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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 fluorogenic reagent fluorescamine has been used to determine the labeling patterns of Type C spinach chloroplast membrane polypeptides. Membrane polypeptides labeled with fluorescamine were detected by scanning high resolution sodium dodecyl sulfate polyacrylamide gradient slab gels for fluorescence emission. Three membrane polypeptides show a decrease in the extent of labeling when chloroplast membranes are labeled in the light compared to when they are labeled in the dark. These polypeptides have apparent molecular weights 0f 32 000, 23 000 and 15 000. The decrease in labeling observed in the light is abolished or reduced by treatments which inactivate the light-generated transmembrane pH gradient. CF1-depleted chloroplasts show neither a light-activated pH gradient nor a light/dark difference in labeling of these three polypeptides. Both a light-activated pH gradient and light/dark difference in labeling are observed in CF1-depleted chloroplasts which have been treated with N,N'-dicyclohexylcarbodiimide. The same ammonium sulfate fractions of a 2% sodium cholate extract, which are believed to be enriched in the membrane-bound sector of the chloroplast ATPase (CFo) are also found to be enriched in the 32 000, 23 000 and 15 000 molecular weight polypeptides. The three polypeptides are believed to be components of CFo, and the light/dark labeling differences may indicate conformational changes within CFo. Such conformational changes may reflect a mechanism which couples light-generated proton gradients to ATP synthesis.
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PMID:Light/dark labeling differences in chloroplast membrane polypeptides associated with chloroplast coupling factor o. 15 23

1. Tightly bound ATP and ADP, found on the isolated mitochondrial ATPase, exchange only slowly at pH 8, but the exchange is increased as the pH is reduced. At pH 5.5, more than 60% of the bound nucleotide exchanges within 2.5 min. 2. Preincubation of the isolated ATPase with ADP leads to about 50% inhibition of ATP hydrolysis when the enzyme is subsequently assayed in the absence of free ADP. This effect, which is reversed by preincubation with ATP, is absent on the membrane-bound ATPase. This inhibition seems to involve the replacement of tightly bound ATP by ADP. 3. Using these two findings, the binding specificity of the tight nucleotide binding sites was determined. iso-Guanosine, 2'-deoxyadenosine and formycin nucleotides displaced ATP from the tight binding sites, while all other nucleotides tested did not. The specificities of the tight sites of the isolated and membrane-bound ATPase were similar, and higher than that of the hydrolytic site. 4. The nucleotide specificities of 'coupled processes' nucleoside triphosphate-driven reversal of electron transfer, nucleoside triphosphate-32Pi exchange and phosphorylation were higher than that of the hydrolytic site of the ATPase and similar to that of the tight nucleotide binding sites.
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PMID:Specificity of nucleotide binding and coupled reactions utilising the mitochondrial ATPase. 15 44

1. The subunit compositions of the F1 (oligomycin-insensitive) and F1--F0 (oligomycin-sensitive) mitochondrial ATPase complexes from Saccharomyces cerevisiae have been examined by the highly resolving technique of sodium dodecyl sulphate-polyacrylamide slab gel electrophoresis using a discontinuous buffer system. When isolated in the presence of protease inhibitors, F1 and F1--F0 contained five and twelve bands, respectively; this contrasts with the four- and ten-band patterns seen previously using the less resolving disc gel method. When isolated in the absence of protease inhibitors both F1 and F1--F0 contain spurious polypeptides produced by proteolytic modification. 2. Endogenous protein turnover in S. cerevisiae was impaired in the presence of protease inhibitors. F1--F0 isolated from cells grown in the presence and absence of inhibitors contained an identical polypeptide composition, suggesting that the subunits are not significantly modified by endogenous proteases prior to cell harvesting. 3. Yeast F1--F0 prepared in the presence of protease inhibitors contains a latent, sodium dodecyl sulphate-activated protease contaminant. Sodium dodecyl sulphate-induced proteolysis is largely confined to the 52 000 dalton alpha subunit which degrades into polypeptides of 40 000 and 10 700 daltons. The 40 000 dalton band is apparently equivalent to the polypeptide previously designated subunit 3. 4. Both F1 and F1--F0 were isolated from Torulopsis glabrata, a yeast with considerably shorter mitochondrial DNA than that in S. cerevisiae. F1--F0 catalysed high rates of ATP--32Pi exchange when reconstituted into phospholipid vesicles, thus demonstrating the presence of a complete coupling mechanism. F1--F0 contained approximately twelve subunits and F1 five, like the S. cerevisiae complexes. It therefore appears that the shorter mitochondrial DNA length does not produce a significantly simpler ATPase subunit structure.
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PMID:The yeast mitochondrial ATPase complex. Subunit composition and evidence for a latent protease contaminant. 15 54

Modification of soluble mitochondrial ATPase (factor F1) by spin-labelled iodoacetamide and spin-labelled methyleneketone does not cause and change in the catalytic properties of the enzyme. The temperature dependence of tau corr. of labels bound to factor F1 testifies to conformational changes in the enzyme at temperatures of 18--20 degrees C and 34--37 degrees C. At these temperature intervals, breaks are observed in the temperature dependence of the ATPase reaction rate in the Arrenius plot. The results obtained indicate that the thermally induced conformational changes in factor F1 affect large areas of the protein molecule. The interaction of factor F1 with the hydrophobic spin probes, namely fatty acid derivatives, was studied. It was shown that the interaction of foctor F1 with Mg2+, Mg-ATP, Mg-ADP and ADP, results in an increase in the ability of the enzyme to adsorb spin probes.
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PMID:[Conformational changes in soluble mitochondrial ATPase by the spin probe method]. 15 72

1. The distribution of ATPase and several marker enzymes was examined after differential and sucrose gradient centrifugation of yeast homogenates. 2. An ATPase activity not sensitive to oligomycin is found exclusively associated with a particulate fraction equilibrating at densities of 1.23-1.25. This particulate material shows the chemical and enzymatic characteristics of the yeast plasma membrane. 3. The pH optimum of the plasma membrane ATPase is 5.6, as compared with 8.5 for the mitochondrial ATPase. In addition to oligomycin, the enzyme is not sensitive to other inhibitors of the mitochondrial ATPase as azide, dicyclohexylcarbodiimide and the mitochondrial ATPase inhibitor protein. It is inhibited by p-chloromercuryphenyl sulfonate, fluoride, quercetin and by the antibiotic Dio-9 but is not affected by ouabain. 4. The plasma membrane ATPase shows a high affinity for ATP (Km = 0.1 mM) and is very specific for this compound, hydrolyzing other nucleotide triphosphates less than 25% as rapidly. No activity was detected with ADP. 5. The enzyme requires a divalent cation for activity and Mg2+ is the most effective. It is not significantly stimulated by K+ or bicarbonate and Ca2+ is inhibitory. 6. The activity cannot be assayed in intact cells unless they are permeabilized with toluene. This suggest that the active site is on the cytoplasmic side of the plasma membrane.
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PMID:Characterization of the plasma membrane ATPase of Saccharomyces cerevisiae. 15 59

The effect of organic solvents on the beef heart mitochondrial ATP-base-catalyzed ATP and ITP hydrolysis was examined. It was observed that numerous organic solvents stimulated ATP hydrolysis while ITP hydrolysis was inhibited. Methanol at 20% (v/v) was found to stimulate ATP hydrolysis by over 300%, while at the same methanol concentration ITP hydrolysis was inhibited approximately 50%. In the presence of 20% methanol, ATP hydrolysis exhibited linear plots of 1/[ATP] vs. 1/v, while in the absence of methanol negative cooperativity was observed. These data can be interpreted to imply that the catalytic and regulatory sites of the mitochondrial ATPase are being dissociated 20% methanol. The effect of methanol on the hydrolysis of ATP and ITP was examined as a function of pH. It was found that, at high pH in totally aqueous solutions, the hydrolysis of ATP and ITP was inhibited, while the presence of 20% methanol either caused the hydrolytic rate to peak and remain constant above pH 8 (with ATP as substrate) or caused the rate of hydrolysis to continue to increase above pH 8 (when ITP was the substrate). These data are interpreted to indicate that an acidic group in the active site may be ionizing, limiting the ATPase-catalyzed hydrolytic rate, and, with 20% methanol, this ionization was inhibited.
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PMID:Effect of organic solvents on the beef heart mitochondrial adenosine triphosphatase. 15 24

An attempt has been made to determine the location of the site at which the metabolism of ethanol interacts with that of choline to produce an increase in the oxidation of choline. The first enzyme in the oxidation pathway for choline, choline dehydrogenase, was assayed using a newly developed spectrophotometric assay and freshly isolated intact rat liver mitochondria. No changes were observed in either 'apparent' V or the 'apparent' Km values of choline dehydrogenase for choline after ethanol ingestion. However, when the choline oxidase system was assayed, a 28% decrease in 'apparent' Km for choline and a 53% increase in 'apparent' V was observed. The effects of ATP on choline oxidase were studied further, and a 29.4% decrease was observed in mitochondrial ATP levels from freshly isolated mitochondria from the ethanol-treated rats. In vitro aging of mitochondria further decreased the level of ATP, and the rate of decrease was considerably faster during the first hour in the mitochondria from the ethanol-treated animals. The decreases in ATP from both control and experimental mitochondria were accompanied by increases in choline oxidase activity. The initial decrease in ATP was correlated with an increase in mitochondrial ATPase activity which may be related to an increase in mitochondria Mg2+. Because chronic ethanol ingestion has resulted in decreased oxidation rates of succinate and beta-hydroxybutyrate while at the same time increasing the oxidation rates of choline, the studies reported here suggest that the effect of chronic ethanol ingestion is primarily on a step that is unique to choline and which probably exists prior to the electron transport chain.
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PMID:A possible mechanism for the increased oxidation of choline after chronic ethanol ingestion. 15 26

1. F1-ATPase has been extracted by the diphosphatidylglycerol procedure from mitochondrial ATPase complexes that differ in ATPase activity, cold stability, ATPase inhibitor and magnesium content. 2. The ATPase activity of the isolated enzymes was dependent upon the activity of the original particles. In this respect, F1-ATPase extracted from submitochondrial particles prepared in ammonia (pH 9.2) and filtered through Sephadex G-50 was comparable to the enzyme purified by conventional procedures (Horstman, L.L. and Racker, E. (1970) J. Biol. Chem. 245, 1336--1344), whereas F1-ATPase extracted from submitochondrial particles prepared in the presence of magnesium and ATP at neutral pH was similar to factor A (Andreoli, T.E., Lam, K.W. and Sanadi, D.R. (1965) J. Biol. Chem. 240, 2644--2653). 3. No systematic relationship has been found in these F1-ATPase preparations between their ATPase inhibitor content and ATPase activity. Rather, a relationship has been observed between this activity and the efficiency of the ATPase inhibitor-F1-ATPase association within the membrane. 4. It is concluded that the ATPase activity of isolated F1-ATPase reflects the properties of original ATPase complex provided a rapid and not denaturing procedure of isolation is employed.
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PMID:F1-ATPase from different submitochondrial particles. 15 27

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.
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PMID:The use of arylazido-beta-alanyl-ATP as a photoaffinity label for the isolated and membrane-bound mitochondrial ATPase complex. 15 61

A heat-stable protein has been purified from rat liver mitochondria which inhibits the ATP hydrolytic activity of both the soluble and membrane-bound mitochondrial F1-ATPase. The overall purification is about 2400-fold with the major purification step consisting of Sephadex "affinity" chromatography. The purified rat liver inhibitor is homogeneous as assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis with an apparent molecular weight of 12,300. Amino acid analysis reveals a high content of glutamic acid, lysine, and arginine and the absence of cysteine, proline and methionine. Whether tested with the rat liver or bovine heart ATPase, the liver inhibitor is equally as potent and specific as the heart inhibitor preparation of Pullman and Monroy (Pullman, M.E., and Monroy, G.C. (1963) J. Biol. Chem. 238, 3762-3769). Although the results presented show that the rat liver ATPase inhibitor resembles closely the ATPase inhibitors from other tissues with respect to specific activity and reaction specificity, it is important to note that the rat liver inhibitor is almost 2000 daltons larger than the bovine heart inhibitor, about 5000 daltons larger than ATPase inhibitors of yeast, and contains significantly more lysine residues than both the bovine heart and yeast inhibitors.
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PMID:A protein inhibitor of the mitochondrial adenosine triphosphatase complex of rat liver. Purification and characterization. 15 68


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