The Membrane Association and Physiological Function of Rat Renal γ-Glutamyltranspeptidase

Summary

The interaction of γ-Glutamyltranspeptidase with membranes was studied by characterizing the association and orientation of this enzyme within lecithin vesicles. It was found that only γ-Glutamyltranspeptidase purified following solubilization with Triton X-100 can associate with single-layered [¹⁴C] lecithin vesicles. This was established by comparing enzyme activity and radioactivity profiles during Sepharose 4B chromatography and isopycnic sucrose gradient centrifugation. The enzyme-vesicle complex exhibits a density corresponding to that of a single enzyme molecule bound to a single vesicle. γ-Glutamyltranspep-tidase purified following solubilization with papain does not bind to vesicles. In addition, papain treatment of vesicles containing the Triton-purified transpeptidase results in the release of 95% of the transpeptidase activity without release of Internally trapped [³H] sucrose. The released transpeptidase is chromatographically identical to the papain-purified transpeptidase. γ-Glutamyltranspeptidase activity associated with both native membranes and with lecithin vesicles exhibits a temperature-induced transition in its energy of activation. In contrast, the proteolytic and detergent solubilized forms of the enzyme exhibit a single energy of activation over the entire temperature range. These results suggest that γ-Glutamyltranspeptidase binding to vesicles is due to a papain-sensitive sequence of amino acids and that the enzyme-vesicle complex closely approximates the interaction and orientation of γ-Glutamyltranspeptidase with brush border membranes. These results are inconsistent with the hypothesis that this enzyme participates in amino acid transport via the γ-Glutamyl cycle, but instead supports the proposal that the principal function of γ-glutamyl-transpeptidase is the extracellular degradation of glutathione.