Characteristics of adenylyl imidodiphosphate- and ADP-binding sites insoluble and particulate mitochondrial ATPase. Studies with methanol

The characteristics of the binding sites for ADP and adenylyl imidodiphosphate have been studied in soluble and particulate F1-ATPase from bovine heart mitochondria. ADP, but not electrochemical gradients, removes the inhibitory effect of adenylyl imidodiphosphate on ATPase activity in coupled submitochondrial particles. In soluble F1-ATPase, methanol at 20% concentration diminishes the ability of ATP and adenylyl imidodiphosphate to inhibit ATP and ITP hydrolysis; these findings suggest that ADP and adenylyl imidodiphosphate inhibit hydrolysis by acting on the same site. Methanol at 20% stimulates the hydrolytic activity of soluble F1-ATPase, but fails to stimulate significantly the activity of the particulate enzyme, even though in particulate F1-ATPase methanol markedly diminishes the inhibiting action of added ADP and adenylyl imidodiphosphate on ATP and ITP hydrolysis. This is consistent with the idea that in the particulate system there are two inhibitory binding sites for ADP, one accessible to methanol, and another which is inaccessible to methanol; the latter is transitorily occupied by ADP arising from ATP hydrolysis. Indeed, experiments on the effect of ADP in ITP hydrolysis by submitochondrial particles show the existence of two ADP inhibitory sites.     

Purification and properties of the adenosine triphosphatase released from the liver mitochondrial membrane by chloroform

1. Soluble ATPase (adenosine triphosphatase) activity is released when rat liver submitochondrial particles are shaken with chloroform, provided that ATP or glycerol is present in the suspending medium. The extraction is very rapid and appears to be complete. 2. The ATPase of the chloroform extract is about 50% pure and can be readily purified to a specific activity of 60-70mumol/min per mg of protein by (NH(4))(2)SO(4) fractionation and column chromatography on Sephadex G-200. 3. The particulate and soluble ATPases have many similar properties, including their K(m) values for ATP, activation by various metal ions, hydrolytic activity with other nucleotides and stimulation by bicarbonate ions. 4. Unlike the particulate enzyme, the soluble enzyme is cold-labile and insensitive to oligomycin. 5. The molecular weight indicated by the mobility of the soluble ATPase on Sepharose 6B is 360000. 6. The soluble ATPase combines very readily with liver submitochondrial particles depleted of ATPase by salt extraction, and oligomycin-sensitivity is restored. Very little recombination of the enzyme occurs with chloroform-extracted particles. 7. The soluble enzyme contains orcinol-reactive material, suggesting that it may be a glycoprotein. The carbohydrate content was estimated to be 1-2% by weight. 8. It is concluded that the liver ATPase obtained by the chloroform extraction method of Beechey, Hubbard, Linnett, Mitchell & Munn [(1975) Biochem. J.148, 533-537] is similar to other preparations described previously and that this method is superior in simplicity and speed.     

ATPase Activity of Pea Cotyledon Submitochondrial Particles: ACTIVATION, SUBSTRATE SPECIFICITY, AND ANION EFFECTS

Submitochondrial particles freshly prepared by sonication from pea cotyledon mitochondria showed low ATPase activity. Activity increased 20-fold on exposure to trypsin. The pea cotyledon submitochondrial particle ATPase was also activated by “aging” in vitro. At pH 7.0 addition of 1 millimolar ATP prevented the activation. ATPase of freshly prepared pea cotyledon submitochondrial particles had a substrate specificity similar to that of the soluble ATPase from pea cotyledon mitochondria, with GTPase > ATPase. “Aged” or trypsin-treated particles showed equal activity with the two substrates. NaCl and NaHCO₃, which stimulate the ATPase but not the GTPase activity of the soluble pea enzyme, were stimulatory to both the ATPase and GTPase activities of freshly prepared submitochondrial particles. However, they were stimulatory only to the ATPase activity of trypsin-treated or “aged” submitochondrial particles. In contrast, the ATPase activity of rat liver submitochondrial particles was stimulated by HCO₃⁻, but inhibited by Cl⁻, indicating that Cl⁻ stimulation is a distinguishing property of the pea mitochondrial ATPase complex.     

Kinetics of interaction of adenosine diphosphate and adenosine triphosphate with adenosine triphosphatase of bovine heart submitochondrial particles.

The short preincubation of submitochondrial particles with low concentrations of ADP in the presence of Mg2+ results in a complete loss of their ATPase and inosine triphosphatase activities. Other nucleoside diphosphates (IDP and GDP) do not affect the ATPase activity. The ADP-inhibited ATPase can be activated in a time-dependent manner by treatment of submitochondrial particles with the enzyme converting ADP into ATP (phosphoenolpyruvate plus pyruvate kinase). The activaton is a first-order reaction with rate constant 0.2 min-1 at 25 degrees C. The rate constant of activation is increased in the presence of ATP up to 2 min-1, and this increase shows saturation kinetics with Km value equal to that for ATPase reaction itself (10(-4) M at 25 degrees C at pH 8.0). The experimental results obtained are consistent with the model where two alternative pathways of ADP dissociation from the inhibitory site of ATPase exist; one is spontaneous dissociation and the second is ATP-dependent dissociation through the formation of the ternary complex between ADP, the enzyme and ATP. ADP-induced inactivation and ATP-dependent activation of ATPase activity of submitochondrial particles is accompanied by the same directed change of their ability to catalyse the ATP-dependent reverse electron transport from succinate to NAD+. The possible implication of the model suggested is discussed in terms of functional role of the inhibitory high-affinity binding site for ADP in the mitochondrial ATPase.     

Mixed anhydrides of nucleotides and mesitylenecarboxylic acid as new specific inhibitors of mitochondrial adenosien triphosphatase.

Mixed anhydrides of nucleoside triphosphates and mesitylenecarboxylic acid inhibit soluble mitochondrial ATPase (adenosine triphosphatase), but do not inhibit ATPase of submitochondrial particles. Inhibition of soluble mitochondrial ATPase by the mixed anhydride of epsilon-ATP and mesitylenecarboxylic acid is followed by the covalent binding of one nucleotide residue to a molecule of the protein. It is suggested that this covalent binding occurs in the catalytic site of the mitochondrial ATPase. The mixed anhydride of ADP and mesitylenecarboxylic acid inhibits the ATPase activity of submitochondrial particles and has no effect on the activity of soluble mitochondrial ATPase. After separation of the submitochondrial particles from the mixed anhydride of ADP and mesitylenecarboxylic acid, their ATPase activity is restored to its original value (half-time of reactivation 3--4 min). Incubation of submitochondrial particles or soluble mitochondrial ATPase with the mixed anhydride of ADP and mesitylenecarboxylic acid results in AMP formation.     

3' Esters of ADP as energy-transfer inhibitors and probes of the catalytic site of oxidative phosphorylation.

1. A large series of 3' esters of ADP has been synthesized. Several of these can serve as photoaffinity labels; others exhibit fluorescent properties. The corresponding AMP and ATP derivatives have also been synthesized in some cases. 2. The influence of the 3'-O-acyl nucleotides on energy-linked functions of beef-heart submitochondrial particles has been investigated. The following results were obtained. a) 3'Esters of ADP are powerful and highly specific inhibitors of oxidative phosphorylation. The inhibition is competitive to ADP and Ki values as low as 0.05 microM, for the 3'-O-(1)naphthoyl ester of ADP, could be observed. b) The inhibition of oxidative phosphorylation by 3' esters of ADP appears to be non-competitive versus inorganic phosphate. c) The nucleotide analogs are not phosphorylated themselves. The corresponding ATP analogs can not drive energy-linked process. d) The 3' esters of AMP are ineffective as inhibitors, whereas the ATP derivatives are only comparatively weak inhibitors. e) Uncoupled or solubilized ATPase is almost two orders of magnitude less sensitive against inhibition by 3' esters than coupled systems. The analogs exert maximal inhibition specifically in systems involving an 'energized' state of the coupling device. f) Azido-group-bearing analogs can be used for irreversible photoinactivation of the coupling ATPase. Photoinactivation also is most efficient when carried out with 'energized' particles. g) The inhibitory properties are similar also in ATP-driven NAD+ reduction by succinate, and in the uncoupler-sensitive ATP in equilibrium with Pi exchange. The required concentrations for half-maximal inhibition are somewhat higher than in oxidative phosphorylation, but lower than with uncoupled ATPase. 3. From molecular models, from substituent properties, and from the conditions required for inhibition it is concluded that these highly effective analogs of ADP may act as conformation-specific probes at the catalytic site of oxidative phosphorylation. The results are interpreted in terms of a model suggesting that, in the process of ATP synthesis, a hydrophobic cavity on the enzyme is exposed only in the energized state, accepting the large 3' substituent. The substituent is assumed to inhibit phosphoryl transfer and/or conformational transitions inherent in the process of ADP phosphorylation by steric hinderance.     

Excessive ATP hydrolysis in ischemic myocardium by mitochondrial F1F0-ATPase: effect of selective pharmacological inhibition of mitochondrial ATPase hydrolase activity

Mitochondrial F(1)F(0)-ATPase normally synthesizes ATP in the heart, but under ischemic conditions this enzyme paradoxically causes ATP hydrolysis. Nonselective inhibitors of this enzyme (aurovertin, oligomycin) inhibit ATP synthesis in normal tissue but also inhibit ATP hydrolysis in ischemic myocardium. We characterized the profile of aurovertin and oligomycin in ischemic and nonischemic rat myocardium and compared this with the profile of BMS-199264, which only inhibits F(1)F(0)-ATP hydrolase activity. In isolated rat hearts, aurovertin (1-10 microM) and oligomycin (10 microM), at concentrations inhibiting ATPase activity, reduced ATP concentration and contractile function in the nonischemic heart but significantly reduced the rate of ATP depletion during ischemia. They also inhibited recovery of reperfusion ATP and contractile function, consistent with nonselective F(1)F(0)-ATPase inhibitory activity, which suggests that upon reperfusion, the hydrolase activity switches back to ATP synthesis. BMS-199264 inhibits F(1)F(0) hydrolase activity in submitochondrial particles with no effect on ATP synthase activity. BMS-199264 (1-10 microM) conserved ATP in rat hearts during ischemia while having no effect on preischemic contractile function or ATP concentration. Reperfusion ATP levels were replenished faster and necrosis was reduced by BMS-199264. ATP hydrolase activity ex vivo was selectively inhibited by BMS-199264. Therefore, excessive ATP hydrolysis by F(1)F(0)-ATPase contributes to the decline in cardiac energy reserve during ischemia and selective inhibition of ATP hydrolase activity can protect ischemic myocardium.     

Nickel(II) transport in human blood serum. Studies of nickel(II) binding to human albumin and to native-sequence peptide, and ternary-complex formation with l-histidine

1. The initial rapid phase of ATP hydrolysis by bovine heart submitochondrial particles or by soluble F1-ATPase is insensitive to anion activation (sulphite) or inhibition (azide). 2. The second slow phase of ATP hydrolysis is hyperbolically inhibited by azide (Ki approximately 10(-5) M); the inosine triphosphatase activity of submitochondrial particles or F1-ATPase is insensitive to azide or sulphite. 3. The rate of interconversion between rapid azide-insensitive and slow azide-sensitive phases of ATP hydrolysis does not depend on azide concentration, but strongly depends on ATP concentration. 4. Sulphite prevents the interconversion of the rapid initial phase of the reaction into the slower second phase, and also prevents and slowly reverses the inhibition by azide. 5. The presence of sulphite in the mixture when ADP reacts with ATPase of submitochondrial particles changes the pattern of the following activation process. 6. Azide blocks the activation of ATP-inhibited ATPase of submitochondrial particles by phosphoenolpyruvate and pyruvate kinase. 7. The results obtained suggest that the inhibiting effect of azide on mitochondrial ATPase is due to stabilization of inactive E*.ADP complex formed during ATP hydrolysis; the activation of ATPase by sulphite is also realized through the equilibrium between intermediate active E.ADP complex and inactive E*.ADP complex.     

The mitochondrial adenosine triphosphatase of Acanthamoeba castellanii. Partial characterization and changes in activity during exponential growth

1. The mitochondrial adenosine triphosphatase (ATPase) of Acanthamoeba castellanii is Mg2+-requiring (optimum cation: ATP ratio of 1.5) and has two pH optima of activity (at pH 6.6 and 8.1). 2. ATPase activity of submitochondrial particles is effectively inhibited by twelve different inhibitors of energy conservation suggesting similarities in inhibitor-binding sites to other previously characterized complexes. 3. Gel filtration by passage through Sephadex G-50 increases ATPase activity of submitochondrial particles between 1.5 and 3.5 fold indicating the presence of a low molecular weight inhibitor protein. 4. After removal of the inhibitor protein, sensitivity to inhibitors of energy conservation decreases by between 1.5 and 14 fold. Crude F1-inhibitor preparations from A. castellanii, Schizosaccharomyces pombe, Tetrahymena pyriformis and bovine heart also inhibit ATPase activity. 5. Large variations in ATPase activity, F1-inhibitor protein activity, and amounts of immunologically-determined ATPase protein were observed during exponential growth, and the correlation between changes in these measurements is discussed. 6. The results are also discussed highlighting the similarities between the mitochondrial ATPase of A. castellanii and other mitochondrial ATPases.     

The use of arylazido-beta-alanyl-ATP as a photoaffinity label for the isolated and membrane-bound mitochondrial ATPase complex.

The effects of a photoaffinity derivate of ATP, arylazido-beta-alanyl-ATP, 3'-O-(3-[N-(4-azido-2-nitrophenyl)amino]propionyl) adenosine 5'-triphosphate, on submitochondrial particles and the partially purified ATPase complex of beef heart mitochondria have been investigated. In the absence of light the ATP analogue has been found to be a substrate for the E132PA1P1-ATP exchange reaction of submitochondrial particles. When photoirradiated in the presence of arylazido-beta-alanyl-ATP, the ATPase activity and the the the [32P]Pi-ATP exchange reaction are inhibited maximally 80%. Arylazido-beta-alanyl-ATP following photolysis is a noncompetitive inhibitor with respect to ATP while arylazido-beta-alanine, the azido-containing adjunct of the ATP analogue, has no inhibitory effect under the same conditions. The inactivating effect of arylazido-beta-alanyl-ATP is prevented in part by the presence of ATP, or ADP and pyrophosphate. Photolabeling produces a covalent binding of the derivative with the F1ATPase being the major protein labeled. The binding of 0.22 mumol of arylazido-beta-alanyl-ATP/mg of mitochondrial protein is associated with a maximal inhibitory effect. The ATPase activity of the partially purified ATPase complex is also sensitive to photoirradiation in the presence of arylazido-beta-alanyl-ATP. When the ATPase complex is associated with liposomes there is an increase in the specific ATPase activity with a 10-fold increase in Vmax and a 4-fold decrease in KmATP associated with a parallel increase in the apparent affinity and maximal inhibitory effect of the arylazido-beta-alanyl-ATP. The photoinhibition of the ATPase complex in the presence of arylazido-beta-alanyl-ATP results in covalent binding of 1.6 mumol of arylazido-beta-alanyl-ATP/mg of protein. The alpha and beta subunits are the only components of the ATPase complex labeled by the [3H]arylazido-beta-alanyl-ATP. The relationship between the arylazido-beta-alanyl-ATP-labeled sites and the nucleotide binding sites on the mitochondrial ATPase is discussed.     

On the mechanism of action of alkylguanidines in oxidative phosphorylation: their action on soluble F1.

Octylguanidine inhibits the adenosine triphosphatase (ATPase) activity of bovine heart submitochondrial particles and soluble F₁. The characteristics of the inhibition as a function of octylguanidine and Mg²⁺ concentrations and pH are very similar in submitochondrial particles and soluble F₁. Only those guanidines that possess an alkyl chain of more than six carbons inhibit the ATPase activity of submitochondrial particles and F₁. The inhibiting action of octylguanidine on F₁ is fully reversible. Octylguanidine prevents the cold-induced inactivation of F₁ at concentrations similar to those that inhibit ATPase activity. Guanidines that inhibit ATPase activity also prevent the cold-induced inactivations of F₁.     

Substrate specificity of soluble mitochondrial ATPase]

The parameters of the hydrolysis of ATP and several analogs by soluble mitochondrial ATPase were determined. Vmax of the reaction decreases within the range: 2'-desoxy-ATP greater than ATP greater than etheno-ATP greater than GTP greater than 3'-O-methylATP greater than UTP. ATP, 2'-desoxypATP, 3'O-methyl-ATP, GTP, and etheno-ATP are hydrolysed by soluble mitochondrial ATPase with close Km(app) values. CTP is not hydrolysed by the enzyme and does not inhibit the ATPase reaction at a concentration of 10(-2) M. Nucleoside triphosphate derivatives with an "open" ribose cycle 9-[1',5'-dihydroxy-4-(S)-hydroxymethyl-3'-oxapent-2' (R)-yl]adenyl-5'-triphosphate, and 1-[1',5'-dihydroxy-4'-(S)-hydroxymethyl-3'-oxapent-2'(R)-yl[cytosine-5'-triphosphate are effective inhibitors of ATPase (Ki approximately 5.10(-5)M). Mitochondrial ATPase binds the ATP analogs that have hydrocarbon radicals-(CH2)2-, -(CH2)3-, and (CH2)4- instead of the ribose residues: 9-(2'hydroxyethyl)adenyl-2'-triphosphate, 9-(3'-hydroxypropyl)-adenine-3'-triphosphate, and 9-(4'-hydroxybutyl)adenine-4'-triphosphyl)adenine-4'-triphosphate were not hydrolysed by the enzyme, although they inbibit the ATPase reaction (Ki 2.10(-4)M). 9-(2'-hydroxyethyl)adenine-2'-triphosphate is hydrolysed by ATPase eight times more slowly than ATP. It is suggested that the hydrolysis of the substrates of mitochondrial ATPase is- preceded by the binding of the substrates in a tense conformation in the active site of the enzyme.     

Inhibitors of the slow stage of proton transfer in the link connecting respiration with mitochondrial phosphorylation]

Electrophilic agents--derivatives of carbonic acids--are found to inhibit respiration, ATP synthesis and reverse electrone transport in intact mitochondria. The inhibition of respiration and ATPase was observed in intact mitochondria at 3 and 3u states (by Chance). Inhibitors concentrations, which caused 50% inhibition, were approximately the same. Sharp decrease of the effect of electrophilic inhibitors on respiration and ATPase activity in mitochondria and submitochondrial particles with substantially impaired coupling system was observed. The following conclusions are drawn on the basis of the data obtained: 1) electrophilic inhibitor attack the coupling site of respiration and ATP synthesis in mitochondria; 2) the reaction of the proton transport from the respiration proton pump to ATP synthetase is one of the slowest steps of the process of ATP-synthesis in mitochondria. A scheme of working the coupling system is suggested which includes the step of proton lateral diffusion.     

Esterase activity of the mitochondria oligomycin-sensitive ATPase complex]

It was found that mitochondrial oligomycin-sensitive ATPase (OS-ATPase) possesses the esterase activity with respect to some carboxylic acid esters with phenols and arylalcane alcohols. The substrate specificity of the esterase found was studied. The effects of some inhibitors and activators of ATPase on the enzyme activity were demonstrated. It was found that ADP inhibits the enzyme from submitochondrial particles containing factor F1 and does not inhibit the enzyme from the particles devoid of this factor. The data obtained suggest that esterase is localized in the hydrophobic part of the oligomycin-sensitive ATPase complex and are indicative of the functional interrelationship between the esterase and ATPase activities.     

On the mechanism of action of alkylguanidines on oxidative phosphorylation in mitochondria.

The effect of octylguanidine and oligomycin on the oxygen uptake of rat liver mitochondria and on the ATPase activity of "sonic" submitochondrial particles has been studied. 1. Octylguanidine inhibits state 3 respiration with glutamate-malate and succinate as substrates, but much lower concentrations are required to inhibit oxygen uptake with the former substrates. State 4 respiration is unaffected by octylguanidine. 2. The titration-curve for the octylguanidine inhibition of glutamate-malate oxidation is hyperbolic and apparently biphasic, half-maximal inhibition is obtained at 30 muM octylguanidine. The octylguanidine-curve for inhibition of succinate oxidation is sigmoid with half-maximal inhibition at about 250 muM. 3. Octylguanidine and oligomycin show additive inhibitory action on state 3 respiration with both glutamate plus malage and succinate as respiratory substrates. 4. Concentrations of oligomycin or octylguanidine, which added separately are ineffective on state 3 respiration, become inhibitory when the two inhibitors are added together. 5. Octylguanidine inhibits the ATPase activity of sonic submitochondrial particles with a hyperbolic titration-curve analogous to that obtained for oligomycin inhibition. The inhibitory actions of octylguanidine and oligomycin on the ATPase activity are additive. 6. It is concluded that octylguanidine acts directly on the ATPase complex and that its binding at the action site is mutually exclusive with the binding of oligomycin. A kinetic explanation is given for the reported higher sensitivity of site I phosphorylation to octylguanidine.     

Tightly bound nucleotides of the energy-transducing ATPase, and their role in oxidative phosphorylation. II. The beef heart mitochondrial system.

1. Beef heart mitochondrial ATPase, in both the membrane-bound and isolated form, contains tightly bound ATP and ADP. Each mol of ATPase contains about 2.2 mol ATP and 1.3 mol ADP. 2. In the absence of ATPase activity, these nucleotides exchange only slowly with nucleotides in solution. The exchange rate is increased during coupled ATPase activity, but not when the ATPase is uncoupled. 3. Oligomycin and dicyclohexylcarbodiimide inhibit exchange of the bound nucleotides, as does the ATPase inhibitor protein, although in each case some residual exchange occurs. Aurovertin, although inhibiting phosphorylation, does not inhibit the exchange. This is discussed in terms of the reversibility of these inhibitors. 4. The stimulation of exchange seen during coupled ATPase activity requires energisation of the ATPase molecule. Using the exchange reaction as a probe of energisation, it is deduced that energy can be transferred between different ATPase molecules. 5. It is proposed that coupled ATPase activity and phosphorylation in submitochondrial particles involve the tight nucleotide binding sites and the (weak) ATPase site, while uncoupled ATPase activity involves only the weak site.     

Three adenine nucleotide binding sites in F1-F0 mitochondrial ATPase as revealed by presteady-state and steady-state kinetics of ATP hydrolysis. Evidence for two inhibitory ADP-specific noncatalytic sites

Preincubation of submitochondrial particles with ADP in the presence of Mg2+ results in the complete inhibition of ATPase which is slowly reactivated in the assay mixture containing ATP and the ATP regenerating system. Significantly, the rate of activation increases as the concentration of ADP in the preincubation mixture rises from 1 microM to 20 microM and reaches a constant value at higher ADP concentrations. The first-order rate constant for the activation process in the assay mixture is ATP-dependent at any level of inhibitory ADP. The data obtained strongly suggest that two ADP-specific inhibitory sites and one ATP-specific hydrolytic site are present in F1-F0 ATPase. Taking into account the (3 alpha.3 beta).gamma.delta.epsilon structure of F1, it is concluded that the synchronous discharge of ADP from two inhibitory sites during the activation occurs after ATP binds to the ATPase catalytic site.     

Characterization of the interaction of parathyroid hormone with the mitochondrial ATPase

Parathyroid hormone (PTH) has been shown to bind specifically to the beta subunit of the mitochondrial ATPase on nitrocellulose blots. We have now examined this interaction further, using intact mitochondria, submitochondrial particles, and the purified F1 ATPase. With intact mitochondria, 1 microM concentrations of PTH and its biologically active 1-34 fragment activate the ATPase about 3-fold. This effect was reduced to a 1.4-fold activation with 3-34 and 7-34 fragments of the hormone, and oxidized PTH gave no detectable activity. Activation could only be observed below pH 7. PTH had no significant effect on the activity of the purified enzyme or on submitochondrial particles. However, specific binding of an iodinated PTH analog, [Nle 8,18-Tyr 34] bPTH (1-34) amide, was found with submitochondrial particles and the purified ATPase. Binding affinity with the purified enzyme was about 10(-3) that of the plasma membrane receptor, and the molar stoichiometry was close to 1:1 (PTH:intact enzyme). With submitochondrial particles the affinity was about 10-fold higher than with the purified enzyme. This binding was further examined with PTH derivatives and fragments, and compared to that seen in the plasma membrane receptor. Oxidation of methionine 18 in PTH reduced the affinity about 50%, oxidation of methionine 8 reduced the affinity 95%, and oxidation of both methionines further decreased affinity in both membranes and submitochondrial particles. However, when compared to the native hormone, the 3-34 and 7-34 PTH fragments had much higher affinity for the submitochondrial particles than for the plasma membranes. PTH also reduced chemical crosslinking of the ATP analog, p-fluorosulfonyl benzoyl 5'-adenosine, to the alpha subunit of this enzyme, but did not alter labeling of the enzyme with 3'-O-(4'-benzoyl) benzoyl ATP, suggesting that the hormone binds near a regulatory nucleotide binding site. Direct chemical crosslinking of PTH to the beta-subunit of the enzyme was attained with a cleavable, photoactivate crosslinker, sulfosuccinimidyl 2-(p-azidosalicylamido) ethyl-1,3-dithiopropionate. The crosslinked protein was cleaved with cyanogen bromide and the labeled fragments were sequenced. The labeled fragments were found to be segments of the protein which have previously been implicated as being close to the noncatalytic ATP binding sites.     

Tightly bound nucleotides of the energy-transducing ATPase,and their role in oxidative phosphorylation. II. The beef heart mitochondrial system

1. Beef heart mitochondrial ATPase, in both the membrane-bound and isolated form, contains tightly bound ATP and ADP. Each mol of ATPase contains about 2.2 mol ATP and 1.3 mol ADP.2. In the absence of ATPase activity, these nucleotides exchange only slowly with nucleotides in solution. The exchange rate is increased during coupled ATPase activity, but not when the ATPase is uncoupled.3. Oligomycin and dicyclohexylcarbodiimide inhibit exchange of the bound nucleotides, as does the ATPase inhibitor protein, although in each case some residual exchange occurs. Aurovertin, although inhibiting phosphorylation, does not inhibit the exchange. This is discussed in terms of the reversibility of these inhibitors.4. The stimulation of exchange seen during coupled ATPase activity requires energisation of the ATPase molecule. Using the exchange reaction as a probe of energisation, it is deduced that energy can be transferred between different ATPase molecules.5. It is proposed that coupled ATPase activity and phosphorylation in submitochondrial particles involve the tight nucleotide binding sites and the (weak) ATPase site, while uncoupled ATPase activity involves only the weak site.     

Inhibition of mitochondrial F1 ATPase and sarcoplasmic reticulum ATPase by hydrophobic molecules

The hydrophobic nature of the active site of two energy-transducing ATPases was explored by comparing interactions between Pi and each of three hydrophobic drugs in the absence and presence of organic solvents. The drugs tested were the Fe . bathophenanthroline complex and the anticalmodulin drugs, calmidazolium and trifluoperazine. All inhibit the Pi in equilibrium with ATP exchange reaction catalyzed by submitochondrial particles and the ATPase activity of both submitochondrial particles and soluble F1 ATPase. The inhibition by the three drugs is reversed by either raising the Pi concentration or by adding organic solvent (dimethylsulfoxide, ethyleneglycol or methanol) to the medium. The inhibition of the Pi in equilibrium with ATP exchange by trifluoperazine becomes more pronounced when the electrochemical proton gradient formed across the membrane of the submitochondrial particles is decreased by the addition to the medium of the proton ionophore carbonylcyanide p-trifluoromethoxyphenylhydrazone. The ATPase activity and the Ca2+ uptake by sarcoplasmic reticulum vesicles are inhibited by the Fe . bathophenanthroline complex, calmidazolium and trifluoperazine. Phosphorylation of the ATPases by Pi, synthesis of ATP from ADP and Pi and the fast efflux of Ca2+ observed during reversal of the Ca2+ pump are inhibited by the three drugs. The inhibition is reversed by raising the concentration of Pi or dimethylsulfoxide. The three drugs tested appear to compete with Pi for a common binding site on the Ca2+-ATPase. The data presented are interpreted according to the proposal that the catalytic site of an enzyme involved in energy transduction undergoes a hydrophobic-hydrophilic transition during the catalytic cycle.