Biosynthesis and Degradation of γ-Glutamyltranspeptidase of Rat Kidney1

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Abstract
γ-Glutamyltranspeptidase (γGTP) of rat kidney is an intrinsic glycoprotein bound to the plasma membrane and composed of two nonidentical subunits and an amino-terminal portion of the heavy subunit anchors the enzyme to the membrane. The mechanisms of biosynthesis, post-translational processing and degradation of the enzyme were studied using mono-specific antibody raised to γ-glutamyltranspep-tidase purified from rat kidney. The following results were obtained. 1. Double isotope labeling in vivo showed that γglutamyltranspeptidase is synthesized as a precursor form with a single polypeptide chain of 78,000 daltons, and then processed post-translationally by limited proteolysis, resulting in two subunits of 50,000 and 23,000 daltons. 2. Incorporation of [3H]leucine or [35S]methionine into the precursor form increased until 60 min after their intravenous injection, and a pulse-chase experiment showed that the half life of the precursor form was 53 min. 3. [3H]Fucose and PHJglucosamine could also be incorporated into the precursor form, showing that glycosylation of the enzyme occurs at the stage of the precursor form. 4. Rat kidney labeled with PHJfucose was subjected to subcellular fractionation. The Golgi fraction contained the glycosylated precursor form and a small amount of subunits, and the plasma membrane fraction contained mostly subunits with a significant amount of precursor, suggesting that post-translational processing of the precursor occurs on the plasma membrane. 5. The apparent half lives of the native enzyme and the heavy and light subunits were all estimated as 4.3 ±0.5 days by labeling with [3H]leucine or 3fucose. 6. γ-Glutamyltranspeptidase has a different turnover rate from aminopeptidase M, which is located in the microvillus membrane close to γ-glutamyltranspeptidase.