γ‐Glutamyl Transpeptidase: Sidedness of Its Active Site on Renal Brush‐Border Membrane

Sidedness of γ‐glutamyl transpeptidase, an integral glycoportein of rat renal brusch border membrane, was investigated and the active siyte of the membrane‐bound enzyme was shown to face to the luminal side of the renal tubules on the basis of the two following observations. First, γ‐glutamyl transpeptidase activity exhibited by purified brush border membrane vesicles was effectively inhibited by an S ‐acetyldextran polymer (molecular weight: 215 000) derivative ofglutathione, which is believed to be impermeable through plasma membranes. Secondly, the membrane‐bound γ‐glutamyl transpeptidase was quantitatively released by treating vesicles with papain immobilized to Sephadex G‐10 beads. Strict stoichiometry of affinity labeline of rat renal γ‐glutamyl transpeptidase, which possesses one active site per molecule [Inoue, M., Horiuchi, S., and Morino, Y. (1977) Eur. J. Biochem. 73 , 335–342; Tate, S. S., and Meister, A. (1977) Proc. Natl Acad. Sci. U.S.A. 74 , 931–935], was utilized to compare the molecular size of the detergent‐solubilized enzyme with that of the papain‐solubilized enzyme. Thus, 1 mol redioactive 6‐diazo‐5‐oxo‐ l ‐norleucine was covalently incorporated into 102000 g detergent‐solubilized enzyme and 64000 g papain‐solubilized enzyme. Both enzyme foorms of γ‐glutamyl transpeptidase are composed of two non‐identical subunits, the large and the small subunit. Molecular weightgs of these subunits were determined in sodium dodecylsulfate/polyacrylamide gel electrophresis from the retardation coefficients obtained from Ferguson plots. The result indicated the size difference between these two enzyme forms could be largely accounted for by the size difference in the large subunits. Thus, iot is likely that the oligomeric γ‐glutamyl transpeptidase was anchored into the brush nborder membrane mainly via te large subunit of tghe enzyme.