TY - JOUR
T1 - 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
AU - Crocker, Peter J.
AU - Watt, David S.
AU - Drake, Richard R.
AU - Abramova, Margaret
AU - D'Souza, Crislyn
AU - Elbein, Alan D.
AU - Slama, James T.
AU - Wall, Katherine A.
PY - 1992/1/1
Y1 - 1992/1/1
N2 - 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.
AB - 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.
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U2 - 10.1021/bc00013a011
DO - 10.1021/bc00013a011
M3 - Article
C2 - 1616952
AN - SCOPUS:0026488963
SN - 1043-1802
VL - 3
SP - 69
EP - 73
JO - Bioconjugate Chemistry
JF - Bioconjugate Chemistry
IS - 1
ER -