Purification and enzymatic properties of gamma-glutamyltransferase from bovine colostrum.

Abstract

γ-Glutamyltransferase was purified 10,000-fold from bovine colostrum and was found to resemble the mammalian kidney enzymes with respect to molecular constitution and substrate specificity. The purification process involved separation of membrane material from colostral whey, treatment with papain, and column chromatography on DEAE-Sephadex and Sephadex G-150. The final preparation was apparently homogeneous on polyacrylamide gel electrophoresis, isoelectric focusing, and double immunodiffusion. It had a molecular weight (as determined by gel filtration) of about 80,000, a sedimentation coefficient of 5.0S20W, and an isoelectric point of pH 3.85, and was composed of two non-identical glycopeptides (with molecular weights of 55,000 and 25,000). Treatment with neuraminidase yielded a more negatively charged variant with intact enzymatic activity. The reaction with γ-glutamyl-p-nitroanilide in the presence of glycylglycine as an acceptor was optimal at about pH 8.5 and at about pH 9.0 in its absence. Hydrolytic reaction, as assessed in terms of glutamate release, was practically absent at high pH. The activation profile by various amino acids and peptides was similar to that observed with the enzymes from other sources; glycylglycine was the best acceptor so far tested. The phosphate-independent glutaminase activity of the colostral enzyme was much lower than that of the human kidney enzyme either in the presence or absence of maleate; glutamate liberation from glutamine in the presence of maleate proceeded at only about 0.2% of the rate observed for transpeptidation between γ-glutamyl-p-nitroanilide and glycylglycine. Initial velocity measurements at various substrate concentrations yielded results which were consistent with a ping-pong mechanism modified by an autotranspeptidation shunt.