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S M Schuster

Publications and source records attributed to S M Schuster.

At least 109 records · Page 6Linked to original sources
PubMed1981

Control of beef heart submitochondrial particle-catalyzed Pi goes to and comes from ATP exchange by nucleotides and the ATPase inhibitor protein.

F1I, the specific ATPase inhibitor protein, and the chromium(III) analogs of ATP and ADP, CrATP and CrADP, were used to study the inhibition of Pi goes to and comes from ATP exchange reaction catalyzed by beef heart submitochondrial particles. F1I was found to be an uncompetitive inhibitor of the exchange reaction. CrATP and CrADP, both competitive inhibitors of ATP hydrolysis in isolated F1 (Schuster, S. M., Ebel, R. E., and Lardy, H. A. (1975) ARch. Biochem. Biophys. 171, 656-661) were shown to be competitive and noncompetitive inhibitors of Pi goes to and comes from ATP exchange, respectively. Dual inhibitor studies were done using combinations of F1I and the chromium nucleotides, or the nucleotide analogs in combination. All cases show sets of intersecting Dixon plots indicative of interacting inhibitors. Upward curvature is also evident on some of the plots. This phenomenon was explained using the concept of multiple synergistic binding of the inhibitors. Binding mechanisms and their relevant kinetic equations were postulated to explain the results of the dual inhibitor studies. They support the notion that in addition to the catalytic site, there are two types of regulatory binding sites on F1, one specific for nucleotides and one specific for F1I. When one of these sites is occupied, other sites are either opened or other inhibitors become more potent.

Adenosine Diphosphate↗
PubMed1981

The effect of nitration and D2O on the kinetics of beef heart mitochondrial adenosine triphosphatase.

The role of tyrosine in the catalytic mechanism of nucleoside triphosphate hydrolysis by beef heart mitochondrial ATPase is explored. We compare the rates of the ATPase reaction by both nitrated and native F1 at both pH 8 and pH 6. The pH-activity profile of nitrated F1 is compared to the pH-activity profile of the unmodified enzyme. These data indicate that the phenolic group of an active-site tyrosine must be protonated during the hydrolysis reaction. Deuterium oxide is used in the reaction buffer to explore the role of protons in the ATPase reaction. Kinetic constants of the nucleoside triphosphates are obtained at various levels of D2O using both the nitrated and native forms of F1. Several nucleoside diphosphates are used as inhibitors of F1-catalyzed ITP hydrolysis. Dissociation constants of these inhibitors are obtained at both low and high concentrations of D2O for both the nitrated and native F1. We explore the possibility that a tyrosine and an arginine lie in close proximity in the F1 active site by studying the effects of sequential modification of arginine and tyrosine. These results are interpreted in terms of possible ATP hydrolysis mechanisms. Two possible roles for tyrosine in the hydrolysis of nucleoside triphosphates by F1 are suggested.

Adenosine Triphosphatases↗
PubMed1981

Catalysis of partial reactions of ATP synthesis by beef heart mitochondrial adenosine triphosphatase.

We have found that when the ATP hydrolysis activity of beef heart mitochondrial adenosine triphosphatase (F1) is eliminated by either cold treatment or chemical modification, the enzyme attains the ability to catalyze the Pi in equilibrium ATP exchange reaction. The ATP hydrolysis activity of isolated F1 was lost upon chemical modification by phenyglyoxal, butanedione, or 7-chloro-4-nitrobenzene-2-oxa-1,3-diazole. The F1 thus chemically modified was able to catalyze an ADP-dependent Pi in equilibrium ATP exchange reaction. In addition F1 that had been cold-treated to eliminate ATP hydrolysis activity, also catalyzed the Pi in equilibrium ATP exchange reaction. The Pi in equilibrium ATP exchange catalyzed by modified F1 was shown to be totally inhibited by the F1-specific antibiotic efrapeptin. We have previously shown that isolated beef heart mitochondrial ATPase will catalyze the formation of a transition state analog of the ATP synthesis reaction (Bossard, M. J., Vik, T. A., and Schuster, S. M. (1980) J. Biol. Chem. 255, 5342-5346). While the F1-catalyzed ATP hydrolysis activity was lost rapidly upon chemical modification or cold treatment, the ability of the enzyme to produce Pi . adenosine 5'-diphosphate (chromium(III) salt) from phosphate and monodentate adenosine 5'-diphosphate (chromium(III) salt) was unimpaired. The implications of these data with regard to the mechanism of ATP synthesis are discussed.

Adenosine Diphosphate↗
PubMed1980

Beef heart mitochondrial adenosine triphosphatase-catalyzed formation of a transition state analog in ATP synthesis.

The mechanism of beef heart mitochondrial ATPase (F1) was studied using chromium(III)-substituted substrate analogs. Incubation of F1 with monodentate Cr(III)ADP and 32Pi followed by chromatography on Sephadex G-25 resulted in 32Pi counts in the F1 protein peak. The appearance of radioactivity from inorganic phosphate in the protein peak was dependent upon the presence of monodentate Cr(III)ADP and F1, and was inhibited by MgADP. Removal of the enzyme from the reaction mixture containing Cr(III)ADP and 32Pi by acid precipitation followed by chromatography on Sephadex G-10 indicated the net formation and slow release of a radioactive product. High voltage electrophoresis showed that this product was 32Pi . Cr(III)ADP. Incubation of F1 with bidentate Cr(III)ATP also resulted in formation of this product. These results indicated that non-membrane-bound F1 was capable of net synthesis of what may be an ATP synthesis and hydrolysis transition state analog. The F1-dependent formation of the complex was taken as evidence that the soluble ATPase can function in ATP synthesis as well as ATP hydrolysis.

Adenosine Diphosphate↗
PubMed1980

Kinetic and thermodynamic properties of beef heart mitochondrial ATPase: effect of co-solvent systems.

The effects of glycerol and methanol upon beef heart mitochondrial ATPase (F1) were studied. Glycerol was found to be a potent reversible inhibitor of the F1-catalyzed hydrolysis of ATP and ITP. The inhibition of ATP hydrolysis was linear with respect to glycerol concentrations, while that of ITP was not. From the temperature dependence of Vmax for F1-catalyzed ATP and ITP hydrolysis in glycerol or methanol solutions, the energy of activation and the enthalpy of activation were calculated. The inhibitory effect of ADP on F1 hydrolytic activity was studied in three solvent systems (totally aqueous, 20% methanol, and 20% glycerol). Compared to the aqueous system, methanol decreased the potency of ADP as an inhibitor, and glycerol enhanced the potency.

Adenosine Triphosphatases↗
PubMed1980

A new assay for L-asparagine synthetase.

A fast, relatively inexpensive method of measuring the enzymatic formation of L-asparagine from L-aspartate is presented. This radiochemical assay requires simple manipulations making it ideal for working with large numbers of samples, while maintaining high sensitivity and reproducibility. A mechanism similar to the enzymatic beta-decarboxylation of aspartate is utilized but in a nonenzymatic reaction. In the presence of pyridoxal and A13+ ions, the 14C of L-[4-14C]aspartate is decarboxylated while L-[4-14C]asparagine remains intact. This assay is shown to be suitable for measuring mammalian L-asparagine synthetase activity, while not requiring the isolation of assay enzymes.

Aluminum↗
PubMed1979

Effect of organic solvents on the beef heart mitochondrial adenosine triphosphatase.

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.

Adenosine Triphosphatases↗
PubMed1979

Regulation of carbohydrate metabolism in mouse liver. Effect of glucagon on gluconeogenic/glycolytic flux in isolated perfused livers.

1. Glucagon stimulated gluconeogenesis from both [U-14C]lactate and [14C]xylitol in isolated perfused mouse liver. 2. Addition of cyclic AMP also stimulated gluconeogenesis from [U-14C]lactate. 3. Glucagon caused a rapid (2.5 min) 12-fold increase in hepatic cyclic AMP but not cyclic GMP concentration. 4. Glucagon caused a rapid and stable decrease in hepatic fructose 1,6-diphosphatase activity measured in vitro. 5. The results are interpreted to indicate that glucagon stimulates hepatic gluconeogenesis in mice via cyclic AMP by two different mechanisms: (a) increased substrate uptake (i.e. utilization) and (b) increased gluconeogenic efficiency (i.e. inhibition of alternate substrate fates).

Animals↗
PubMed1978

A catalytic generator for the production of H2 14O and H2 15O.

A technique is described for the continuous production of water labelled with 14O or 15O. Total activities are achieved which are virtually independent of the distance separating the accelerator from the user site, limited only by beam current and energy. The specific activity is determined by the user's needs, since the entire 100-mCi inventory can be collected in liquid volumes on the order of microliters. The studies triggered by this resource range from basic investigations in physics and biochemistry to practical nuclear medical procedures.

Isotope Labeling↗
PubMed1977

Studies on the kinetic mechanism of oxidative phosphorylation.

The kinetics of the synthesis of ATP from ADP and Pi by beef heart submitochondrial particles were examined. When Pi was the variable substrate positive cooperativity was observed, whereas if ADP was varied, linear double reciprocal plots were obtained. The analog of Pi, thiophosphate, was a noncompetitive inhibitor of ATP synthesis with respect to ADP, while the analog of ADP, AMP (CH2)P, was an uncompetitive Pi leads to ATP exchange inhibitor. The kinetics of the initial velocity isotopic exchanges of oxidative phosphorylation were also examined. When the Pi leads to ATP exchange was examined, it was found that if ADP concentration was held constant while ATP and Pi concentrations were varied at a constant ratio, linear double reciprocal plots were obtained. However, if Pi concentration was held constant and ADP and ATP concentrations were varied at constant ratio, apparent substrate inhibition was observed. The 2, 4-dinitrophenol-sensitive ADP leads to ATP exchange showed linear double reciprocal plots regardless of which components were varied. These results are interpreted to indicate that in the direction of ATP synthesis, the reaction is ordered, with Pi adding to the enzyme before ADP addition.

Adenosine Diphosphate↗
PubMed1976

Effect of inosine 5' -(beta, gamma-imido) triphosphate and other nucleotides on beef heart mitochondrial ATPase.

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.

Adenine Nucleotides↗