Specific labelling of the hydrophobic domain of rat renal gamma-glutamyltransferase.
The amphipathic form of gamma-glutamyltransferase (EC 2.3.2.2) was reconstituted into unilamellar lecithin vesicles and labelled using the membrane-soluble reagent, 3-trifluoromethyl-3-(m-[125I]iodophenyl)diazirine ( [125I]TID), which can be photoactivated. Label was incorporated exclusively into the large subunit of the transferase, but no label was found in the enzyme released from the vesicles by partial proteolysis with papain. Chromatography of the papain-treated vesicles on Sephadex LH-60 indicated that the [125I]TID was bound to two peptides of low relative molecular mass. The [125I]-TID-labelled peptides should constitute the hydrophobic membrane-binding domain. The transferase was also labelled by reductive methylation (Lys residues) or with immobilized galactose oxidase and NaB3H4 (Gal residues), incorporated into lecithin vesicles and treated with papain. Both procedures yielded a single [3H]-labelled hydrophobic peptide that remained associated with the vesicles. After chromatography on Sephadex LH-60, the peptide labelled by the latter two procedures corresponds to the larger of the two [125I]TID-labelled peptides. These results suggest that papain may cleave the hydrophobic domain into two peptides. The observed labelling is also consistent with the alternative possibility that the N-terminal hydrophobic domain is preceded by an initial sequence that contains both lysine and carbohydrate residues. The smaller of the two [125I]TID-labelled peptides could be generated by removal of the hydrophilic segment from the transferase molecules that are reconstituted with the N-terminus exposed on the external surface of the vesicles.
D000595:Amino Acid Sequence; D000818:Animals; D001389:Azirines / Q000378:metabolism; D002462:Cell Membrane / Q000201:enzymology; D007668:Kidney / Q000201:enzymology*; D008745:Methylation; D008871:Microvilli / Q000201:enzymology; D008954:Models, Biological; D051381:Rats; D005723:gamma-Glutamyltransferase / Q000378:metabolism*
D001389:Azirines; C033174:3-(trifluoromethyl)-3-(3-iodophenyl)diazirine; D005723:gamma-Glutamyltransferase