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. |
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Authors: | R R Drake J T Slama K A Wall M Abramova C D'Souza A D Elbein P J Crocker D S Watt |
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Institution: | Department of Chemistry, University of Kentucky, Lexington 40506. |
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Abstract: | In order to investigate the O-mannosyltransferase involved in the initial O-mannosylation of glycoproteins in Saccharomyces cerevisiae, a photoactive hexapeptide, 125I]-N-(4-azido-2,3,5,6-tetrafluorobenzoyl)-3-iodo-Tyr-Asn-Pro-T hr-Ser-Val (125I]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 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 N-(4-azido-2,3,5,6-tetrafluorobenzoyl) moiety to reducing agents in aqueous buffers was also examined. |
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