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.
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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N-(4-叠氮基-2,3,5,6-四氟苯甲酰基)酪氨酸取代肽作为异双功能交联剂在酵母蛋白 O-糖基化研究中的应用。
DOI：
10.1021/bc00013a011
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发表时间：
1992
影响因子：
4.7
通讯作者：
Watt,DS
中科院分区：
文献类型：
--
作者：
Drake,RR;Slama,JT;Wall,KA;Abramova,M;D'Souza,C;Elbein,AD;Crocker,PJ;Watt,DS
为了研究参与酿酒酵母糖蛋白初始 O-甘露糖基化的 O-甘露糖基转移酶，一种光活性六肽，[125I]-AT-(4-azido-2, 3, 5, 6-四氟苯甲酰基)-3-iodo-Tyr-Asn-Pro-Thr-Ser-Val ([125I]叠氮基酪氨酸肽），使用新型光活性交联剂 AT-(4-叠氮基-2,3,5,6-四氟苯甲酰基)酪氨酸和树脂结合的 Asn-Pro-Thr (tBu)-Ser (tBu)-Val 通过固相技术合成。当这种修饰的六肽底物与O-甘露糖基转移酶制剂一起温育时，六肽是来自磷酸长效醇-[14C]甘露糖的[14C]-甘露糖的受体。部分纯化 O-甘露糖基转移酶并用 [125I] 叠氮基酪氨酸肽对这些酶制剂进行光标记后，得到了约 125I 的酶制剂。 82-kDa 蛋白质被证明是唯一受未修饰六肽保护的明显光标记蛋白质。这个约。 82-kDa 蛋白可能是 O-甘露糖基转移酶的催化亚基。还检查了Ar-(4-叠氮基-2,3,5,6-四氟苯甲酰基)部分对水性缓冲液中还原剂的敏感性。
In orderto investigate the O-mannosyltransferase involved in the initial O-mannosylation of glyco-proteins in Saccharomyces cerevisiae, a photoactive hexapeptide,[125I]-AT-(4-azido-2, 3, 5, 6-tetrafluo-robenzoyl)-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) ty-rosine, 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 theonly 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.