Studies on the structure of gamma-glutamyltranspeptidase. III. Evidence that the amino terminus of the heavy subunit is the membrane binding segment.

Abstract

γ-Glutamyltranspeptidase solubilized with Triton X-100 from the membrane fraction of rat kidney was previously shown to contain a hydrophobic domain that anchors the enzyme to the membrane, and which may be localized in the aminoterminal portion of the heavy subunit. The light subunit does not associate with the membrane [Tsuji et al . (1980) J. Biochem . 87, 1567–1571]. In the current work, the membrane binding portion of γ-glutamyltranspeptidase was obtained by treating Triton-solubilized enzyme with papain. This treatment resulted in solubilization of about 8.8% of the protein mass of the Triton-solubilized γ-glutamyltranspeptidase from the heavy subunit. The product split from the heavy subunit had a molecular weight of 6,000 judging from its amino acid composition. The residual enzyme showed no loss of catalytic activity but became hydrophilic. The fragment removed from γ-glutamyltranspeptidase by papain treatment was not particularly hydrophobic and contained carbohydrate. The partial amino-terminal sequences and carboxyl-terminal amino acids of the heavy and light subunits of γ-glutamyltranspeptidase solubilized with Triton X-100 and papain were determined. The light subunits of γ-glutamyltranspeptidase solubilized with Triton X-100 and papain contained the same sequence, Thr-Ala-(X)-Leu, as an amino-terminal portion, but that of the heavy subunit of the form solubilized with Triton X-100 contained the sequence Met-Lys-Asn-Arg-Phe-Leu-Val-Leu-Gly-Leu-Val-Ala-Val-Val-Leu-Val-Phe-Val-Ile-Ile-Gly- and the sequence of the hydrophobic domain resembled that of the signal peptide which contributes to directing newly synthesized secretory protein to associate with the membrane of the endoplasmic reticulum. The papain-solubilized form had a completely different amino-terminal sequence, Gly-Pro-Pro-Leu-. The carboxyl-terminal amino acids of the subunits of the two forms were determined by carboxypeptidase digestion; that of both light subunits was found to be phenylalanine, and that of both heavy subunit to be tyrosine. These results indicate that the amino-terminal region of the heavy subunit of γ-glutamyltranspeptidase is removed by proteolysis with papain and that it is composed af a hydrophobic domain and a hydrophilic domain. The hydrophobic domain contains the amino-terminus of the heavy subunit, includes about 20 hydropholic amino acids and contributes to anchorage of the enzyme to the membrane. The hydropholic domain following the hydrophobic domain probably contains carbohydrate.