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Inhibition of the soluble adenosine triphosphatase from mitochondria by adenylyl imidodiphosphate.

ABSTRACT: 1. Adenylyl imidodiphosphate is an inhibitor with high affinity for the soluble ATPase (adenosine triphosphatase) from mitochondria. 2. The reaction of the inhibitor with the ATPase is slow and estimates for the association and dissociation reaction rate constants are given. 3. The number of binding sites for the inhibitor appears to be doubled in the presence of 2,4-dinitrophenol. 4. Adenylyl imidodiphosphate is less effective as an inhibitor of the ATPase activity of this enzyme than of the inosine triphosphatase activity. It is also less effective on the ATPase of frozen-thawed or intact mitochondria and did not inhibit ADP-stimulated respiration by intact mitochondria.

SUBMITTER: Philo RD

PROVIDER: S-EPMC1168443 | biostudies-other | 1974 Dec

REPOSITORIES: biostudies-other

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A kinetic study of the interaction between mitochondrial F1 adenosine triphosphatase and adenylyl imidodiphosphate and guanylyl imidodiphosphate.

Project description:1. The presence of 5'-adenylyl imidodiphosphate, a non-hydrolysable analogue of ATP, in the solution used to assay the soluble bovine heart mitochondrial F1-ATPase produced slow competitive inhibition. If the enzyme was preincubated with the inhibitor before the substrate, MgATP, was added, a partial re-activation was obtained. 2. The slow inhibitory process showed first-order rate kinetics, and therefore it seems likely that a conformational change of the enzyme occurs following a faster binding process. A reaction scheme is suggested. At pH 7.8 the rate constant for the inhibition reaction was calculated to be 6.7 X 10(-2)s-1 and that for the re-activation 3.8 X 10(-3)s-1, with Keq. 17.6, indicating that the inhibited enzyme-inhibitor complex will be favoured over the non-inhibited enzyme-inhibitor complex. 3. The presence of 5'-guanylyl imidodiphosphate in the solution used to assay F1-ATPase produced rapid competitive inhibition, which was then slowly reversed until a steady state was reached. This might be explained by a rapid but reversible shift of the inhibition pathway induced by this non-hydrolysable analogue of ATP. A complex rate constant for the displacement of the inhibitor by the substrate of 7.6 X 10(-3)s-1 was calculated. 4. The results are discussed in the light of other recent observations about binding of 5'-adenylyl imidodiphosphate to F1-ATPase and with reference to the binding-site-change mechanism of hydrolysis of ATP by F1-ATPase.

| S-EPMC1154284 | biostudies-other

Preparation and general properties of a soluble adenosine triphosphatase from mitochondria.

Project description:1. The purification of an adenosine triphosphatase present in aqueous extracts of acetone-dried ox-heart mitochondria is described. 2. No evidence was found for the presence of more than one protein having adenosine-triphosphatase activity in these extracts. 3. The enzyme is less stable at 0 degrees than at 25 degrees but is stabilized by glycerol. 4. The activity is dependent on the presence of Mg(2+) or certain other bivalent metal cations. 5. The adenosine-triphosphatase activity of the Mg(2+)-activated enzyme is enhanced by 2,4-dinitrophenol. 6. The kinetics of Mg(2+) activation indicate that the ATP-Mg(2+) complex is the important substrate: free ATP and Mg(2+) are inhibitory. 7. This preparation of mitochondrial adenosine triphosphatase has many properties in common with the adenosine triphosphatase coupling factor from mitochondria (Racker, 1961).

| S-EPMC1198299 | biostudies-other

Soluble adenylyl cyclase controls mitochondria-dependent apoptosis in coronary endothelial cells.

Project description:The cAMP signaling pathway plays an essential role in modulating the apoptotic response to various stress stimuli. Until now, it was attributed exclusively to the activity of the G-protein-responsive transmembrane adenylyl cyclase. In addition to transmembrane AC, mammalian cells possess a second source of cAMP, the ubiquitously expressed soluble adenylyl cyclase (sAC). However, the role of this cyclase in apoptosis was unknown. A mitochondrial localization of this cyclase has recently been demonstrated, which led us to the hypothesis that sAC may play a role in apoptosis through modulation of mitochondria-dependent apoptosis. To prove this hypothesis, apoptosis was induced by simulated in vitro ischemia or by acidosis, which is an important component of ischemia. Suppression of sAC activity with the selective inhibitor KH7 or sAC knockdown by small interfering RNA transfection abolished endothelial apoptosis. Furthermore, pharmacological inhibition or knockdown of protein kinase A, an important cAMP target, demonstrated a significant anti-apoptotic effect. Analysis of the underlying mechanisms revealed (i) the translocation of sAC to mitochondria under acidic stress and (ii) activation of the mitochondrial pathway of apoptosis, i.e. cytochrome c release and caspase-9 cleavage. sAC inhibition or knockdown abolished the activation of the mitochondrial pathway of apoptosis. Analysis of mitochondrial co-localization of Bcl-2 family proteins demonstrated sAC- and protein kinase A-dependent translocation of Bax to mitochondria. Taken together, these results suggest the important role of sAC in modulating the mitochondria-dependent pathway of apoptosis in endothelial cells.

| S-EPMC2685657 | biostudies-literature

Effects of triphenylsulphonium ions on mitochondria. Inhibition of adenosine triphosphatase activity.

Project description:Triphenylsulphonium ions inhibit mitochondrial oxidative phosphorylation and adenosine triphosphatase activity. The site of action is on the soluble F1 adenosine triphosphatase component. Triphenylsylphonium ions also inhibit electron transfer in the NAD-cytochrome b region of the respiratory chain. In both types of inhibition, triphenylsulphonium ions are effective at low concentrations, half-maximal inhibition being produced by a concentration of about 20-30 muM. These effects resemble the effects of alkylguanidines on mitochondria and are discussed in relation to the effects of alkylguanidines and other lipophilic cations such as ethidium and dibenzyldimethylammonium ions. A modification of the purification procedure for the soluble mitochondrial adenosine triphosphatase [Beechey, Hubbard, Linnett, Mitchell & Munn (1975) Biochem. J. 148, 533-537] IS DESCRIBED, WHICH YIELDS A PREPARATION WITH A HIGHER SPECIFIC ACTIVITY AND SHOWING FEWER BANDS IN GEL ELECTROPHORESIS.

| S-EPMC1163751 | biostudies-other

MgATP-induced inhibition of the adenosine triphosphatase activity of the chloroform-released mitochondrial adenosine triphosphatase.

Project description:1. Preincubation of the ox heart chloroform-released mitochondrial ATPase with MgATP results in a time-dependent inhibition of ATPase activity. No re-activation occurs when MgATP remains in the preincubation medium. The enzyme activity returns when all the MgATP in the preincubation system has been hydrolysed. 2. The mechanism of the MgATP-induced inhibition was examined. Inhibition occurs on incubation with MgATP or other hydrolysable nucleotides. Incubation with MgADP or Pi does not cause any inhibition. Neither freshly bound adenine nucleotide nor Pi is associated with inhibited enzyme. The rate of MgATP-induced inhibition correlates with the rate of ATP hydrolysis in the preincubation medium. Changing the rate of ATP hydrolysis at a fixed concentration of ATP also changes the rate of MgATP-induced inhibition by the same proportion. The inhibition is thus related to the ATP-hydrolysis process itself. 3. We propose that intermediate enzyme species of the ATP-hydrolytic sequence can undergo a conformational change to form inhibited species. The kinetics of the inhibition suggest that a substrate-activation step is involved in ATP hydrolysis and MgATP-induced inhibition. 4. The effects of the nature of the preincubation medium on the process of MgATP-induced inhibition and its reversal were examined.

| S-EPMC1163014 | biostudies-other

Involvement of the endogenous inhibitor protein in the MgATP-induced inhibition of soluble mitochondrial adenosine triphosphatase activity.

Project description:Chloroform-released ATPase from ox heart mitochondria contains significant amounts of inhibitor protein. There is a correlation between processes that affect the interactions between the inhibitor protein and the ATPase molecule and the ability of MgATP to induce an inhibition of ATPase activity. Evidence is presented suggesting that the endogenous inhibitor protein is involved in the process of MgATP-induced inhibition of soluble ATPase activity.

| S-EPMC1163588 | biostudies-other

"Soluble" adenylyl cyclase-generated cyclic adenosine monophosphate promotes fast migration in PC12 cells.

Project description:In a model for neuronal movement, PC12 cells undergo fast migration in response to nerve growth factor (NGF) and phorbol ester (PMA). We previously showed that NGF increases intracellular cAMP via activation of soluble adenylyl cyclase (sAC). In this report, we demonstrate that sAC activation is an essential component of NGF- + PMA-induced fast migration in PC12 cells. Interestingly, PMA also raises intracellular cAMP but does so by stimulating transmembrane adenylyl cyclases (tmAC); however, this tmAC-generated cAMP does not contribute to fast migration. Therefore, cells must possess independent pools of cAMP capable of modulating distinct functions.

| S-EPMC2587045 | biostudies-other

Use of progress curves to investigate product inhibition in enzyme-catalysed reactions. Application to the soluble mitochondrial adenosine triphosphatase.

Project description:1. Several methods of analysing progress curves of enzyme-catalysed reactions are discussed briefly in relation to their usefulness in a situation where a reaction product has a K(i) much lower than the K(m) for the substrate 2. A comparison is made of different methods of estimating initial rates in this situation. 3. The use of a computer curve-fitting routine capable of handling functions of more than one variable for the extraction of kinetic parameters from progress curves is described. 4. This method and that of fitting time as a polynomial in product concentration are applied to progress curves for the soluble mitochondrial adenosine triphosphatase and the results are compared with values obtained by more conventional methods.

| S-EPMC1165854 | biostudies-other

MgATP-induced inhibition of the adenosine triphosphatase activity of submitochondrial particles.

Project description:1. The ATP-hydrolytic activity of ox heart submitochondrial particles can be increased from 2-3 mumol/min per mg of protein to 10-12 mumol/min per mg of protein by incubation in media containing 50 mM-Na2B4O7. This process appears to be due to the partial release of inhibitor protein from the particles. 2. The ATPase activity of submitochondrial particles can be inhibited by incubation with the substrate, MgATP. This inhibition is not due to the accumulation of the hydrolysis products, MgADP and Pi, but could involve the process of ATP hydrolysis. 3. The mechanism of MgATP-induced inhibition of ATPase activity is proposed to involve a conformational change in one of the intermediate enzyme species of the ATP-hydrolytic sequence. 4. MgATP inhibits the ATPase activity of control submitochondrial particles at a higher rate and to a greater extent than it does that of inhibitor-protein-depleted submitochondrial particles, suggesting that the conformational change involves the endogenous inhibitor protein.

| S-EPMC1163015 | biostudies-other

Soluble adenylyl cyclase is essential for proper lysosomal acidification.

Project description:Lysosomes, the degradative organelles of the endocytic and autophagic pathways, function at an acidic pH. Lysosomes are acidified by the proton-pumping vacuolar ATPase (V-ATPase), but the molecular processes that set the organelle's pH are not completely understood. In particular, pH-sensitive signaling enzymes that can regulate lysosomal acidification in steady-state physiological conditions have yet to be identified. Soluble adenylyl cyclase (sAC) is a widely expressed source of cAMP that serves as a physiological pH sensor in cells. For example, in proton-secreting epithelial cells, sAC is responsible for pH-dependent translocation of V-ATPase to the luminal surface. Here we show genetically and pharmacologically that sAC is also essential for lysosomal acidification. In the absence of sAC, V-ATPase does not properly localize to lysosomes, lysosomes fail to fully acidify, lysosomal degradative capacity is diminished, and autophagolysosomes accumulate.

| S-EPMC5037342 | biostudies-literature

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