Biosynthesis of terminal Gal alpha 1----3Gal beta 1----4GlcNAc-R oligosaccharide sequences on glycoconjugates. Purification and acceptor specificity of a UDP-Gal:N-acetyllactosaminide alpha 1----3-galactosyltransferase from calf thymus.
Biosynthesis of terminal Gal alpha 1----3Gal beta 1----4GlcNAc-R oligosaccharide sequences on glycoconjugates. Purification and acceptor specificity of a UDP-Gal:N-acetyllactosaminide alpha 1----3-galactosyltransferase from calf thymus.
复制标题
末端 Gal α 1----3Gal beta 1----4GlcNAc-R 寡糖序列在糖缀合物上的生物合成。
DOI：
10.1016/s0021-9258(17)38814-2
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发表时间：
1985
期刊：
影响因子：
--
通讯作者：
D. H. van den Eijnden
中科院分区：
文献类型：
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作者：
W. M. Blanken;D. H. van den Eijnden
在小牛胸腺Triton X-100提取液中，用亲和层析法分别纯化了一种UDP-Gal:Gal β 1—4GlcNAc-R α 1—3和一种UDP-Gal:GlcNAc-R β 1—4-半乳糖基转移酶，分别达到44,000倍和101,000倍，产率为25-40%。在还原条件下，十二烷基硫酸钠凝胶电泳显示，α 1—3-半乳糖基转移酶的主要多肽种分子量为40,000，α 1—3-半乳糖基转移酶的次要形式分子量为42,000，β 1—4-半乳糖基转移酶的主要多肽分子量为51,000。在Sephadex G-100上进行分析凝胶过滤，每一种半乳糖转移酶的Mr分别为43,000和59,000，此外在较高分子量的活性峰。α 1—3-半乳糖转移酶的等电聚焦显示出明显的电荷异质性，其pI值在5.0和6.5之间变化。受体特异性研究表明，纯化的α 1—3-半乳糖基转移酶不受半乳糖基转移酶活性的污染，如合成Gal β 1—4GlcNAc-R和Gal β 1—3GalNAc-R序列、B型血决定因子、Pk抗原、三己糖神经酰胺和神经节苷脂GM1。α 1- 3-半乳糖转移酶对糖蛋白、低聚糖和末端有Gal β 1- 4GlcNAc β 1-单位的糖脂具有高度活性，如asialo- α 1-酸性糖蛋白（Km = 1.25 mM）、Gal β 1- 4GlcNAc β 1- 2Man α 1- 3Man β 1- 4GlcNAc （Km = 0.57 mM）和副叶皂苷。研究发现，牛初乳α 1—3-半乳糖转移酶的作用与NeuAc:Gal β 1—4GlcNAc-R α 2—6-唾液转移酶的作用是相互排斥的。此外，首选二糖受体结构中n -乙酰氨基葡萄糖残基的α 1—3-聚焦完全阻断α 1—3-半乳糖基转移酶的半乳糖基化。
A UDP-Gal:Gal beta 1—-4GlcNAc-R alpha 1—-3- and a UDP-Gal:GlcNAc-R beta 1—-4-galactosyltransferase have been purified 44,000- and 101,000-fold, respectively, from a Triton X-100 extract of calf thymus by affinity chromatography on UDP-hexanolamine-Sepharose and alpha-lactalbumin-Sepharose in a yield of 25-40%. Sodium dodecyl sulfate gel electrophoresis under reducing conditions revealed a major polypeptide species with a molecular weight of 40,000 and a minor form at Mr 42,000 for the alpha 1—-3-galactosyltransferase and a major polypeptide with Mr 51,000 for the beta 1—-4-galactosyltransferase. Analytical gel filtration on Sephadex G-100 yielded a monomeric form for each of the galactosyltransferases with Mr 43,000 and 59,000 respectively, in addition to peaks of activity at higher molecular weights. Isoelectric focussing of the alpha 1—-3-galactosyltransferase revealed a significant charge heterogeneity with forms varying in pI values between 5.0 and 6.5. Acceptor specificity studies indicated that the purified alpha 1—-3-galactosyltransferase was free from contaminating galactosyltransferase activities such as those involved in the synthesis of Gal beta 1—-4GlcNAc-R and Gal beta 1—-3GalNAc-R sequences, the blood group B determinant, the Pk antigen, trihexosylceramide, and ganglioside GM1. The alpha 1—-3-galactosyltransferase appeared to be highly active with glycoproteins, oligosaccharides, and glycolipids having a terminal Gal beta 1—-4GlcNAc beta 1—-unit such as asialo-alpha 1-acid glycoprotein (Km = 1.25 mM), Gal beta 1—-4GlcNAc beta 1—-2Man alpha 1—-3Man beta 1—-4GlcNAc (Km = 0.57 mM), and paragloboside. The action of the alpha 1—-3-galactosyltransferase was found to be mutually exclusive with that of the NeuAc:Gal beta 1—-4GlcNAc-R alpha 2—-6-sialyltransferase from bovine colostrum. In addition alpha 1—-3-fucosylation of the N-acetylglucosamine residue in the preferred disaccharide acceptor structure completely blocked galactosylation of the alpha 1—-3-galactosyltransferase.