Glutamine synthetase. IX. Purification and characterization of the enzyme from sheep spleen.
Glutamine synthetase (L-glutamate: ammonia ligase (ADP-forming), EC 6.3.1.2) has been purified about 550-fold from sheep spleen. The subunit weight 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 molecular weight calculated from the S value is 378500. In addition, electron micrographs of the enzyme show an "H" shape. Hence, the protein appears to have eight 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 five 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, such as 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. On the other hand, 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 of Mn2+ and ATP and its reversal by Mg2+ have been discussed as a means of regulating the enzyme activity in mammalian tissues.
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D004789:Enzyme Activation; D005974:Glutamate-Ammonia Ligase / Q000302:isolation & purification / Q000378:metabolism; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D008345:Manganese / Q000494:pharmacology; D008854:Microscopy, Electron; D008970:Molecular Weight; D012756:Sheep; D013154:Spleen / Q000201:enzymology* / Q000648:ultrastructure; D013347:Subcellular Fractions / Q000201:enzymology; D005723:gamma-Glutamyltransferase / Q000378:metabolism
D000596:Amino Acids; D008345:Manganese; D005723:gamma-Glutamyltransferase; D005974:Glutamate-Ammonia Ligase; D008274:Magnesium