Characterization of the membrane binding domain of gamma-glutamyltranspeptidase by specific labeling techniques.
The amphipathic form of gamma-glutamyltranspeptidase was labeled either by reductive methylation of primary amino groups or by galactose oxidase/NaB3H4 labeling of galactose residues. The labeled enzyme was asymmetrically reconstituted into unilamellar phosphatidylcholine vesicles and subjected to partial papain proteolysis, and the resulting products were resolved by Sepharose 4B chromatography. Chromatography of the vesicle-associated material on Sephadex LH-60 yields an 8700 molecular weight peptide which is labeled by both techniques. This peptide, therefore, contains lysine residues and/or the NH2-terminus of the large subunit and a galactose-containing oligosaccharide side chain. This peptide appears to be identical with peptide I which is labeled by 3-(trifluoromethyl)-3-(m-[125I]iodophenyl)diazirine [Frielle, T., Brunner, J., & Curthoys, N.P. (1982) J. Biol. Chem. 257, 14979-14982]. A second hydrophobic peptide (peptide II) which is also labeled by the membrane-permeable, photoactivatable probe is not significantly labeled either by reductive methylation or by galactose oxidase/NaB3H4 labeling. Sephadex G-25 chromatography of the 3H-labeled hydrophilic peptides released from the vesicles by papain proteolysis yields a [3H]galactose-labeled peptide of 2600 molecular weight (peptide III) and a 1300 molecular weight peptide labeled by reductive methylation (peptide IV). Peptide I, but not peptide IV, partitions into a series of primary aliphatic alcohols and can be reconstituted into vesicles. The hydrophilic peptides are probably derived either from a peripheral sequence of the membrane binding domain or from the region which connects the hydrophilic domain of the large subunit with the membrane binding domain.
D001665:Binding Sites; D002850:Chromatography, Gel; D002854:Chromatography, Paper; D008566:Membranes / Q000378:metabolism; D008970:Molecular Weight; D010206:Papain / Q000378:metabolism; D010446:Peptide Fragments / Q000032:analysis; D010713:Phosphatidylcholines; D005723:gamma-Glutamyltransferase / Q000378:metabolism*
D010446:Peptide Fragments; D010713:Phosphatidylcholines; D005723:gamma-Glutamyltransferase; D010206:Papain