Chinese Hamster Liver Glutamine Synthetase

Abstract

Purification procedures to prepare homogeneous glutamine synthetase from Chinese hamster liver and a sensitive radioisotope assay for the enzyme are described. The enzyme has a molecular weight of 335,000 as determined by sedimentation in a glycerol gradient, and consists of 8 identical subunits with molecular weights of 42,000 as determined by mobility in sodium dodecyl sulfate polyacrylamide gels. The enzyme displays Michaelis-Menten kinetics. The apparent Michaelis constants determined for glutamate, ATP, and ammonia are 3.1 mm, 1.2 mm, and 0.16 mm, respectively. Alanine and serine are noncompetitive inhibitors of glutamate. ATP is competitively inhibited by adenosine diphosphate, noncompetitively inhibited by phosphate, and uncompetitively inhibited by cytidine triphosphate. The maximum velocity of the reaction is increased by α-ketoglutarate. Preincubation of Chinese hamster glutamine synthetase with ATP protects the enzyme from inactivation by heat and by the sulfhydryl-specific reagent N-ethylmaleimide. However, preincubation of glutamine synthetase with ATP is required for inactivation of the enzyme by the glutamine analogs 6-diazo-5-oxo-l-norleucine and 5-diazo-4-oxo-l-norvaline, suggesting that ATP combines with the enzyme to form part of the glutamate binding site.