Exploring sites on mitochondrial ATPase for catalysis,regulation, and inhibition

Evidence is presented that mitochondrial ATPase has two types of sites that bind adenine nucleotides. The catalytic site, C, binds the substrates ATP, GTP, or ITP and the inhibitor guanylyl imidodiphosphate (GMP-PNP). A second type of site, R, binds ATP, ADP, adenylyl imidodiphosphate (AMP-PNP), and the chromium complexes of ATP or ADP. All of these substances binding to the R site inhibit the hydrolysis of ATP in a competitive manner; their inhibition of hydrolysis of ITP and GTP is noncompetitive. GMP-PNP inhibits oxidative phosphorylation in submitochondrial particles but AMP-PNP does not. The localization on mitochondrial membranes of sites for the binding of various antibiotics that inhibit oxidative phosphorylation is discussed.     

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.     

Adenosine triphosphatase from rat liver mitochondria: separate sites involved in ATP hydrolysis and in the reversible, high affinity binding of ADP.

Homogeneous ATPase from rat liver mitochondria binds one mole of ADP per mole of enzyme reversibly, and with high affinity (K_D = 1–2 μM). The high affinity binding site is highly specific for ADP and dADP. AMP does not bind. Agents which inhibit ATP hydrolysis have little inhibitory effect on the high affinity binding of ADP. These agents include adenylyl imidodiphosphate (AMP-PNP), azide, sucrose, and the divalent cation Mg⁺⁺. AMP-PNP inhibits ATPase activity in phosphorylating membrane preparations of rat liver mitochondria by about 90 percent, but is without effect on ATP synthesis. These results are consistent with the view that the purified soluble, and the membrane-bound ATPase of rat liver mitochondria contain separate sites involved in ATP hydrolysis and in the reversible, high affinity binding of ADP.     

Kinetic mechanism of mitochondrial adenosine triphosphatase. ADP-specific inhibition as revealed by the steady-state kinetics.

1. A substantial increase of the initial rate of ATP hydrolysis was observed after preincubation of bovine heart submitochondrial particles with phosphoenolpyruvate and pyruvate kinase. 2. The activation was accompanied by an increase of Vmax, without change of Km for ATP. 3. The activated particles catalysed the biphasic hydrolysis of ATP in the presence of an ATP-regenerating system; the initial rapid phase was followed by a second, slower, phase in a time-dependent fashion. 4. The higher the ATP concentration used as a substrate, the higher is the rate of transition between these two phases. 5. The particles catalysed the hydrolysis of ITP with a lag phase; after preincubation with phosphoenolpyruvate and pyruvate kinase, ITP was hydrolysed at a constant rate. 6. Qualitatively the same phenomena were observed when soluble mitochondrial ATPase (F1-ATPase) prepared by the conventional method in the presence of ATP was used as nucleotide triphosphatase. 7. A kinetic scheme is proposed, in which the intermediate active enzyme-product complex (E.ADP) formed during ATP hydrolysis is in slow equilibrium with the inactive E*.ADP complex forming as a result of dislocation of ADP from the active site of ATPase to the other site, which is not in rapid equilibrium with the surrounding medium.     

Mitochondrial H+-ATPase activation by an amine oxide detergent

Lauryl dimethylamine oxide activates ATP hydrolysis by the mitochondrial H+-ATPase. Activation is observed in systems with a high content of inhibitor protein as described by Pullman and Monroy (Pullman, M.E., and Monroy, G.C. (1963) J. Biol. Chem. 238, 3762-3769), i.e. Mg-ATP submitochondrial particles and a Triton X-100-solubilized H+-ATPase from the same particles. Detergent activation of ATP hydrolysis is also present in inhibitor-reconstituted systems, i.e. submitochondrial particles, Triton extracts, and soluble F1-ATPase. In submitochondrial particles depleted of inhibitor protein, lauryl dimethylamine oxide induced a biphasic response which is characterized by a drop-in activity induced by relatively low concentrations of LDAO; at higher concentrations the detergent activates to an extent never greater than the initial activity. In inhibitor protein-depleted oligomycin-sensitive Triton extracts, lauryl dimethylamine oxide stimulates ATP hydrolysis to very high values (30 mumol min-1 mg-1). These findings suggest that in addition to the inhibitor protein ATP hydrolysis is controlled by other subunit interactions.     

Effect of citreoviridin and isocitreoviridin on beef heart mitochondrial ATPase

Citreoviridin is a toxic metabolite from fungus that has been shown to be an inhibitor of mitochondrial F1-ATPases. Studies of citreoviridin, however, have been compromised by the light-dependent isomerization that it undergoes. The isomerization is a potential source of extensive variability in the studies, if citreoviridin and isocitreoviridin have different kinetic effects and binding properties. Both citreoviridin and isocitreoviridin recently have been purified and have been shown to be stable in the dark. Using the purified isomers, the effects of both citreoviridin and isocitreoviridin on soluble and membrane-bound beef heart mitochondrial F1-ATPase activity were investigated. It was found that citreoviridin was an uncompetitive inhibitor of ATP hydrolysis, and a non-competitive inhibitor of ITP hydrolysis catalyzed by soluble F1-ATPase. Isocitreoviridin had no effect on the hydrolysis of either of the triphosphates catalyzed by soluble F1-ATPase. The inhibition constant, Ki for citreoviridin was determined as 4.5 microM for ATP hydrolysis. The inhibition constants Kii and Kis for ITP hydrolysis were determined as 4.3 and 1.03 microM, respectively. Citreoviridin was an uncompetitive inhibitor of ATP hydrolysis and a noncompetitive inhibitor of ATP synthesis catalyzed by membrane-bound F1-ATPase. The inhibition constant, Ki, for ATP hydrolysis was around 4 microM. For ATP synthesis the inhibition constants were determined as 0.12 and 0.16 microM for Kis and Kii, respectively, when ADP concentration was kept saturating. Isocitreoviridin had no effect on either activity of the membrane-bound enzyme.     

Binding of adenine nucleotides to the F1-inhibitor protein complex of bovine heart submitochondrial particles.

The binding of ATP radiolabeled in the adenine ring or in the gamma- or alpha-phosphate to F1-ATPase in complex with the endogenous inhibitor protein was measured in bovine heart submitochondrial particles by filtration in Sephadex centrifuge columns or by Millipore filtration techniques. These particles had 0.44 +/- 0.05 nmol of F1 mg-1 as determined by the method of Ferguson et al. [(1976) Biochem. J. 153, 347]. By incubation of the particles with 50 microM ATP, and low magnesium concentrations (less than 0.1 microM MgATP), it was possible to observe that 3.5 mol of [gamma-32P]ATP was tightly bound per mole of F1 before the completion of one catalytic cycle. With [gamma-32P]ITP, only one tight binding site was detected. Half-maximal binding of adenine nucleotides took place with about 10 microM. All the bound radioactive nucleotides were released from the enzyme after a chase with cold ATP or ADP; 1.5 sites exchanged with a rate constant of 2.8 s-1 and 2 with a rate constant of 0.45 s-1. Only one of the tightly bound adenine nucleotides was released by 1 mM ITP; the rate constant was 3.2 s-1. It was also observed that two of the bound [gamma-32P]ATP were slowly hydrolyzed after removal of medium ATP; when the same experiment was repeated with [alpha-32P]ATP, all the label remained bound to F1, suggesting that ADP remained bound after completion of ATP hydrolysis. Particles in which the natural ATPase inhibitor protein had been released bound tightly only one adenine nucleotide per enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Energy-dependent transformation of the catalytic activities of the mitochondrial F0 x F1-ATP synthase

The ADP(Mg2+)-deactivated, azide-trapped F0 x F1-ATPase of coupled submitochondrial particles is capable of ATP synthesis being incapable of ATP hydrolysis and ATP-dependent delta muH+ generation [FEBS Lett. (1995) 366, 29-32]. This puzzling phenomenon was studied further. No ATPase activity of the submitochondrial particles catalyzing succinate-supported oxidative phosphorylation in the presence of azide was observed when ATP was added to the assay mixture after an uncoupler. Rapid ATP hydrolysis was detected in the same system when ATP followed by an uncoupler was added. Less than 5% of the original ATPase activity was seen when the reaction (assayed with ATP-regenerating system) was initiated by the addition of ATP to the azide-trapped coupled particles oxidizing succinate either in the presence or in the absence of the uncoupler. High ATP hydrolytic activity was revealed when the reaction was started by the simultaneous addition of the ATP plus uncoupler to the particles generating delta muH+. The energy-dependent conversion of the enzyme into latent uncoupler-activated ATPase was prevented by free ADP (Ki approximately 20 microM) and was greatly enhanced after multiple turnovers in oxidative phosphorylation. The results suggest that the catalytic properties of F0 x F1 are delta muH+-dependent which is in accord with our hypothesis on different conformational states of the enzyme participating in ATP synthesis or hydrolysis.     

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 phosphorylation potential generated by respiring bovine heart submitochondrial particles.

A phosphorylation potential deltaGp, where deltaGp = deltaGo' + RT2.303 log ([ATP]/([ADP][Pi])), of approx. 44.3 kJ.mol-1 (10.6 kcal.mol-1) was generated by submitochondrial particles that were oxidizing either NADH or succinate. Addition of adenylyl imidodiphosphate, which should suppress adenosine triphosphatase activity of any uncoupled particles, did not raise the phosphorylation potential. Raising the Pi concentration slightly increased the magnitude of the value for [ATP]/[ADP], but this did not fully compensate for the increased Pi concentration, so that the phosphorylation potential decreased slightly as the Pi concentration was raised. The phosphorylation potential developed by submitochondrial particles is lower than that generated by phosphorylating membrane vesicles from some bacteria, and is also less than that developed externally by mitochondria, but is strikingly close to the phosphorylation potential that is generated internally by mitochondria.     

Presence of two enzymes, different from the F1F0-ATPase, hydrolyzing nucleotides in human term placental mitochondria

The hydrolysis of ATP, ADP or GTP was characterized in mitochondria and submitochondrial particles since a tightly-bound ATPase associated with the inner mitochondrial membrane from the human placenta has been described. Submitochondrial particles, which are basically inner membranes, were used to define the location of this enzyme. Mitochondria treated with trypsin and specific inhibitors were also used. The oxygen consumption stimulated by ATP or ADP was 100% inhibited in intact mitochondria by low concentrations of oligomycin (0.5 microgram/mg) or venturicidine (0.1 microgram/mg), while the hydrolysis of ATP or ADP was insensitive to higher concentrations of these inhibitors but it was inhibited by vanadate. Oligomycin or venturicidine showed a different inhibition pattern in intact mitochondria in relation to the hydrolysis of ATP, ADP or GTP. When submitochondrial particles were isolated from mitochondria incubated with oligomycin or venturicidine, no further inhibition of the nucleotide hydrolysis was observed, contrasting with the partial inhibition observed in the control. By incubating the placental mitochondria with trypsin, a large fraction of the hydrolysis of nucleotides was eliminated. In submitochondrial particles obtained from mitochondria treated with trypsin or trypsin plus oligomycin, the hydrolysis of ATP was 100% sensitive to oligomycin at low concentrations, resembling the oxygen consumption; however, this preparation still showed some ADP hydrolysis. Native gel electrophoresis showed two bands hydrolyzing ADP, suggesting at least two enzymes involved in the hydrolysis of nucleotides, besides the F1F0-ATPase. It is concluded that human placental mitochondria possesses ADPase and ATP-diphosphohydrolase activities (247).     

Fluoroaluminum and fluoroberyllium nucleoside diphosphate complexes as probes of the enzymatic mechanism of the mitochondrial F1-ATPase

The mechanism by which fluoride and aluminum or beryllium in combination with ADP inhibit beef heart mitochondrial F1-ATPase was investigated. The kinetics of inhibition depended on the nature of the anion present in the F1-ATPase assay medium. Inhibition required the presence of Mg2+ and developed more rapidly with sulfite and sulfate than with chloride, i.e., with anions which activate F1-ATPase activity. The ADP-fluorometal complexes were bound quasi-irreversibly to F1, and each mole of the inhibitory nucleotide-fluorometal complex was tightly associated with 1 mol of Mg2+. One mole of nucleotide-fluorometal complex was able to inhibit the activity of 1 mol of catalytic site in F1. Direct measurements of bound fluoride, aluminum, beryllium, and ADP indicated that the F1-bound ADP-fluorometal complexes are of the following types: ADP1A11F4, ADP1Be1F1, ADP1Be1F2, or ADP1Be1F3. Fluoroaluminates or fluoroberyllates are isomorphous to Pi, and the inhibitory nucleotide-fluorometal complexes mimicked transient intermediates of nucleotides that appeared in the course of ATP hydrolysis. On the other hand, each mole of fully inhibited F1, retained 2 mol of inhibitory complexes. The same stoichiometry was observed when ADP was replaced by GDP, a nucleotide which, unlike ADP, binds only to the catalytic sites of F1. These results are discussed in terms of a stochastic model in which the three cooperative catalytic sites of F1 function in interactive pairs.     

Inhibition of mitochondrial F1-ATPase activity by an anti-alpha subunit monoclonal antibody which modifies interactions between catalytic and regulatory sites

A monoclonal antibody, 7B3, specific to the alpha subunit of the mitochondrial ATPase-ATP synthase inhibited the rate of ATP hydrolysis by either soluble F1 or electron transport particles up to a maximum of 75%. However, 7B3 did not modify the rate of ITP hydrolysis. In addition, the apparent Km for MgATP extrapolated at high ATP concentrations had the same value in the absence as in the presence of 7B3. The antibody did not change the inactivation rate of F1-ATPase induced by dicyclohexylcarbodiimide or 4-chloro-7-nitro-2,1,3-benzoxadiazole. These observations indicate that 7B3 did not directly interfere with the catalytic sites of ATP or ITP hydrolysis. On the contrary, 7B3 modified the interaction between nucleotide sites and therefore the regulation of the rate of ATP hydrolysis. Indeed, 7B3 changed into a positive cooperativity the negative cooperativity observed when measuring the rate of ATP hydrolysis as a function of ATP concentration. 7B3 also increased the binding of ADP to F1. 7B3 prevented the rapid phase of inactivation of F1 by 5'-p-fluorosulfonylbenzoyladenosine. This phase has been correlated to the binding of 5'-p-fluorosulfonylbenzoyladenosine to regulatory sites (Di Pietro, A., Godinot, C., Martin, J. C., and Gautheron, D. C. (1979) Biochemistry 18, 1738-1745). The inhibition of ATP hydrolysis is concomitant with the binding of 1 mol of IgG or of 2 mol of Fab fragments per mol of F1. However, by further increasing the ratio Fab/F1, only 1 mol of Fab remained bound to F1 without change in inhibition of ATPase activity. All these experiments strongly support the suggestion that F1 conformational changes occurring upon binding of 7B3 to alpha subunit induce a modification of interactions between nucleotide sites. This modification would be consecutive to a change in the normal interaction between the alpha and beta subunits which is required to observe an active rate of ATP hydrolysis or synthesis. In conclusion, the use of this monoclonal antibody demonstrates for the first time in mammalian F1 the role of the conformation of the alpha subunit in the regulation of the ATPase activity.     

Kinetic studies on bacterial plasma membrane ATPase (F1). Nucleotide-induced long term inactivation of ATP hydrolyzing activity is linked to the formation of multiple "tight" enzyme nucleotide complexes.

ADP and the ATP analogs Nb-S6ITP (6-[(3-carboxy-4-nitrophenyl)thio]-9-beta-D-ribofuranosylpurine 5'-triphosphate) and AMP-P(NH)P (adenyl-5'-yl imidodiphosphate) interact with soluble plasma membrane ATPase (F1) from Micrococcus species in two ways: (i) at short incubation times, these inhibitors exhibit the kinetics of competitive inhibition, (ii) at long incubation times, these inhibitors induce an inactivation of the ATPase which can be reversed only in the case of AMP-P(NH)P. Kinetic treatment of the long term inactivation by ADP or Nb-S6ITP reveals a pseudo-first order process via the formation of an enzyme-inhibitor complex for which a Km analogous constant is obtained that is identical with the corresponding Ki value of the competitive inhibition. The long term inactivation by ADP and Nb-S6ITP involves the successive "tight" binding of 6 +/- 1 nucleotides/F1 molecule. One additional ADP molecule/F1 complex which is also "tightly" bound has no effect on the ATPase activity. The long term inactivation by ADP and Nb-S6ITP is inhibited at higher inhibitor concentrations according to a kinetics analogous to a substrate excess inhibition. Evidence is presented indicating that the mechanism of ATP hydrolysis by F1 and the long term inactivation by ADP or Nb-S6ITP are related processes. The mechanism of long term inactivation by AMP-P(NH)P appears to be different from that of ADP or Nb-S6ITP.     

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.     

Cold lability of membrane-bound F1-ATPase.

1. Preincubation of MgATP submitochondrial particles with EDTA or Tris.HCl liberated a measurable amount of ATPase inhibitor that could be rapidly purified using only trichloroacetic acid precipitation and heat treatment. 2. In spite of the emergence of high ATPase activity, a considerable amount of ATPase inhibitor was left in the particles. Comparative analysis of other submitochondrial preparations indicated that only AS-particles were effectively depleted. 3. The high ATPase activity of inhibitor-deficient particles, was labile at low temperature provided that the exposure to cold was done in the presence of MgATP. Other nucleotides could not substitute for ATP. Glycerol inhibited and salts enhanced the cold inactivation of membrane-bound F1-ATPase. Isolation of F1-ATPase from cold-inactivated particles yielded a soluble preparation of correspondingly lower activity. 4. It is concluded that together with the increase of ATPase activity, the ATP-dependent cold lability of membrane-bound F1-ATPase and the dislocation of ATPase inhibitor at non operative sites reveal the extent of ATPase complex disorganization.     

Kinetics of ATP synthesis in pea cotyledon submitochondrial particles

A kinetic study of oxidative phosphorylation by pea submitochondrial particles gave two K_m values for ADP, one low, the other high. The high value probably reflected a damaged site or a population of leaky mitochondria. Only the high affinity site with a low K_m for ADP was involved in ATP synthesis. α,β-Methylene ADP was found to be a competitive inhibitor of ATP synthesis. The inorganic phosphate analog, thiophosphate, decreased the apparent K_m of ADP while the rate of the reaction remained approximately the same. Adenyl imidodiphosphate, a specific inhibitor of ATP hydrolysis activity, had little effect on oxidative phosphorylation. A slight decrease in the K_m of the high affinity binding site for ADP was noted. Aurovertin was found to be a potent inhibitor of oxidative phosphorylation in pea submitochondrial particles. The K_m of the high affinity site was increased 10-fold. Also, the inhibition normally exerted by ADP on ATPase activity was severely reduced by aurovertin. In contrast, increasing the concentration of aurovertin only slightly affected the level of inhibition caused by adenyl imidodiphosphate on ATP hydrolysis.     

The extent of mitochondrial F1-ATPase and adenine nucleotide carrier activity with epsilon-ATP.

1. The use of 1,N6-ethenoadenosine 5'-triphosphate (epsilon-ATP), a synthetic, fluorescent analog of ATP, by whole rat liver mitochondria and by submitochondrial particles produced via sonication has been studied. 2. Direct [3H]adenine nucleotide uptake studies with isolated mitochondria, indicate the epsilon-[3H]ATP is not transported through the inner membrane by the adenine nucleotide carrier and is therefore not utilized by the 2,4-dinitrophenol-sensitive F1-ATPase (EC 3.6.1.3) that functions in oxidative phosphorylation. However, epsilon-ATP is hydrolyzed by a Mg2+-dependent, 2,4-dinitrophenol-insensitive ATPase that is characteristic of damaged mitochondria. 3. epsilon-ATP can be utilized quite well by the exposed F1-ATPase of sonic submitochondrial particles. This epsilon-ATP hydrolysis activity is inhibited by oligomycin and stimulated by 2,4-dinitrophenol. The particle F1-ATPase displays similar Km values for both ATP and epsilon-ATP; however, the V with ATP is approximately six times greater than with epsilon-ATP. 4. Since epsilon-ATP is a capable substrate for the submitochondrial particle F1-ATPase, it is proposed that the fluorescent properties of this ATP analog might be employed to study the submitochondrial particle F1-ATPase complex, and its response to various modifiers of oxidative phosphorylation.     

Na+, K+ -ATPase: causes of activation of K+ -phosphatase reaction by nucleotides

Hydrolysis of umbelliferone phosphate is inhibited by sodium ions and ATP added to the incubation medium together with Na+ activates it, CTP, ITP, UTP and GTP produce the same effect. Acetyl phosphate, p-nitrophenyl phosphate, ADP and adenylyl imidodiphosphate beta, gamma-NH-ATP, ATP nonhydrolyzable analog, have no activating effect. ATP produces a mixed inhibition of umbelliferone phosphate hydrolysis both in the presence and absence of Na+. A scheme is suggested which explains the mechanism of the nucleotide activating effect.