CONTROL OF GLYCOPROTEIN-SYNTHESIS - THE USE OF OLIGOSACCHARIDE SUBSTRATES AND HPLC TO STUDY THE SEQUENTIAL PATHWAY FOR N-ACETYLGLUCOSAMINYLTRANSFERASES-I, N-ACETYLGLUCOSAMINYLTRANSFERASES-II, N-ACETYLGLUCOSAMINYLTRANSFERASES-III, N-ACETYLGLUCOSAMINYLTRANSFERASES-IV, N-ACETYLGLUCOSAMINYLTRANSFERASES-V, N-ACETYLGLUCOSAMINYLTRANSFERASES-AND N-ACETYLGLUCOSAMINYLTRANSFERASES-VI IN THE BIOSYNTHESIS OF HIGHLY BRANCHED N-GLYCANS BY HEN OVIDUCT MEMBRANEU
CONTROL OF GLYCOPROTEIN-SYNTHESIS - THE USE OF OLIGOSACCHARIDE SUBSTRATES AND HPLC TO STUDY THE SEQUENTIAL PATHWAY FOR N-ACETYLGLUCOSAMINYLTRANSFERASES-I, N-ACETYLGLUCOSAMINYLTRANSFERASES-II, N-ACETYLGLUCOSAMINYLTRANSFERASES-III, N-ACETYLGLUCOSAMINYLTRANSFERASES-IV, N-ACETYLGLUCOSAMINYLTRANSFERASES-V, N-ACETYLGLUCOSAMINYLTRANSFERASES-AND N-ACETYLGLUCOSAMINYLTRANSFERASES-VI IN THE BIOSYNTHESIS OF HIGHLY BRANCHED N-GLYCANS BY HEN OVIDUCT MEMBRANEU
复制标题
DOI：
10.1139/o88-131
复制
发表时间：
1988-10-01
影响因子：
2.9
通讯作者：
SCHACHTER, H
中科院分区：
文献类型：
--
作者：
BROCKHAUSEN, I;CARVER, JP;SCHACHTER, H
从母鸡输卵管中分离的糖蛋白含有高度分支的天冬酰胺连接的寡糖（N-聚糖）。六个N-乙酰葡糖胺转移酶参与启动这些分支的合成。高效液相色谱法已被用来研究这些GlcNAc-T的底物特异性和参与高度分支的N-聚糖在鸡输卵管中的生物合成的顺序途径。通过肼解-再-N-乙酰化从各种糖蛋白制备具有游离还原GlcNAc末端的寡聚体，并用作GlcNAc-T底物和HPLC标准品。酶测定组分在AG 1X上分离。8，然后在胺键合的硅胶柱上进行HPLC，用乙腈洗脱。水的混合物。监测195 nm处的吸光度和洗脱化合物的放射性。通过比较底物和产物与已知结构的寡糖的保留时间来鉴定底物和产物。HPLC法测定酶活性比以往用凝集素柱电泳法测定GlcNAc-T酶活性更快速、方便。由于一些底物产生多种产物，因此这些可用于在同一孵育中测定多于一种GlcNAc-T。显示GlcNAc-T VI作用于二等分和非二等分GlcNAc-终止四触角寡糖底物; GlcNAc-T II、IV和V对二等分底物作用较差或根本不作用。GlcNAc-IV是所研究的六种转移酶中唯一可以在不存在Mn 2+的情况下测定的酶。
Glycoproteins isolated from hen oviduct contain highly branched asparagine-linked oligosaccharides (N-glycans). Six N-acetylglucosaminyltransferases are involved in initiating the synthesis of these branches. HPLC has been used to study the substrate specificities of these GlcNAc-T and the sequential pathways involved in the biosynthesis of highly branched N-glycans in hen oviduct. Oligosaccharides with free reducing GlcNAc termini were prepared from various glycoproteins by hydrazinolysis-re-N-acetylation and used as GlcNAc-T substrates and HPLC standards. Enzyme assay components were separated on AG1 .times. 8, followed by HPLC on amine-bonded silica columns eluted with acetonitrile .sbd. water mixtures. Absorbance at 195 nm and radioactivity of eluted compounds were monitored. Substrates and products were identified by comparison of their retention times with those of oligosaccharides with known structures. Enzyme assay by HPLC is more rapid and convenient than previous GlcNAc-T assays using lectin columns of electrophoresis. Since some substrates yielded multiple products, these could be used to assay more than one GlcNAc-T in the same incubation. GlcNAc-T VI was shown to act on both bisected and nonbisected GlcNAc-terminating tetraantennary oligosaccharide substrates; GlcNAc-T II, IV, and V acted poorly or not at all on bisected substrates. GlcNAc-T V was the only enzyme among the six transferases studied that could be assayed in the absence of Mn2+.