Glutamine synthetase IX. Purification and characterization of the enzyme from sheep spleen

Abstract

Glutamine synthetase (EC 6.3.1.2) was purified about 550-fold from sheep spleen. The subunit MW of the enzyme is estimated to be 48,000. Sedimentation coefficient determination by density gradient centrifugation gives a value of 15.0 S. The approximate MW calculated from the S value is 378,500. Electron micrographs of the enzyme show an H shape. The protein appears to have 8 subunits. In sheep spleen, the enzyme resides chiefly in the soluble fraction of the cell. The amino acid composition of the enzyme from spleen shows similarity to that from other sources. The enzyme activity is nearly 5 times as high in Mg2+ as in Mn2+. ATP inhibits the enzyme; the inhibition is competitive with respect to Mg2+ ATP. A number of compounds, e.g., D-alanine, AMP, creatine phosphate, arsenite in combination with 2,3-dimercaptopropanol, and 2-amino-4-phosphonobutyrate, also inhibit the enzyme. The inhibition by the last compound is competitive with respect to glutamate. D-Glutamate and .alpha.-methyl-DL-glutamate can serve as substrates in the synthesis reaction, but N-methyl-DL-glutamate cannot. Neither D-glutamine nor N-acetyl-L-glutamine can replace L-glutamine as a substrate in the .gamma.-glutamyl transfer reaction of the enzyme. Inhibition by Mn2+ and ATP and its reversal by Mg2+ are discussed as a means of regulating the enzyme activity in mammalian tissues.