Purification and characterization of gamma-glutamyltransferase from rat pancreas.
gamma-Glutamyltransferase ((5-glutamyl)-peptide:amino-acid 5-glutamyltransferase, EC 2.3.2.2.) from rat pancreas has been purified to homogeneity and shown to be a glycoprotein of apparent molecular weight 68000, composed of one heavy and one light subunit, with respective molecular weights 43000 and 25000. At the optimum pH 8.0 the specific activity of the purified enzyme is 630 units/mg protein, with L-gamma-glutamyl-p-nitroanilide as substrate (Km = 0.9 mM) and 20 mM glycylglycine as acceptor. The enzyme is inactivated by the active-site modifying agent and glutamine analogue, 6-diazo-5-oxo-L-norleucine, through a specific and stoichiometric reaction with the light subunit (Ki = 1.2 mM); both the inactivation and the modification of the light subunit are accelerated by maleate and prevented by S-methylglutathione. The enzyme is also inactivated by the fluorescent alkylating agent 5-iodoacetamidofluorescein, by specific and stoichiometric incorporation of the fluorescent moiety into the light subunit, which is likewise prevented by S-methylglutathione, but is unaffected by maleate. Antiserum to rat kidney gamma-glutamyltransferase cross-reacts with the pancreas enzyme in immunodiffusion and inhibits its activity in the p-nitroanilide assay. Despite structural, enzymological and immunological similarities between the pancreas and kidney enzymes, their amino acid compositions are markedly different. The rat pancreas enzyme shows an interesting ontological development, being present in minimal amounts in the fetus, and increasing dramatically on birth and during the following 2 days.
D000217:Acyltransferases / Q000302:isolation & purification* / Q000378:metabolism; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D006801:Humans; D005779:Immunodiffusion; D007668:Kidney / Q000201:enzymology; D007700:Kinetics; D046911:Macromolecular Substances; D008297:Male; D008970:Molecular Weight; D009928:Organ Specificity; D010179:Pancreas / Q000201:enzymology*; D051381:Rats; D011919:Rats, Inbred Strains; D013045:Species Specificity; D011503:Transglutaminases
D000596:Amino Acids; D046911:Macromolecular Substances; D000217:Acyltransferases; D011503:Transglutaminases