Evidence that the hydrophobic domain of rat renal gamma-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 gamma-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, and small subunit of the gamma-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. These experiments, the large subunit of the gamma-glutamyltransferase was preferentially labeled. The similarity of the labeling patterns obtained for aminopeptidase M and gamma-glutamyltransferase suggests that the hydrophobic domain of the two amphipathic enzymes are selectively labeled from the internal surface and that the gamma-glutamyltransferase may also be a transmembrane protein.
D000217:Acyltransferases / Q000378:metabolism*; D000626:Aminopeptidases / Q000378:metabolism; D000818:Animals; D018826:CD13 Antigens; D002374:Catalase / Q000494:pharmacology; D002458:Cell Fractionation; D002462:Cell Membrane / Q000201:enzymology*; D005949:Glucose Oxidase; D007672:Kidney Cortex / Q000201:enzymology*; D007784:Lactoperoxidase; D008297:Male; D008871:Microvilli / Q000201:enzymology* / Q000648:ultrastructure; D010206:Papain; D051381:Rats; D011919:Rats, Inbred Strains; D011503:Transglutaminases
D005949:Glucose Oxidase; D007784:Lactoperoxidase; D002374:Catalase; D000217:Acyltransferases; D011503:Transglutaminases; D000626:Aminopeptidases; D018826:CD13 Antigens; D010206:Papain