Studies of human kidney γ glutamyl transpeptidase. Purification and structural, kinetic, and immunological properties

S. P. Miller, Y. C. Awasthi, S. K. Srivastava

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Abstract

γ Glutamyl transpeptidase, present in various mammalian tissues, transfers the γ glutamyl moiety of glutathione to a variety of acceptor amino acids and peptides. This enzyme has been purified from human kidney cortex about 740 fold to a specific activity of 200 units/mg of protein. The purification steps involved incubation of the homogenate at 37° followed by centrifugation and extraction of the sediment with 0.1 M Tris HCl buffer, pH 8.0, containing 1% sodium deoxycholate; batchwise absorption on DEAE cellulose; DEAE cellulose (DE52) column chromatography; Sephadex G 200 gel filtration and affinity chromatography using concanavalin A insolubilized on beaded Agarose. Detergents were used throughout the purification of the enzyme. The purified enzyme separated into three protein bands, all of which had enzyme activity, on polyacrylamide disc electrophoresis in the presence of Triton X 100. The enzyme has an apparent molecular weight of about 90,000 as shown by Sephadex G 200 filtration, and appears to be a tetramer with subunits of molecular weights of about 21,000. The Km for γ glutamyl transpeptidase using the artificial substrate, γ glutamyl p nitroanilide, with glycylglycine as the acceptor amino acid was found to tbe about 0.8 mM. The optimum pH for the enzyme activity is 8.2 and the isoelectric point is 4.5. Both GSH and GSSG competitively inhibited the activity of γ glutamyl transpeptidase when γ glutamyl p nitroanilide was used as the substrate. Treatment of the purified enzyme with papain has no effect on the enzyme activity or mobility on polyacrylamide disc electrophoresis. The purified γ glutamyl transpeptidase. had no phosphate independent glutaminase activity. The ratio of γ glutamyl transpeptidase to phosphate independent glutaminase changed significantly through the initial steps of γ glutamyl transpeptidase purification. These studies indicate that the transpeptidase and phosphate independent glutaminase activities are not exhibited by the same protein in human kidney.

Original languageEnglish (US)
Pages (from-to)2271-2278
Number of pages8
JournalJournal of Biological Chemistry
Volume251
Issue number8
StatePublished - Dec 1 1976

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ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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