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, [¹²⁵I]-AT-(4-azido-2, 3, 5, 6-tetrafluorobenzoyl)-3-iodo-Tyr-Asn-Pro-Thr-Ser-Val ([¹²⁵I]azidoTyr-peptide), was synthesized by solid-phase techniques using a new photoactive cross-linking reagent, AT-(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 O-mannosyltransferase preparations, the hexapeptide was an acceptor of [¹⁴C]-mannose from dolichol phosphate-[14C]mannose. After partially purifying the O-mannosyltransferase and photolabeling these enzyme preparations with [¹²⁵I] azidoTyr-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 Ar-(4-azido-2, 3, 5, 6-tetrafluorobenzoyl) moiety to reducing agents in aqueous buffers was also examined.