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S M Schuster

Publications and source records attributed to S M Schuster.

At least 91 records · Page 5Linked to original sources
PubMed1985

Interaction of azide with beef heart mitochondrial ATPase.

This study examined the inhibition of azide as a probe of the magnesium regulation of beef heart mitochondrial ATPase (F1) catalysis. Azide elicited a slow hysteretic effect on both ATP and ITP hydrolysis of F1. This hysteretic effect was shown to be due to the consecutive binding of magnesium and azide, and to be independent of catalytic turnover. The azide binding site was also shown to be separate from the anion binding HCO3- site on F1. The results presented indicate that metal binding is important in the inhibition of the hydrolytic activity and regulation of F1. A model is presented which is consistent with the hysteretic inhibition of F1 by azide, in which there is a slow equilibration between free enzyme and the enzyme-magnesium-azide complex.

Animals↗
PubMed1985

Purification and characterization of beef pancreatic asparagine synthetase.

Bovine pancreatic asparagine synthetase has been partially purified using ammonium sulfate fractionation, DEAE ion-exchange, Cibacron Blue affinity chromatography, and HPLC anion-exchange chromatography to a specific activity of 170 nmol asparagine produced min-1 mg protein-1, or 1400-fold, from a crude homogenate. Using HPLC size exclusion chromatography, an apparent molecular weight of 110,000-120,000 was determined. An aspartyl-adenylate intermediate was found to occur by demonstrating an 18O transfer from [18O]Asp to AMP that was detected with 31P NMR. A number of divalent metals were found to be able to replace magnesium with retention of activity, but none produced as high an activity as Mg2+, and the stoichiometry of the ATP/Mg2+ ratio was found to be 1. The chloride ion was found to stimulate the glutamine-dependent and glutaminase reactions, but the ammonia-dependent reaction was inhibited. Chloride appeared to be a competitive inhibitor with respect to ammonia and produced negative cooperativity.

Allosteric Regulation↗
PubMed1985

Rat liver 4-hydroxy-2-ketoglutarate aldolase: purification and kinetic characterization.

The enzyme 4-hydroxy-2-ketoglutarate aldolase (4HKG aldolase), which catalyzes the reversible cleavage of 4-hydroxy-2-ketoglutarate to form pyruvate and glyoxylate, was isolated from rat liver. The purification scheme as well as a study of several of the physical and kinetic properties of the enzyme are presented. The effects of anions, various buffers, and possible physiologically relevant effectors on the kinetic parameters of the aldolase were also investigated. It was found that pyruvate analogs inhibited the aldolase. Oxaloacetate was a competitive inhibitor of the aldolase, and in addition caused synergistic inhibition with respect to pyruvate analogs at low substrate concentration. These results are discussed in terms of possible regulation of the aldolase.

Animals↗
PubMed1985

Amino acid content of L5178Y and L5178Y/L-ASE cells after L-asparaginase treatment.

The amino acid contents of tumor cells that are either sensitive or resistant to treatment with L-asparaginase were measured. These amino acid concentrations were measured as a function of incubation time with L-asparaginase or as a function of the L-asparaginase dose. The cell types compared were the mouse leukemia lines L5178Y (sensitive to L-asparaginase treatment) and L5178Y/L-ASE (resistant to L-asparaginase treatment). Upon L-asparaginase treatment both cell lines lost most of their cellular asparagine but, whereas the resistant cells exhibited the ability to rebound to about 50% of initial values, the sensitive cells did not. While previous work had suggested that asparagine-dependent glycine synthesis was essential for sensitive cells (but not in resistant cells), we found no difference in the glycine content of either of the two cell lines as a function of either time or dose that would support this hypothesis. Major differences between the two cell lines were seen in the content of the essential amino acids before treatment with L-asparaginase. After incubation without L-asparaginase the contents of the two cell lines became similar. These results are discussed in terms of possible mechanisms of L-asparaginase sensitivity and resistance.

Amino Acids↗
PubMed1985

Isolation and Antigenic Characterization of Corn Mitochondrial F(1)-ATPase.

Corn mitochondrial F(1)-ATPase was purified from submitochondrial particles by chloroform extraction. Enzyme stored in ammonium sulfate at 4 degrees C was substantially activated by ATP, while enzyme stored at -70 degrees C in 25% glycerol was not. Enzyme in glycerol remained fully active (8-9 micromoles P(i) released per minute per milligram), while the ammonium sulfate preparations steadily lost activity over a 2-month storage period. The enzyme was cold labile, and inactived by 4 minutes at 60 degrees C. Treatment with octylglucoside resulted in complete loss of activity, while vanadate had no effect on activity. The apparent subunit molecular weights of corn mitochondrial F(1)-ATPase were determined by SDS-polyacrylamide gel electrophoresis to be 58,000 (alpha), 55,000 (beta), 35,000 (gamma), 22,000 (delta), and 12,000 (epsilon). Monoclonal and polyclonal antibodies used in competitive binding assays demonstrated that corn mitochondrial F(1)-ATPase was antigenically distinct from the chloroplastic CF(1)-ATPases of corn and spinach. Monoclonal antibodies against antigenic sites on spinach CF(1)-ATPase beta and gamma subunits were used to demonstrate that those sites were either changed substantially or totally absent from the mitochondrial F(1)-ATPase.

Journal Article↗
PubMed1985

Kinetic Analysis of Corn Mitochondrial F(1)-ATPase.

The activation and catalytic mechanism of corn mitochondrial F(1) were examined for the two distinct forms of the enzyme which appear upon storage in ammonium sulfate or glycerol. Apparently irreversible differences in the stability of the two active forms were found. Nucleosidetriphosphate induced activation of the enzyme was found to produce lasting effects on subsequent catalysis. These effects varied with both the nucleotide used for activation, and the hydrolyzed species. The substrate and metal specificity were examined with the ATP activated enzyme. Mg(2+) and Ca(2+) were found to be the most effective at promoting ATP hydrolysis. The substrates were hydrolyzed in the order GTP > ITP > ATP regardless of which nucleotide was used for activation. While ATP and GTP hydrolysis exhibited kinetics typical of other ATPases, ITP showed a transition from negative to positive cooperativity at low substrate concentrations. Bicarbonate was found to affect primarily the kinetics of ATP hydrolysis. AMP-PNP proved to be a potent inhibitor with respect to ATP hydrolysis. The results are discussed in terms of possible catalytic mechanisms and the similarities of the corn mitochondrial F(1) to other ATPases.

Journal Article↗
PubMed1984

Metal dependence and thermodynamic characteristics of the beef heart mitochondrial adenosine triphosphatase.

A systematic study was done examining the steady state kinetics of F1-catalyzed nucleotide hydrolysis in the presence of various activating divalent metal cations. Values of Km and kcat were obtained from Lineweaver-Burk plots, and kcat/Km values were calculated. With some exceptions, kcat/Km was shown to be independent of the metal present for F1-catalyzed ATP hydrolysis, independent of the nucleotide hydrolyzed with magnesium as the metal cation present, and independent of temperature with most activating metal cations. An average value for kcat/Km of 2.62 X 10(5) M-1 S-1 is calculated as the lower limit to the second order rate constant for binding substrate to enzyme. Changes in steady state kinetic parameters with temperature were also studied. The Ki value for ADP inhibition of F1-catalyzed ATP hydrolysis with magnesium as divalent cation present was found to be temperature-independent. Plots of log kcat versus 1/T showed either abrupt breaks or straight line dependencies depending on the metal ion present. These results may indicate that different rate-limiting steps in the reaction sequence can be operative at different temperatures depending on the divalent cation present.

Animals↗
PubMed1984

A high-performance liquid chromatography assay for asparagine synthetase.

A highly sensitive method for assaying asparagine synthetase and its glutaminase activity is presented. The amino acids L-asparagine, L-aspartate, L-glutamate, and L-glutamine, are separated by derivatization with o-phthaldialdehyde followed by reversed-phase high-performance liquid chromatography on an Altex ultrasphere-ODS C18 column. The elution is isocratic and the mobile phase used is 50 mM sodium acetate buffer (pH 5.9) with 30% methanol. This assay can easily detect picomoles of asparagine, which may be difficult to do with the other assays that have been described.

Amino Acids↗
PubMed1984

Effects of deuterium on the kinetics of beef heart mitochondrial ATPase.

A study was done examining the steady-state kinetics of F1-catalyzed ATP and ITP hydrolyses in the presence or absence of D2O as a function of temperature. The steady-state kinetic parameters kcat and kcat/Km were obtained. For ATP hydrolysis, kcat/Km was independent of temperature in the presence or absence of D2O, while kcat/Km for ITP hydrolysis increased in both cases. The relative magnitudes of change of kcat and kcat/Km in the presence and absence of D2O over the temperature range studied were much different for the cases of ATP and ITP hydrolysis. A normal isotope effect was observed in plots of kcat H2O/kcat D2O versus temperature for ATP hydrolysis, which increased then leveled off as temperature increased. An inverse isotope effect at low temperatures changed to a normal isotope effect and increased dramatically as temperature increased during ITP hydrolysis. The results are discussed in terms of the nature and location of the rate-limiting steps in the reaction mechanisms.

Animals↗
PubMed1984

Pre-steady-state kinetics of beef heart mitochondrial ATPase.

The pre-steady-state kinetics of beef heart mitochondrial ATPase (F1) were examined. F1 was found to exhibit hysteretic behavior when hydrolyzing ATP. The hysteretic property was expressed as an activation process which occurred when the enzyme was mixed with its substrate, MgATP. Many catalytic turnovers were required before the activation was complete. The lag in hydrolysis increased hyperbolically as the concentration of enzyme increased. Passage of F1 through Sephadex G25 eliminated the activation process. Several kinetically distinct possibilities for explaining these data, including multiple nucleotide dissociations, enzyme conformational changes, and regulatory site interactions, are discussed. The enzyme was apparently able to recognize nucleotide in a noncatalytic manner, as evidenced by the fact that F1 preincubated with ADP in the absence of substrate achieved partial activation (smaller lag times) before being introduced to substrate. ADP is also a time-dependent inhibitor, exhibiting a slow hysteretic inhibition in addition to immediate competitive inhibition.

Adenosine Diphosphate↗
PubMed1984

The effects of dietary amino acids on hepatic L-asparagine synthetase.

Rats were fed Roger's synthetic amino acid diets, complete or without asparagine, aspartate, or glutamate, for 2 weeks. Hepatic and serum amino acid content and hepatic asparagine synthetase were assayed before and after the test diets were administered. Special attention was paid to the multiple forms of asparagine synthetase present. Rats fed the test diet without asparagine exhibited high molecular weight (110,000) alpha-form and intermediate molecular weight (57,000) beta-form of asparagine synthetase. Rats fed the complete test diet and the test diet without asparatate exhibited only the beta-form of the enzyme. Rats fed the test diet without glutamate exhibited no enzyme activity. These data are related to the hepatic amino acid levels and to the role of asparagine and asparagine synthetase in amino acid homeostasis.

Amino Acids↗
PubMed1983

Neurospora crassa mutants deficient in asparagine synthetase.

Neurospora crassa mutants deficient in asparagine synthetase were selected by using the procedure of inositol-less death. Complementation tests among the 100 mutants isolated suggested that their alterations were genetically allelic. Recombination analysis with strain S1007t, an asparagine auxotroph, indicated that the mutations were located near or within the asn gene on linkage group V. In vitro assays with a heterokaryon indicated that the mutation was dominant. Thermal instability of cell extracts from temperature-sensitive strains in an in vitro asparagine synthetase assay determined that the mutations were in the structural gene(s) for asparagine synthetase.

Aspartate-Ammonia Ligase↗
PubMed1982

Methotrexate stimulation of asparagine synthetase activity in rat liver.

Methotrexate was found to stimulate asparagine synthetase activity in vivo by approximately six-fold in rat liver. The maximum effect of methotrexate on hepatic asparagine synthetase activity was observed sixteen hours after intraperitoneal injection of the drug. Cycloheximide, like methotrexate, is a protein synthesis inhibitor and was used to determine that asparagine synthetase activity was not preferentially stimulated under stress. As expected, hepatic asparagine synthetase activity falls markedly with the decreased protein synthesis caused by injection of cycloheximide. It is proposed that methotrexate inhibits serine-dependent glycine biosyn-thesis by decreasing the concentration of tetrahydrofolate for serine hydroxymethyltransferase. This leads to a stimulation of asparagine synthetase to provide nitrogen for asparagine-dependent glycine synthesis. This may provide an explanation of the observed chemotherapeutic synergism between asparaginase and methotrexate treatment.

Animals↗
PubMed1982

Kinetics of chromium nucleotide isomer interconversion.

The rates of chromium nucleotide isomer interconversion were studied as a function of pH, ionic strength, and temperature. Nucleotide isomers were separated using high voltage electrophoresis and gel filtration chromatography. The rate of conversion of monodentate adenosine 5'-monophosphate-chromium salt (CrADP) to the bidentate complex increased with increasing pH, temperature, and ionic strength. Optimal stability for CrADP complexes was found to be at pH 3.5 with a temperature of 4 degrees C. It was found that at pH values above 7.0, the chromium complexes rapidly decomposed even at 4 degrees C. It was found that the conversion of monodentate CrADP to binentate CrADP required the removal of one proton by the solvent. The activation energy for the conversion was found to be 7.3 kcal/mol at pH 6.5. The kinetics of the isomer interconversion are described in terms of possible conversion mechanisms.

Adenine Nucleotides↗
PubMed1981

Kinetic mechanism of beef pancreatic L-asparagine synthetase.

The kinetic mechanism of bovine pancreatic asparagine synthetase was deduced from initial velocity studies and product inhibition studies of both the glutamine-dependent and ammonia-dependent reactions. For the glutamine-dependent pathway, parallel lines were observed in the double reciprocal plot of 1/V vs. 1/[glutamine] at varied aspartate concentrations, and in the plot of 1/V vs. 1/[ATP] at varied aspartate concentrations. Intersecting lines were found for the plot of 1/V vs. 1/[ATP] at varied glutamine concentrations. Product inhibition patterns, including dual inhibitor studies for measuring the synergistic effects of multiproduct inhibition, were used to support an ordered bi-uni-uni-ter ping-pong mechanism. Glutamine and ATP sequentially bind, followed by the release of glutamate and the addition of aspartate. Pyrophosphate, AMP, and asparagine are then sequentially released. When the ammonia-dependent reaction was studied, it was found that the mechanism was significantly different. NH3 bound first followed by a random addition of ATP and aspartate. Pyrophosphate, AMP, and asparagine were then sequentially released as in the glutamine-utilizing mechanism. From these studies, a comprehensive mechanism has been proposed through which either glutamine or NH3 can provide nitrogen for asparagine production from aspartate.

Adenosine Triphosphate↗
PubMed1981

Structural preferences for the binding of chromium nucleotides by beef heart mitochondrial ATPase.

The mono- and bidentate forms of adenosine 5'-diphosphate, chromium (III) salt (CrADP) were separated using Sephadex G-10 column chromatography. The isomeric purity of the two forms was monitored using high voltage electrophoresis and column chromatography. The same techniques were employed to assess the purity of the mono-, bi-, and tridentate forms of adenosine 5'-triphosphate, chromium (III) salt (CrATP). Distinct differences in the interaction of beef heart mitochondrial ATPase with the various isomers of chromium nucleotides were seen in kinetic studies. Monodentate CrADP was a competitive inhibitor of the ATP hydrolysis activity of both purified ATPase and submitochondrial particles. However, when ITPase activity was examined, noncompetitive inhibition was observed. The bidentate isomer of CrADP did not affect ATPase activity. Enzymatic synthesis of the transition state analog of ATP synthesis and hydrolysis, Pi-CrADP occurred exclusively with the monodentate isomer of CrADP. It was also found that only the mono- and tridentate forms of CrATP were potent inhibitors of ATP hydrolysis by beef heart mitochondrial ATPase. These results are discussed in terms of possible ATP synthesis and hydrolysis mechanisms.

Adenosine Diphosphate↗