Sheldon M. Schuster

Immunoaffinity chromatography utilizing monoclonal antibodies: Factors which influence antigen-binding capacity

Differences in antigen-binding capacity of a monoclonal antibody coupled to Sepharose under varying conditions were explored. The extent of cyanogen bromide activation, and the pH of the coupling reaction had a profound effect upon the rate of antibody coupling, but only small differences in antigen-binding capacity were observed if the antibody coupling reaction was terminated when 80-90% of the antibody was covalently coupled to Sepharose. However, if antibody was incubated with activated resin until 100% coupling was attained, the antigen-binding capacity of the resulting immunoadsorbent decreased significantly. Monoclonal antibody coupled to Sepharose via an N-hydroxysuccinimide ester linkage and approximately half the antigen-binding capacity of antibody coupled by CNBr activation. Concentrations of monoclonal antibodies as high as 13 mg/ml of packed resin could be used without noticeable steric hindrance.

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.

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.

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.

Effect of citreoviridin and isocitreoviridin on beef heart mitochondrial ATPase

Citreoviridin is a toxic metabolite from fungus that has been shown to be an inhibitor of mitochondrial F1-ATPases. Studies of citreoviridin, however, have been compromised by the light-dependent isomerization that it undergoes. The isomerization is a potential source of extensive variability in the studies, if citreoviridin and isocitreoviridin have different kinetic effects and binding properties. Both citreoviridin and isocitreoviridin recently have been purified and have been shown to be stable in the dark. Using the purified isomers, the effects of both citreoviridin and isocitreoviridin on soluble and membrane-bound beef heart mitochondrial F1-ATPase activity were investigated. It was found that citreoviridin was an uncompetitive inhibitor of ATP hydrolysis, and a non-competitive inhibitor of ITP hydrolysis catalyzed by soluble F1-ATPase. Isocitreoviridin had no effect on the hydrolysis of either of the triphosphates catalyzed by soluble F1-ATPase. The inhibition constant, Ki for citreoviridin was determined as 4.5 microM for ATP hydrolysis. The inhibition constants Kii and Kis for ITP hydrolysis were determined as 4.3 and 1.03 microM, respectively. Citreoviridin was an uncompetitive inhibitor of ATP hydrolysis and a noncompetitive inhibitor of ATP synthesis catalyzed by membrane-bound F1-ATPase. The inhibition constant, Ki, for ATP hydrolysis was around 4 microM. For ATP synthesis the inhibition constants were determined as 0.12 and 0.16 microM for Kis and Kii, respectively, when ADP concentration was kept saturating. Isocitreoviridin had no effect on either activity of the membrane-bound enzyme.

Separation of β,γ-bidentate Cr · ATP diastereomers by reverse-phase high-performance liquid chromatography

The separation of the four diastereomers of β,γ-bidentate Cr · ATP using reverse-phase HPLC techniques is described. This technique provides complete resolution of the diastereomers within 10 min and relies on the use of methanesulfonic acid (in the ionized form) as an ion-pairing agent. To identify the screw sense of these resolved isomers, the CD spectra of each isomer were done, and substrate and inhibition specificities were examined using hexokinase. The results were then correlated with the isomeric assignments made by D. Dunaway-Mariano and W. W. Cleland (1980, Biochemistry19, 1506–1515). Further studies included the monitoring of isomer interconversion at pH 6.2 to an equilibrium concentration of all four, and specific rotation measurements of the pure isomers at pH 2.5, 23°C, and 546 nm.

A graph-theoretic approach to quantitative structure—activity/reactivity studies

Abstract Characterization of molecular species based on the use of suitable graph invariants (graph paths, in particular) can provide a quantitative means of encoding structure; the technique is complementary to commoner approaches to studies of quantitative structure— activity relationships. Graph path encoding is here applied to quantitative studies of relationships between molecular structures and biological activity; the examples are the rates of various substrate reactions with hexoldnase, and the potential opiate-like activity of enkephalin analogs.

Asparagine catabolism in rat liver mitochondria

A large portion of mitochondrial asparagine (Asn) is degraded by asparagine amino-transferase to produce alpha-ketosuccinamate (alpha KSA), which is then hydrolized by omega-amidase to produce oxaloacetate (OAA) and ammonia. This is in contrast to the catabolism in the cytosol, where the main catabolic route for Asn occurs initially via asparaginase-catalyzed hydrolysis to form aspartate and ammonia. Mitochondrial production of OAA from Asn was followed by monitoring the decrease in the rate of succinate oxidation (which is inhibited by OAA) in both coupled and uncoupled mitochondria. Rapid OAA production was found to be dependent on the presence of both Asn and glyoxylate, and was eliminated by the aminotransferase inhibitor, aminooxyacetate (AOX). HPLC separation and quantitation of alpha-keto acids and amino acids allowed direct observation of the proposed mitochondrial pathway. Studies using L-[U-14C]Asn in mitochondria yielded labeled carbon in alpha KSA, OAA, and CO2 when either an alpha-keto acid or glyoxylate was provided. The extent of the labeled carbon in these products was greatly influenced by factors that affected the citric acid cycle and oxidative phosphorylation. Carbon dioxide production from Asn alone, even in the presence of AOX, suggested the existence of at least one additional Asn catabolic pathway in the rat liver mitochondria which does not involve alpha KSA as an intermediate.

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.

Substrates of hydroxyketoglutarate aldolase

Abstract The substrate specificity of the condensation reaction catalyzed by rat liver 4-hydroxy-2-ketoglutarate aldolase has been investigated. It was found that an enzyme-mediated condensation between-glyoxylate and several “activated” carbonyl compounds could be performed. Two classes of these “activated” carbonyls were tested—the first of which are pyruvate analogs differing by substitution at C-3, whereas the second include some C-1 analogs of pyruvate as well as other simple carbonyl compounds. The possible synthetic uses of such a system are discussed as well as possible insights into the structure of the active site of this enzyme.