27] Glutamine synthetase from mammalian tissues

Abstract

This chapter provides an overview of the glutamine synthetase from mammalian tissues. The glutamine synthesis reaction is freely reversible. When the enzyme is incubated with 10 mM concentrations each of L-glutamate, ammonium ions, and ATP in the presence of Mg2+ at pH 7.0 and 37°, equilibrium is attained when about 90% of the L-glutamate is converted to L-glutamine. Substitution of hydroxylamine for ammonia in this system leads to a reaction that goes to greater than 99% of completion. The glutamine synthetases of mammalian origin closely resemble each other with respect to amino acid composition, subunit structure, and molecular weight. They differ substantially in these respects from bacterial glutamine synthetases, some of which exist in adenylylated forms. Glutamine synthetase activity may be followed by measuring the rate of formation of inorganic phosphate, ADP, or glutamine. ADP may be determined by coupling the glutamine synthetase reaction with those catalyzed by pyruvate kinase and lactate dehydrogenase. By use of reaction mixtures containing labeled glutamate, the disappearance of glutamate and the formation of glutamine may be determined. A commonly used procedure for determining glutamine synthetase activity involves replacement of ammonia by hydroxylamine. In this reaction, γ-glutamylhydroxamate is formed, which gives a characteristic color reaction on addition of ferric chloride.