gamma-Glutamylcysteine synthetase. Further purification, "half of the sites" reactivity, subunits, and specificity.
gamma-Glutamylcysteine synthetase was purified from rat liver by an improved method involving chromatography on Sepharose-aminohexyl-ATP to a specific activity of about 1600 units/mg, or approximately twice that previously obtained; it is thus the most active preparation of this enzyme thus far isolated. The earlier preparation, which is homogeneous on polyacrylamide gel electrophoresis, exhibits "half of the sites" reactivity in that it binds a maximum of 0.5 mol of the inhibitor L-methionine-S-sulfoximine phosphate per mol of enzyme. In contrast, the present enzyme preparation binds 1 mol of methionine sulfoximine phosphate per mol of enzyme; it also differs from the enzyme obtained earlier in exhibiting much less ATPase activity and less activity in catalyzing ATP-dependent cyclization of glutamate. gamma-Glutamylcysteine synthetase dissociates in sodium dodecyl sulfate into two nonidentical subunits of apparent molecular weights 74,000 and 24,000; after cross-linking with dimethyl-suberimidate, a species having a molecular weight of about 100,000 was found on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. New information has been obtained about the interaction of the enzyme with glutamate analogs; thus, the enzyme is active with such glutamate analogs as beta-glutamate, N-methyl-L-glutamate, and threo-beta-hydroxy-L-glutanate, and it is effectively inhibited by cis-1-amino-1,3-dicarboxycyclonexane, 2-amino-4-phosphonobutyrate, and gamma-methylglutamate.
D000251:Adenosine Triphosphatases / Q000378:metabolism; D000818:Animals; D001665:Binding Sites; D005721:Glutamate-Cysteine Ligase / Q000302:isolation & purification / Q000378:metabolism; D005971:Glutamates; D007668:Kidney / Q000201:enzymology*; D007700:Kinetics; D046911:Macromolecular Substances; D008297:Male; D008970:Molecular Weight; D010453:Peptide Synthases / Q000302:isolation & purification; D051381:Rats; D013329:Structure-Activity Relationship
D005971:Glutamates; D046911:Macromolecular Substances; D000251:Adenosine Triphosphatases; D010453:Peptide Synthases; D005721:Glutamate-Cysteine Ligase