Nonidentity of sulfhydryl oxidase and gama-glutamyltransferase in bovine milk

Sulfhydryl oxidase (glutathione-oxidizing activity) is closely associated with γ-glutamyltransferase (γ-glutamyl transpeptidase) in skim milk membranes. Similar close association of the two enzymatic activities in kidney membranes has led to the recent proposal that glutathione-oxidizing activity can be attributed to the action of γ-glutamyltransferase, itself, in generating cysteinylglycine which, in turn, catalyzes sulfhydryl group oxidation (O. W. Griffith and S. S. Tate, 1980, J. Biol. Chem.255, 5011–5014). However, a previously published procedure for the isolation of highly purified sulfhydryl oxidase from skim milk membranes (V. G. Janolino and H. E. Swaisgood, 1975, J. Biol. Chem.250, 2532–2538) leads to the effective separation of the two activities. Quantitative chromatographic analyses of GSH, GSSG, and Glu levels revealed that the highly purified sulfhydryl oxidase preparation catalyzes the direct oxidation of GSH to GSSG without detectable cleavage of the γ-glutamyl peptide bond. These results were confirmed by monitoring the time course of substrate disappearance and product formation using high-performance liquid chromatography. Conversely, a supernatant fraction enriched in γ-glutamyltransferase activity displayed no sulfhydryl group-oxidizing activity. 6-Diazo-5-oxo-l-norleucine selectively inhibited the transferase in crude preparations containing both sulfhydryl oxidase and γ-glutamyltransferase. It is concluded that sulfhydryl oxidase and γ-glutamyltransferase activities are distinct and separable.