APPLICATION OF AN N-(4-AZIDO-2,3,5,6-TETRAFLUOROBENZOYL)TYROSINE-SUBSTITUTED PEPTIDE AS A HETEROBIFUNCTIONAL CROSS-LINKING AGENT IN A STUDY OF PROTEIN O-GLYCOSYLATION IN YEAST

In order to investigate the O-mannosyltransferase involved in the initial O-mannosylation of glycoproteins in Saccharomyces cerevisiae, a photoactive hexapeptide, [I-125]-N-(4-azido-2,3,5,6-tetrafluorobenzoyl)-3-iodo-Tyr-Asn-Pro-Thr-Ser-Val ([I-125]azidoTyr-peptide), was synthesized by solid-phase techniques using a new photoactive cross-linking reagent, N-(4-azido-2,3,5,6-tetrafluorobenzoyl)tyrosine, and resin-bound Asn-Pro-Thr(tBu)-Ser(tBu)-Val. When this modified hexapeptide substrate was incubated with 0-mannosyltransferase preparations, the hexapeptide was an acceptor of [C-14]-mannose from dolichol phosphate-[C-14]mannose. After partially purifying the O-mannosyltransferase and photolabeling these enzyme preparations with [I-125]azido Tyr-peptide, a ca. 82-kDa protein was shown to be the only apparent photolabeled protein that was protected by unmodified hexapeptide. This ca. 82-kDa protein may be the catalytic subunit of the O-mannosyltransferase. The susceptibility of the N-(4-azido-2,3,5,6-tetrafluorobenzoyl) moiety to reducing agents in aqueous buffers was also examined.