title: In vitro translation and processing of rat kidney gamma-glutamyl transpeptidase.
abstract: Rat kidney gamma-glutamyl transpeptidase is composed of two nonidentical glycosylated subunits. The enzyme is localized on the lumenal surface of the brush-border membranes of proximal tubule epithelial cells; it is attached to the membranes via an NH2-terminal segment of the larger of the two subunits. Tissue-labeling experiments followed by immunoprecipitation with antibodies directed against the enzyme and its two subunits demonstrate that a glycosylated single chain precursor (Mr = 78,000), containing the elements of both the subunits, is initially synthesized. Pulse-chase studies in the presence of pactamycin, and inhibitor of protein synthesis initiation, indicate that the larger of the two subunits is located at the NH2 terminus of the Mr = 78,000 precursor. The initial events in the biosynthesis and processing of gamma-glutamyl transpeptidase were investigated by in vitro translation of rat kidney mRNA. Such translation results in the synthesis of a Mr = 63,000 unglycosylated polypeptide which has been shown immunologically to contain the domains for both subunits. The Mr = 63,000 species is processed to a Mr = 78,000 core-glycosylated polypeptide when translation of mRNA is carried out in the presence of dog pancreas microsomes. This processing does not appear to be associated with cleavage of an NH2-terminal leader sequence. The Mr = 78,000 polypeptide is integrated into the microsomal membranes with an orientation that is analogous to that found on the brush-border membranes. Glycosylation and membrane integration of transpeptidase are cotranslational events. Upon longer incubation, the Mr = 78,000 species sequestered within the microsomal vesicles is cleaved to species corresponding in size to the two subunits of the kidney enzyme.
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D004285:Dogs; D066298:In Vitro Techniques; D007668:Kidney / Q000201:enzymology*; D008861:Microsomes / Q000378:metabolism; D008970:Molecular Weight; D017830:Octoxynol; D010179:Pancreas / Q000378:metabolism; D011092:Polyethylene Glycols / Q000494:pharmacology; D014176:Protein Biosynthesis; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D005723:gamma-Glutamyltransferase / Q000096:biosynthesis* / Q000276:immunology
chemical_list: D012333:RNA, Messenger; C017474:Lubrol WX; D011092:Polyethylene Glycols; D017830:Octoxynol; D005723:gamma-Glutamyltransferase