Abstract
This chapter presents the general assay methods and the properties of a representative, well-characterized enzyme from an Acinetobacter soil organism. Four assay methods are commonly used to measure the hydrolysis of glutamine or asparagine: (1) release of ammonia and direct nesslerization; (2) release of ammonia, distillation, and assay with a phenol-hypochlorate reagent; (3) isolation of the radiolabeled dicarboxylic acid on ion-exchange columns; and (4) coupling the formation of dicarboxylic acid to the disappearance of NADH. Asparagine is usually used as a substrate because of its greater stability. The purification procedure consists of several steps: (1) preparation of the crude extracts, (2) CM-Sephadex chromatography, (3) ammonium sulfate fractionation, and (4) DEAE-Sephadex chromatography. Several enzymes with glutaminase activity, optimal activity at physiologic pH, and antitumor activity have been isolated. These enzymes have an identical 8 amino acid segment that contains the threonine residue binding site for 6-diazo-5-oxonorleucine.
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