Studies on the Structure of γ-Glutamyltranspeptidase

II. Location of the Segment Anchoring γ-Glutamyltranspeptidase to the Membrane

Abstract

The amino acid compositions of the subunits of γ-glutamyltranspeptidase solubilized with Triton X-100 (T-γGTP) and papain (P-γGTP) were compared. The results showed that the light subunits of the two forms are identical in amino acid composition, but that the heavy subunit of T-γGTP contains about 52 amino acid residues more than that of P-γGTP.
The susceptibilities of the two forms to endopeptidase and the endo-groups of the two forms were studied to determine whether the membrane binding segment was located near the amino-terminus or the carboxyl-terminus. Leucine amino peptidase released amino acids from T-γGTP, converting it to a hydrophilic form like P-γGTP without loss of activity, and the composition of the amino acids released was similar to that of the difference in the amino acid compositions of the heavy subunits of T-γGTP and P-γGTP. No amino acids were released from P-γGTP by treatment with this enzyme. The amino-terminal residues of the heavy and light subunits of T-γGTP were methionine and threonine, respectively, while those of P-γGTP were glycine and threonine, respectively. Neither T-γGTP nor P-γGTP was affected by treatment with carboxypeptidase Y. Amino acids were released from both forms by treatment with carboxypeptidase Y in the presence of sodium dodecyl sulfate, and the patterns of these released amino acids were very similar.
These results indicate that the amino terminal portion of the heavy subunit of γ-glutamyltranspeptidase contains the portion that anchors the enzyme to the membrane.