Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.6.3.14 (ATP synthase)
7,042 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The effects of various substrates and alternative substrates on the hydrolytic activity of beef heart mitochondrial ATPase was examined. It was found that ATP or ADP, ITP hydrolysis showed positive cooperativity. IDP inhibited ITP hydrolysis and caused positive cooperativity. When ITP was present during an ATP hydrolysis assay, the rate of ATP hydrolysis was stimulated. IDP had no effect on ATP hydrolysis rates. A nonhydrolyzable ITP analog, inosine 5'-(beta, gamma-imido)triphosphate (IMP-P(NH)P), was synthesized and purified. It was found to be a potent competitive inhibitor of ITP and GTP hydrolytic activity. However, this beta-gamma-imido-bridged ITP analog was found to change the ITP and GTP hydrolysis kinetics from linear to positively cooperative. This compound inhibited ATP hydrolysis at substrate concentrations of 100 muM and lower, and stimulated ATP hydrolysis at substrate concentrations between 100 muM and 2 mM. IMP-P(NH)P had no effect on ATP hydrolysis when the substrate concentration was above 2 mM. In the presence of the activating anion, bicarbonate, IMP-P(NH)P inhibited ATP hydrolysis competitively, and induced positive cooperativity. IMP-P(NH)P had no effect on the ATP equilibrium Pi exchange, the ITP equilibrium Pi exchange, or ATP synthesis catalyzed by beef heart submitochondrial particles.
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PMID:Effect of inosine 5' -(beta, gamma-imido) triphosphate and other nucleotides on beef heart mitochondrial ATPase. 13 64

The bovine heart mitochondrial F1-ATPase (MF1) is inactivated by 5'-p-fluorosulfonylbenzoylethenoadenosine (FSB epsilon A) with pseudo-first order kinetics. The dependence of the rate of inactivation on the concentration of FSB epsilon A revealed an apparent Kd of 0.25 mM. ATP and ADP, and to a lesser extent, ITP and IDP provide partial protection against inactivation by the reagent. Isolation and sequence analysis of major radioactive fragments in peptic or cyanogen bromide digests of MF1 inactivated with [3H]FSB epsilon A indicate that modification of Tyr-alpha 244 is associated with the loss of activity observed. Assessment of the amount of Tyr-alpha 244 derivatized with [3H]FSB epsilon A at specific points during inactivation of the ATPase indicates that maximal inactivation is achieved on modification of this residue in slightly greater than one copy of the alpha subunit. The following characteristics of inactivation of MF1 by FSB epsilon A have also been determined. (a) The rate of inactivation of ITPase activity by FSB epsilon A is 1.4 times greater than that observed for inactivation of ATPase activity under identical conditions. (b) After maximally inactivating the capacity of MF1 to hydrolyze saturating ATP with FSB epsilon A, the modified enzyme retained its capacity to hydrolyze substoichiometric ATP. (c) Inactivation of the ATPase by FSB epsilon A is accelerated by Pi. In each of the above characteristics, MF1 modified by FSB epsilon A resembles enzyme inactivated with 5'-p-fluorosulfonylbenzoyladenosine (FSBA) more than it does enzyme inactivated with 5'-p-fluorosulfonylbenzoylinosine (FSBI). Furthermore, prior inactivation of MF1 with FSBA completely prevents labeling of Tyr-alpha 244 with [3H]FSB epsilon A, whereas prior inactivation of the enzyme with FSBI does not. Since a single catalytic site is modified when FSBI inactivates MF1 whereas three noncatalytic sites are modified when it is maximally inactivated with FSBA, it is concluded that FSB epsilon A also modifies noncatalytic sites.
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PMID:Tyrosine alpha 244 is derivatized when the bovine heart mitochondrial F1-ATPase is inactivated with 5'-p-fluorosulfonylbenzoylethenoadenosine. 213 76

The stoichiometry of nucleotide binding to the isolated alpha- and beta-subunits of Escherichia coli F1-ATPase was investigated using two experimental techniques: (a) titration with fluorescent trinitrophenyl (TNP) derivatives of AMP, ADP, and ATP and (b) the centrifuge column procedure using the particular conditions of Khananshvili and Gromet-Elhanan (Khananshvili, D., and Gromet-Elhanan, Z. (1985) FEBS Lett. 178, 10-14). Both procedures showed that alpha-subunit contains one nucleotide-binding site, confirming previous work. TNP-ADP and TNP-ATP bound to a maximal level of 1 mol/mol beta-subunit, consistent with previous equilibrium dialysis studies which showed isolated beta-subunit bound 1 mol of ADP or ATP per mol (Issartel, J. P., and Vignais, P. V. (1984) Biochemistry 23, 6591-6595). However, binding of only approximately 0.1 mol of ATP or ADP per mol of beta-subunit was detected using centrifuge columns. Our results are consistent with the conclusion that each of the alpha- and beta-subunits contains one nucleotide-binding domain. Because the subunit stoichiometry is alpha 3 beta 3 gamma delta epsilon, this can account for the location of the six known nucleotide-binding sites in E. coli F1-ATPase. Studies of in vitro assembly of isolated alpha-, beta-, and gamma- subunits into an active ATPase showed that ATP, GTP, and ITP all supported assembly, with half-maximal reconstitution of ATPase occurring at concentrations of 100-200 microM, whereas ADP, GDP, and IDP did not. Also TNP-ATP supported assembly and TNP-ADP did not. The results demonstrate that (a) the nucleotide-binding site on beta-subunit has to be filled for enzyme assembly to proceed, whereas occupancy of the alpha-subunit nucleotide-binding site is not required, and (b) that enzyme assembly requires nucleoside triphosphate.
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PMID:Trinitrophenyl-ATP and -ADP bind to a single nucleotide site on isolated beta-subunit of Escherichia coli F1-ATPase. In vitro assembly of F1-subunits requires occupancy of the nucleotide-binding site on beta-subunit by nucleoside triphosphate. 289 69

This communication presents the results obtained in tubular aggregates of 24 enzyme histochemical techniques for demonstrating activity of oxidoreductases, transferases, hydrolases and isomerases. The activity characteristics of the tubular aggregates in m. gluteus medius of 18 patients with diseases of the neuromuscular system were almost identical. A high activity of the mitochondrial enzymes, NADPH: tetrazolium oxidoreductase, NADH:tetrazolium oxidoreductase and cytochrome c oxidase, could be shown in the pathological structures, whereas the activity of the mitochondrial enzymes, glycerol-3-phosphate:menadione oxidoreductase, succinate:PMS oxidoreductase, malate:NAD+ oxidoreductase and isocitrate:NAD+ oxidoreductase, and the partial mitochondrial enzymes, malate:NADP+ oxidoreductase and isocitrate:NADP+ oxidoreductase, was very slight or even absent. There was a moderate to strong activity of the glycolytic enzymes lactate:NAD+ oxidoreductase, glyceraldehyde-3-phosphate:NAD+ oxidoreductase, phosphofructokinase, phosphoglucomutase and glucose phosphate isomerase. In contrast, the activity of alpha-glucan phosphorylase was slight. The activity of phosphogluconate:NADP+ oxidoreductase, glucose-6-phosphate:NADP+ oxidoreductase and 5'-nucleotidase was slight, whereas there was no activity of myosin ATPase and mitochondrial ATPase, acid phosphatase or alkaline phosphatase. The high activity of AMP-deaminase was very striking. The activity of peroxidase was moderate. Results obtained with adsorption studies point to adsorption of some of the enzymes studied to the tubular aggregates in vivo and this phenomenon very probably determined the histochemical characteristics of these structures.
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PMID:Histochemical features of tubular aggregates in diseased human skeletal muscle fibres. 317 98

A soluble Mg-dependent ATPase, similar to the mitochondrial ATPase from beef heart, has been isolated from heart mitochondria of salmon (Salmo salar). The salmon heart ATPase has 5 subunits with molecular weights similar to the beef heart enzyme, but the Stoke's radius of the intact salmon enzyme is larger. The salmon heart ATPase is less temperature labile than the beef heart enzyme. The salmon heart ATPase is strongly inhibited by ADP, and the inhibition is highly temperature dependent. The ITPase activity is also inhibited by IDP (Ki = 180 micron). 2,4-Dinitrophenol in small concentrations stimulates the ITPase activity as well as the ATPase activity of the "washed" salmon heart enzyme. However, in an enzyme preparation which had been freed of most of the bound nucleotides by dialysis in the presence of glycerol (Roveri et al., 1980) the ITPase activity is not stimulated by 2,4-dinitrophenol.
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PMID:Some properties of isolated mitochondrial ATPase from salmon heart. 615 Aug 4

The microsomal fraction (13 000 -60 000 x g pellet) from pea stem shows a very high divalent cation-dependent, diethylstilbestrol- and orthovanadate-inhibited ATPase and ADPase activity. No detectable inorganic or organic pyrophosphatase and adenylate kinase and almost negligible activities of mitochondrial ATPase and of other phosphomonoesterases are present in this preparation. The ATPase and ADPase activities of microsomes have been solubilized with NaClO4 and then purified by gel filtration and DEAE-Sephadex fractionation to a final specific activity of 71.5 and 102 mumol Pi/min per mg for ATP and ADP, respectively. The purified enzyme hydrolyzes triphosphonucleosides (ATP, CTP, GTP, UTP) and diphosphonucleosides (ADP and to a lesser extent CDP, UDP, IDP) and presents pH optima of 6 for ATP and 7 for ADP. It requires Mg2+, Mn2+ or Ca2+ and is inhibited by diethylstilbestrol and orthovanadate. The conclusion that the ATPase and ADPase activities belong to the same enzyme is based on the following results: (1) parallel effects of diethylstilbestrol and orthovanadate on both ATPase and ADPase; (2) parallel behavior of ATPase and ADPase throughout all the purification steps; (3) non-additivity of ATPase and ADPase and (4) lack of dilution of beta-32P formed from [beta-32P]-ATP by unlabelled ADP.
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PMID:Purification and characterization of a divalent cation-activated ATP-ADPase from pea stem microsomes. 626 9

During net nucleoside triphosphate synthesis by chloroplast ATP synthase the extent of water oxygen incorporation into each nucleoside triphosphate released increases with decrease in ADP, GDP or IDP concentration. Likewise, during net ATP hydrolysis by the Mg2+-activated chloroplast ATPase, the extent of water oxygen incorporation into each Pi released increases as the ATP, GTP, or ITP concentration is decreased. However, the concentration ranges in which substrate modulation occurs differs with each nucleotide. Modulation of oxygen exchange during synthesis and hydrolysis of adenine nucleotides, as measured by variation in the extent of water oxygen incorporation into products, occurs below 250 microM. In contrast, guanosine and inosine nucleotides alter the extent of exchange at higher and much wider concentration ranges. Activation of the chloroplast ATPase by either heat or trypsin results in similar catalytic behavior as monitored by ATP modulation of oxygen exchanges during hydrolysis in the presence of Mg2+. More exchange capacity is evident with octylglucoside-activated enzyme at all ATP concentrations. High levels of tentoxin were also found to alter the catalytic exchange parameters resulting in continued water oxygen exchange into Pi released during hydrolysis at high ATP concentrations. Little or no oxygen exchange accompanies ATP hydrolysis in the presence of Ca2+. The [18O]Pi species formed from highly gamma-18O-labeled ATP at lower ATP concentrations gives a distribution as expected if only one catalytic pathway is operative at a given ATP concentration. This and other results support the concept of catalytic cooperativity between alternating sites as explanation for the modulation of oxygen exchange by nucleotide concentration.
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PMID:Probes of catalytic site cooperativity during catalysis by the chloroplast adenosine triphosphate and the adenosine triphosphate synthase. 630 19

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.
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PMID:Kinetics of interaction of adenosine diphosphate and adenosine triphosphate with adenosine triphosphatase of bovine heart submitochondrial particles. 645 Dec 17

Tryptophan fluorescence was investigated as a tool to study the noncatalytic nucleotide-binding sites of Escherichia coli F1-ATPase. Site-directed mutagenesis, affinity labeling, and lin-benzo-ATP binding studies had shown that residues alpha R365 and beta Y354 are located close to the base moiety of bound nucleotide; here, we mutagenized each to tryptophan. The new tryptophans gave a fluorescence signal indicating an environment of high (alpha W365) or intermediate (beta W354) polarity in unoccupied sites. alpha W365 fluorescence was completely quenched by binding of ATP or ADP, providing a direct, specific probe of noncatalytic site nucleotide occupancy. Using this signal, we measured binding parameters for ATP and ADP, showed that nucleotide binding was magnesium-dependent, and showed that GTP and ITP did bind to some extent, but AMP, GDP, and IDP did not. It was possible to follow initial rates of MgATP hydrolysis and noncatalytic site binding under identical conditions; the results indicated that occupancy of noncatalytic sites was not required for catalysis. Fluorescence from beta W354 was quenched completely by lin-benzo-ATP, but only slightly by ATP or ADP. Probably, residue beta 354 is not as closely juxtaposed to the adenine ring of bound ATP and ADP as is residue alpha 365. With either alpha W365 or beta W354 as donor and catalytic site-bound lin-benzo-ADP as acceptor, no fluorescence resonance energy transfer was detected, indicating that the distance between non-catalytic and catalytic sites is > or = 27 A.
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PMID:Tryptophan fluorescence provides a direct probe of nucleotide binding in the noncatalytic sites of Escherichia coli F1-ATPase. 815 56

The natural developmental gradient of light-grown primary leaves of barley (Hordeum vulgare L.) was used to analyze the biogenesis of mitochondrial proteins in relation to the age and physiological changes within the leaf. The data indicate that the protein composition of mitochondria changes markedly during leaf development. Three distinct patterns of protein development were noted: group A proteins, consisting of the E1 beta-subunit of the pyruvate dehydrogenase complex, ORF156, ORF577, alternative oxidase, RPS12, cytochrome oxidase subunits II and III, malic enzyme, and the alpha- and beta-subunits of F1-ATPase; group B proteins, consisting of the E1 alpha-subunit of the pyruvate dehydrogenase complex, isocitrate dehydrogenase, HSP70A, cpn60C, and cpn60B; and group C proteins, consisting of the four subunits of the glycine decarboxylase complex (P, H, T, and L proteins), fumarase, and formate dehydrogenase. All of the proteins increased in concentration from the basal meristem to the end of the elongation zone (20.0 mm from the leaf base), whereupon group A proteins decreased, group B proteins increased to a maximum at 50 mm from the leaf base, and group C proteins increased to a maximum at the leaf tip. This study provides evidence of a marked heterogeneity of mitochondrial protein composition, reflecting a changing function as leaf cells develop photosynthetic and photorespiratory capacity.
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PMID:Heterogeneity of mitochondrial protein biogenesis during primary leaf development in barley 980 54


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