Evidence that the hydrophobic domain of rat renal γ-glutamyltransferase spans the brush border membrane

Lactoperoxidase and glucose oxidase catalyzed ¹²⁵I-iodination was used to specifically label isolated rat renal brush border membrane vesicles from either side of the membrane. Autoradiography of total membrane proteins demonstrated that asymmetric labeling was achieved. Specific immunoprecipitates of aminopeptidase M, an established transmembrane protein, and of γ-glutamyltransferase were isolated from vesicles solubilized with Triton X-100 or with papain. Following electrophoresis and autoradiography, the immunoprecipitates of the two solubilized forms of each enzyme derived from externally labeled vesicles exhibited the same intensity of labeling. In these experiments, the small subunit of the γ-glutamyltransferase was preferentially labeled suggesting that, compared to the large subunit, it is more exposed on the external surface of the membrane. With the samples derived from internally labeled vesicles, the Triton-solubilized form of each enzyme was intensely labeled, whereas the papain-solubilized forms contained insignificant amounts of radioactivity. Thus, the extent of contramembrane labeling was minimal. In these experiments, the large subunit of the γ-glutamyltransferase was preferentially labeled. The similarity of the labeling patterns obtained for aminopeptidase M and γ-glutamyltransferase suggests that the hydrophobic domain of the two amphipathic enzymes are selectively labeled from the internal surface and that the γ-glutamyltransferase may also be a transmembrane protein.