid in the formation of the nucleotide binding site or render it accessible. Incubation of the ATPase with Mg2+ plus vanadate, which locks the enzyme in a conformation resembling the transition state of the enzyme dephosphorylation reaction, completely protects against inhibition by oAMP, suggesting that in this transition state conformation the nucleotide site either does not exist, or is inaccessible to oAMP. Labeling studies with [14C] oAMP indicate that the incorporation of 1 mol of oAMP is sufficient to cause complete inactivation of the ATPase.     

Inactivation and phosphorylation of sarcoplasmic reticulum Ca(2+)-ATPase by Mg.ATP analogues Rh(III)-ATP and Co(III)-ATP.

The interaction of sarcoplasmic reticulum Ca(2+)-ATPase with the Mg.ATP analogues Rh(H2O)4ATP and Co(NH3)4ATP have been examined. Co(NH3)4ATP slowly inactivates Ca(2+)-ATPase in a first order process, with a rate constant of 1.13 x 10(-3) s-1 and an apparent inactivation constant, KI, of 32 mM. Rh(H2O)4ATP likewise inactivates sarcoplasmic reticulum Ca(2+)-ATPase, but the plot of reciprocal apparent inactivation rate constants versus 1/[Rh(H2O)4ATP] is biphasic. The chi-intercepts of this plot yield apparent inactivation constants for the inhibition of Ca(2+)-ATPase by Rh(H2O)4ATP of KI1 = 30 microM and KI2 = 221 microM. The corresponding values of k2, the maximal first-order rate constant for inhibition in these two phases, are 1.16 and 2.19 x 10(-4)s-1. Tridentate Rh(H2O)3ATP also inhibits Ca(2+)-ATPase, but only after much longer incubation times. Ca(2+)-ATPase inactivation is accompanied by incorporation of radioactivity from gamma-32P into an acid-precipitable enzyme. Both processes were dependent on the presence of Ca2+ ions and were quenched by excess ATP. The first-order rate constant for inactivation of Ca(2+)-dependent ATPase activity in this experiment was 2.19 x 10(-4)s-1, and the first-order rate constant for Ca(2+)-dependent E-P formation was 2.07 x 10(-4)s-1, in excellent agreement with the value for inactivation. A linear relationship is observed between ATPase inactivation and E-P formation. Moreover, atomic absorption analysis demonstrates that the phosphorylation of Ca(2+)-ATPase by Rh(H2O)4ATP is accompanied by incorporation and tight binding of rhodium, with a stoichiometry of one rhodium incorporated per ATPase molecule phosphorylated. The characteristics of ATPase inactivation and phosphorylation (i.e., Ca2+ dependence, ATP competition, agreement of rate constants, and stoichiometric rhodium incorporation) suggest that Rh(H2O)4ATP is binding to the catalytic nucleotide site on Ca(2+)-ATPase and producing a highly stable, phosphorylated intermediate.     

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.     

Conformational states of the (Na+ + K+)-transporting ATPase. Formation of 240 000-Mr and 116 000-Mr polypeptides in the presence of a bifunctional thiol probe.

Interpeptide cross-linking of alpha-subunits with concomitant loss of Na+ + K+-transporting ATPase (Na+, K+-ATPase) activity was found when the purified lamb kidney enzyme was treated with the bifunctional thiol reagent 4,4'-difluoro-3,3'-dinitrodiphenyl sulphone (F2DNS). Several forms of the enzyme could be clearly distinguished: one binding ATP (non-phosphorylated enzyme, E1 X ATP), a phosphorylated form (E2-P) and a phosphoenzyme-ouabain complex (E2P X ouabain). A polypeptide of approx. Mr 240 000 and probable alpha 2 composition comprised up to 5-20% of the total polypeptides after reaction of the lamb kidney Na+, K+-ATPase with F2DNS. The amount of this polypeptide formed was related to the conformational state of the enzyme. The presence of adenine nucleotide greatly diminished the amount of 240 000-Mr polypeptide formed and provides evidence for an enzyme-adenine-nucleotide complex under conditions where the enzyme is not phosphorylated. F2DNS reacted with the enzyme in the presence of Mg2+, Pi and ouabain to form a new polypeptide with an approx. Mr of 116 000, and comprised 23% of the total, whereas the 240 000-Mr polypeptide comprised 9% of the total. This suggests that the 116 000-Mr polypeptide is a characteristic marker of the E2P X ouabain complex. By using specific antibodies it was established that both the 240 000- and 116 000-Mr polypeptides contained alpha-, but not beta-, subunits of the Na+, K+-ATPase.     

Effect of the epsilon-subunit on nucleotide binding to Escherichia coli F1-ATPase catalytic sites.

The influence of the epsilon-subunit on the nucleotide binding affinities of the three catalytic sites of Escherichia coli F1-ATPase was investigated, using a genetically engineered Trp probe in the adenine-binding subdomain (beta-Trp-331). The interaction between epsilon and F1 was not affected by the mutation. Kd for binding of epsilon to betaY331W mutant F1 was approximately 1 nM, and epsilon inhibited ATPase activity by 90%. The only nucleotide binding affinities that showed significant differences in the epsilon-depleted and epsilon-replete forms of the enzyme were those for MgATP and MgADP at the high-affinity catalytic site 1. Kd1(MgATP) and Kd1(MgADP) were an order of magnitude higher in the absence of epsilon than in its presence. In contrast, the binding affinities for MgATP and MgADP at sites 2 and 3 were similar in the epsilon-depleted and epsilon-replete enzymes, as were the affinities at all three sites for free ATP and ADP. Comparison of MgATP binding and hydrolysis parameters showed that in the presence as well as the absence of epsilon, Km equals Kd3. Thus, in both cases, all three catalytic binding sites have to be occupied to obtain rapid (Vmax) MgATP hydrolysis rates.     

Epsilon subunit of Escherichia coli F1-ATPase: effects on affinity for aurovertin and inhibition of product release in unisite ATP hydrolysis

The epsilon subunit of Escherichia coli F1-ATPase is a tightly bound but dissociable partial inhibitor of ATPase activity. The effects of epsilon on the enzyme were investigated by comparing the ATPase activity and aurovertin binding properties of the epsilon-depleted F1-ATPase and the epsilon-replete complex. Kinetic data of multisite ATP hydrolysis were analyzed to give the best fit for one, two, or three kinetic components. Each form of F1-ATPase contained a high-affinity component, with a Km near 20 microM and a velocity of approximately 1 unit/mg. Each also exhibited a component with a Km in the range of 0.2 mM. The velocity of this component was 25 units/mg for epsilon-depleted ATPase but only 4 units/mg for epsilon-replete enzyme. The epsilon-depleted enzyme also contained a very low affinity component not present in the epsilon-replete enzyme. In unisite hydrolysis studies, epsilon had no effect on the equilibrium between substrate ATP and product ADP.P1 at the active site but reduced the rate of product release 15-fold. These results suggest that epsilon subunit slows a conformational change that is required to reduce the affinity at the active site, allowing dissociation of product. It is suggested that inhibition of multisite hydrolysis by epsilon is also due to a reduced rate of product release. epsilon-depleted F1-ATPase showed little of no modulation of aurovertin fluorescence by added ADP and ATP. Aurovertin fluorescence titrations in buffer containing ethylenediaminetetraacetic acid (EDTA) revealed that epsilon-depleted enzyme had high affinity for aurovertin (Kd less than 0.1 microM) regardless of the presence of nucleotides.(ABSTRACT TRUNCATED AT 250 WORDS)     

The binding mechanism of the yeast F1-ATPase inhibitory peptide: role of catalytic intermediates and enzyme turnover

The mechanism of inhibition of yeast mitochondrial F(1)-ATPase by its natural regulatory peptide, IF1, was investigated by correlating the rate of inhibition by IF1 with the nucleotide occupancy of the catalytic sites. Nucleotide occupancy of the catalytic sites was probed by fluorescence quenching of a tryptophan, which was engineered in the catalytic site (beta-Y345W). Fluorescence quenching of a beta-Trp(345) indicates that the binding of MgADP to F(1) can be described as 3 binding sites with dissociation constants of K(d)(1) = 10 +/- 2 nm, K(d2) = 0.22 +/- 0.03 microm, and K(d3) = 16.3 +/- 0.2 microm. In addition, the ATPase activity of the beta-Trp(345) enzyme followed simple Michaelis-Menten kinetics with a corresponding K(m) of 55 microm. Values for the K(d) for MgATP were estimated and indicate that the K(m) (55 microm) for ATP hydrolysis corresponds to filling the third catalytic site on F(1). IF1 binds very slowly to F(1)-ATPase depleted of nucleotides and under unisite conditions. The rate of inhibition by IF1 increased with increasing concentration of MgATP to about 50 mum, but decreased thereafter. The rate of inhibition was half-maximal at 5 microm MgATP, which is 10-fold lower than the K(m) for ATPase. The variations of the rate of IF1 binding are related to changes in the conformation of the IF1 binding site during the catalytic reaction cycle of ATP hydrolysis. A model is proposed that suggests that IF1 binds rapidly, but loosely to F(1) with two or three catalytic sites filled, and is then locked in the enzyme during catalytic hydrolysis of ATP.     

The mechanism of stimulation of MgATPase activity of chloroplast F1-ATPase by non-catalytic adenine-nucleotide binding. Acceleration of the ATP-dependent release of inhibitory ADP from a catalytic site.

The presence of ATP at non-catalytic sites of the chloroplast F1-ATPase (CF1) eliminates a considerable lag in onset of enzyme activity that otherwise occurs in the presence of bicarbonate [Milgrom, Y. M., Ehler, L. & Boyer, P. D. (1991) J. Biol. Chem. 266, 11551-11558]. Sulfite is known to be much more effective than bicarbonate in stimulating ATPase activity CF1. Results reported here show that when assayed in the presence of sulfite, CF1, with some non-catalytic sites empty or filled with GT(D)P, is able to hydrolyze both ATP and GTP. Thus, the presence of adenine nucleotides at non-catalytic sites is not necessary for catalytic turnover of CF1. However, even though CF1 with empty non-catalytic sites shows a significant initial activity, the prior binding of adenine nucleotides at non-catalytic site(s) results in further activation of MgATPase and MgGTPase activities, even at relatively high sulfite and substrate concentrations. Although extensive activation of CF1 results from the presence of sulfite, with or without nucleotide binding at non-catalytic sites, the Km remains constant, at about 50 microM for MgATP and 400 microM for MgGTP. The results obtained show that the ATPase activity of CF1 is determined by the fraction of the active enzyme. The inactive CF1.ADP.Mg2+ formed during MgATP hydrolysis can be rapidly trapped by azide to provide a measure of the fraction of inactive enzyme. Increasing the concentration of sulfite increases the fraction of active CF1 in the assay medium. Measurements with radioactively labeled nucleotides show that the presence of ATP at non-catalytic sites promotes the ATP-dependent release of inhibitory ADP from a catalytic site. The activating effect of ATP binding at non-catalytic sites results from increasing the portion of CF1 in an active state during steady-state ATP hydrolysis.     

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.     

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].     

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)     

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.     

Stabilization of rat liver mitochondrial F1-adenosine triphosphatase during chloroform-induced solubilization.

1. Isolation of ATPase from rat liver submitochondrial particles by chloroform treatment requires the presence of ATP or ADP during enzyme solubilization. In the absence of adenine nucleotides the enzyme activity is very low although all protein components of F1-ATPase are released. The low concentrations of ATP or ADP required (5 microM) indicate that the high affinity nucleotide-binding sites are involved in enzyme stabilization. Other nucleotides tested (ITP, GTP, UTP, CTP) were found to be less effective. 2. Polyacrylamide gel electrophoresis and immunodiffusion in agar plates revealed that in the absence of adenine nucleotides a fraction of F1-ATPase released by chloroform treatment is split into fragments. The part of the dissociated enzyme molecule has a molecular weight identical with that of a beta-subunit of F1-ATPase. 3. Dissociation of the F1-ATPase molecule could also be prevented by aurovertin. 4. Crude F1-ATPase solubilized by chloroform treatment can be further purified by Sepharose 6B gel filtration. Specific ATPase activity of the purified enzyme was 90 mumol Pi/min per mg protein and the enzyme was composed of five protein subunits (alpha, beta, gamma, delta, epsilon) with molecular weights 58 000, 55 000, 28 000, 13 000 and 8000, respectively. 5. Chloroform-released F1-ATPase from rat liver mitochondria displayed immunochemical cross-reactivity with that isolated from beef heart mitochondria.     

The interaction of mitochondrial F1-ATPase with the natural ATPase inhibitor protein

The interaction of soluble mitochondrial ATPase from beef heart with the natural ATPase inhibitor was studied. It was found that the phosphorylation of small amounts of ADP by phosphoenolpyruvate and pyruvate kinase, and an ensuing catalytic cycle supports the binding of the inhibitor to the enzyme. The association of the inhibitor with F1-ATPase does not increase the content of ATP in the F1-ATPase-inhibitor complex. The inhibitor of catalytic activity bathophenanthroline-Fe2+ chelate prevents the interaction, while the association of the inhibitor with F1-ATPase is delayed if the reaction is carried out in 2H2O. The date indicate that a transient state involved in the catalytic cycle is the form of the enzyme that interacts with the inhibitor. The proton-motive force-induced dissociation of the inhibitor from particulate ATPase is prevented by bathophenanthroline-Fe2+ chelate and nitrobenzofurazan chloride, which indicates that a functional catalytic (beta) subunit is required for the proton-motive force-induced release of the inhibitor. The data suggest a direct involvement of catalytic (beta) subunit in the mechanism by which the F1-ATPase senses the proton-motive force.     

Properties of the Mg2+-induced low-affinity nucleotide binding site of (Na+ + K+)-activated ATPase

The Mg2+-induced low-affinity nucleotide binding by (Na+ + K+)-ATPase has been further investigated. Both heat treatment (50-65 degrees C) and treatment with N-ethylmaleimide reduce the binding capacity irreversibly without altering the Kd value. The rate constant of inactivation is about one-third of that for the high-affinity site and for the (Na+ + K+)-ATPase activity. Thermodynamic parameters (delta H degree and delta S degree) for the apparent affinity in the ATPase reaction (Km ATP) and for the true affinity in the binding of AdoPP[NH]P (Kd and Ki) differ greatly in sign and magnitude, indicating that one or more reaction steps following binding significantly contribute to the Km value, which thus is smaller than the Kd value. Ouabain does not affect the capacity of low-affinity nucleotide binding, but only increases the Kd value to an extent depending on the nucleotide used. GTP and CTP appear to be most sensitive, ATP and ADP intermediately sensitive and AdoPP[NH]P and AMP least sensitive to ouabain. Ouabain reduces the high-affinity nucleotide binding capacity without affecting the Kd value. The nucleotide specificity of the low-affinity binding site is the same for binding (competition with AdoPP[NH]P) and for the ATPase activity (competition with ATP): AdoPP[NH]P greater than ATP greater than ADP greater than AMP. The low-affinity nucleotide binding capacity is preserved in the ouabain-stabilized phosphorylated state, and the Kd value is not increased more than by ouabain alone. It is inferred that the low-affinity site is located on the enzyme, more specifically its alpha-subunit, and not on the surrounding phospholipids. It is situated outside the phosphorylation centre. The possible functional role of the low-affinity binding is discussed.     

Reversible binding of Pi by beef heart mitochondrial adenosine triphosphatase.

Beef heart mitochondrial ATPase (F1) exhibited a single binding site for Pi. The interaction with Pi was reversible, partially dependent on the presence of divalent metal ions, and characterized by a dissociation constant at pH 7.5 of 80 micronM. A variety of substances known to influence oxidative phosphorylation or the activity of the soluble ATPase (F1) also influenced Pi binding by the enzyme. Thus aurovertin, an inhibitor of oxidative phosphorylation, which was bound tightly by F1 and inhibited ATPase activity, enhanced Pi binding via a 4-fold increase in the affinity of the enzyme for Pi (KD = 20 micronM) but did not alter binding stoichiometry. Anions such as SO4(2-), SO3(2-), chromate, and 2,4-dinitrophenolate, which stimulated ATPase activity of F1, also enhanced Pi binding. Inhibitors of ATPase activity such as nickel/bathophenanthroline and the protein ATPase inhibitor of Pullman and Monroy (Pullman, M. E., and Monroy, G. C. (1963) J. Biol. Chem. 238, 3762-3769) inhibited Pi binding. The adenine nucleotides ADP, ATP, and the ATP analog adenylyl imidodiphosphate as well as the Pi analog arsenate, also inhibited Pi binding. The observations suggest that the Pi binding site was located in or near an adenine nucleotide binding site on the molecule.     

Activation and inhibition of the Escherichia coli F1-ATPase by monoclonal antibodies which recognize the epsilon subunit

The properties of two monoclonal antibodies which recognize the epsilon subunit of Escherichia coli F1-ATPase were studied in detail. The epsilon subunit is a tightly bound but dissociable inhibitor of the ATPase activity of soluble F1-ATPase. Antibody epsilon-1 binds free epsilon with a dissociation constant of 2.4 nM but cannot bind epsilon when it is associated with F1-ATPase. Likewise epsilon cannot associate with F1-ATPase in the presence of high concentrations of epsilon-1. Thus epsilon-1 activates F1-ATPase which contains the epsilon subunit, and prevents added epsilon from inhibiting the enzyme. Epsilon-1 cannot bind to membrane-bound F1-ATPase. The epsilon-4 antibody binds free epsilon with a dissociation constant of 26 nM. Epsilon-4 can bind to the F1-ATPase complex, but, like epsilon-1, it reverses the inhibition of F1-ATPase by the epsilon subunit. The epsilon subunit remains crosslinkable to both the beta and gamma subunits in the presence of epsilon-4, indicating that it is not grossly displaced from its normal position by the antibody. Presumably the activation arises from more subtle conformational effects. Antibodies epsilon-4 and delta-2, which recognizes the delta subunit, both bind to F1F0 in E. coli membrane vesicles, indicating that these subunits are substantially exposed in the membrane-bound complex. Epsilon-4 inhibits the ATPase activity of the membrane-bound enzyme by about 50%, and Fab prepared from epsilon-4 inhibits by about 40%. This inhibition is not associated with any substantial change in the major apparent Km for ATP. These results suggest that inhibition of membrane-bound F1-ATPase arises from steric effects of the antibody.