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 membrane-bound coupling factor from Mycobacterium phlei was solubilized from membrane vesicles by washing with low ionic strength buffer or 0.25 M sucrose. The solubilized enzyme exhibited coupling factor, latent ATPase, and succinate oxidation-stimulating activity. Purification by affinity chromatography using Sepharose coupled to ADP yielded a homogeneous preparation of latent ATPase which was purified about 200-fold with an 84% yield in a single step. Purified latent ATPase exhibited coupling factor activity but no succinate oxidation-stimulating activity. The molecular weight of latent ATPase was determined to be 250,000 +/- 10,000 by Sephadex G-200 chromatography. The ATPase was unmasked by trypsin treatment and activated by Mg2+ ion. However, trypsin treatment inactivated the coupling factor activity in the purified enzyme, indicating that the catalytic sites for ATPase and coupling activity are different. Unlike mitochondrial ATPase, latent ATPase from M. phlei was not cold-labile. Of the nucleoside triphosphates, UTP, ITP, and epsilon-ATP (1-N6-ethenoadenosine triphosphate) were hydrolyzed to a lesser extent compared to ATP. Kinetic data showed that ADP acted as a competitive inhibitor of latent ATPase activity with a Ki of 5 x 10(-3) M. Uncouplers of oxidative phosphorylation and respiratory inhibitors did not affect the latent ATPase activity, while sodium azide (0.1 mM) inhibited the latent ATPase activity.
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PMID:Energy-transducing membrane-bound coupling factor-ATPase from Mycobacterium phlei. I. Purification, homogeneity, and properties. 12 54

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.
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PMID:Exploring sites on mitochondrial ATPase for catalysis, regulation, and inhibition. 12 84

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 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

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.
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PMID:Stabilization of rat liver mitochondrial F1-adenosine triphosphatase during chloroform-induced solubilization. 15 60

Three ATP-dependent reactions catalyzed by the inner membrane of rat liver mitochondria and the ATPase reaction catalyzed by purified mitochondrial ATPase (F1), were studied with respect to kinetic properties, substrates specificity, and sensitivity to bicarbonate. The ATP-dependent transhydrogenase reaction (reduction of NADP+ by NADH) catalyzed by inner membrane vesicles displays typical Michaelis-Menten kinetics in both Tris-Cl and Tris-bicarbonate buffers, with Km (ATP) values of 0.035 mM and 0.054 mM respectively. The Vmax of transhydrogenase activity (25 nmol min-1 mg-1) is the same in Tris-bicarbonate or Tris-Cl buffer. ITP and GTP readily substitute for ATP in the transhydrogenase reaction. The ATP-P1 exchange reaction catalyzed by inner membrane vesicles displays typical Michaelis-Menten kinetics in both Tris-Cl and Tris-bicarbonate buffers with Km (ATP) values of 1.0 mM and 1.4 mM respectively. The Vmax of exchange (200 nmol min-1 mg-1) is the same in either buffer. ITP and GTP do not effectively replace ATP in the exchange reaction.
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PMID:ATP-dependent reactions catalyzed by inner membrane vesicles of rat liver mitochondria. Kinetics, substrate specificity, and bicarbonate sensitivity. 17 67

Treatment of either beef heart or rat liver mitochondrial ATPase with the arginine reagent, 2,3-butanedione, resulted in enzyme inactivation. The reaction followed pseudo-first order kinetics until 90 to 95% of the enzyme had been inactivated, and prolonged incubation with butanedione resulted in complete inactivation. When the modification reaction was performed in the presence of ATP, the rate of inactivation was significantly decreased. The kinetics of inactivation indicates that the reaction of 1 molecule of reagent per active site of beef heart mitochondrial ATPase is necessary for inactivation. The loss of ATPase activity was also observed when submitochondrial particles were treated with butanedione. Studies with beef heart mitochondrial ATPase indicated that the inactivation was not due to enzyme dissociation into subunits. Kinetic studies with partially inactivated enzyme demonstrated that the Km values of ITP and of ATP in the presence of HCO3-were similar to the same constants for the control enzyme. When ATP was used as the substrate in the absence of anion activator, the partially inactivated enzyme still exhibited negative cooperativity. Inactivation was also observed when beef heart mitochondrial ATPase was treated with another arginine reagent, phenylglyoxal. The loss of ATPase activity was analyzed in terms of [14C]phenylglyoxal incorporation. From the present studies it is concluded that arginyl residues play an essential role in mitochondrial ATPase, probably at the hydrolytic site.
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PMID:Essential arginyl residues in mitochondrial adenosine triphosphatase. 17 62

Spinach leaf mitochondrial F0F1 ATPase has been purified and is shown to consist of twelve polypeptides. Five of the polypeptides constitute the F1 part of the enzyme. The remaining polypeptides, with molecular masses of 28 kDa, 23 kDa, 18.5 kDa, 15 kDa, 10.5 kDa, 9.5 kDa and 8.5 kDa, belong to the F0 part of the enzyme. This is the first report concerning identification of the subunits of the plant mitochondrial F0. The identification of the components is achieved on the basis of the N-terminal amino acid sequence analysis and Western blot technique using monospecific antibodies against proteins characterized in other sources. The 28-kDa protein crossreacts with antibodies against the subunit of bovine heart ATPase with N-terminal Pro-Val-Pro- which corresponds to subunit F0b of Escherichia coli F0F1. Sequence analysis of the N-terminal 32 amino acids of the 23-kDa protein reveals that this protein is similar to mammalian oligomycin-sensitivity-conferring protein and corresponds to the F1 delta subunit of the chloroplast and E. coli ATPases. The 18.5-kDa protein crossreacts with antibodies against subunit 6 of the beef heart F0 and its N-terminal sequence of 14 amino acids shows a high degree of sequence similarity to the conserved regions at N-terminus of the ATPase subunits 6 from different sources. ATPase subunit 6 corresponds to subunit F0a of the E. coli enzyme. The 15-kDa protein and the 10.5-kDa protein crossreact with antibodies against F6 and the endogenous ATPase inhibitor protein of beef heart F0F1-ATPase, respectively. The 9.5-kDa protein is an N,N'-dicyclohexylcarbodiimide-binding protein corresponding to subunit F0c of the E. coli enzyme. The 8.5-kDa protein is of unknown identity. The isolated spinach mitochondrial F0F1 ATPase catalyzes oligomycin-sensitive ATPase activity of 3.5 mumol.mg-1.min-1. The enzyme catalyzes also hydrolysis of GTP (7.5 mumol.mg-1.min-1) and ITP (4.4 mumol.mg-1.min-1). Hydrolysis of ATP was stimulated fivefold in the presence of amphiphilic detergents, however the hydrolysis of other nucleotides could not be stimulated by these agents. These results show that the plant mitochondrial F0F1 ATPase complex differs in composition from the other mitochondrial, chloroplast and bacterial ATPases. The enzyme is, however, more closely related to the yeast mitochondrial ATPase and to the animal mitochondrial ATPase than to the chloroplast enzyme. The plant mitochondrial enzyme, however, exhibits catalytic properties which are characteristic for the chloroplast enzyme.
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PMID:Plant mitochondrial F0F1 ATP synthase. Identification of the individual subunits and properties of the purified spinach leaf mitochondrial ATP synthase. 131 68

The beta-subunit of the mitochondrial ATP synthase complex comprises the bulk, if not all, of the catalytic nucleotide binding site on the enzyme. A region of homologous sequence rich in glycines (G) and containing a basic lysine (K) and a threonine (T) is found in the beta-subunit as well as many other purine nucleotide binding proteins. The consensus sequence of this region is Gx4GKT, where x represents any amino acid, and is called the A region or glycine-rich loop. The related function of these proteins implies that the glycine-rich loop is directly involved in nucleotide binding. Here we directly test the involvement of the beta-subunit's glycine-rich region in adenine nucleotide binding using two independent approaches. A synthetic fifty amino acid peptide, PP-50, containing the glycine-rich region and the surrounding sequence was assessed for secondary structure and interaction with potential ligands. Circular dichroism spectropolarimetry indicates that PP-50 assumes a predominantly beta-sheet conformation in solution. Significantly, the peptide precipitates from solution when ATP, ADP, GTP, ITP, and pyrophosphate are added, but not when AMP or phosphate are included. Magnesium is not required for the interaction with the purine nucleotides. Complimentary to these studies, the sequence around the Gx4GKT motif was deleted from a recombinant rat liver beta-subunit overexpressed in E. coli. While the wild type beta-subunit showed specificity for the tri- and diphosphonucleotides, the deletion mutant bound tri-, di-, and monophosphate nucleotides with equal affinity.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Mitochondrial F-type ATPases: the glycine-rich loop of the beta-subunit is a pyrophosphate binding domain. 133 55

1. This paper is the first detailed report of the purification of a mitochondrial ATPase from an avian species. 2. The Gallus gallus liver mitochondrial F1-ATPase was purified by chloroform extraction and ion-exchange chromatography. 3. The enzyme shows the five alpha, beta, tau, delta, and epsilon subunits characteristic of mitochondrial F1-ATPases. 4. The Km for ATP is 1 mM and for Mg 0.5 mM with a specific activity of 25.2 mu moles of ATP hydrolyzed x min-1 x mg-1. 5. Unlike mammals enzymes the chicken mitochondrial ATPase shows maximal activity with ITP as substrate, and is strongly inhibited by Cu.
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PMID:Purification and properties of the F1-ATPase from liver mitochondria of Gallus gallus. 145 35


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