Solubilization of mannosyltransferase activities for the biosynthesis of mammary glycoproteins. Elongation of tetrasaccharide-lipid to heptasaccharide-lipid by a solubilized enzyme preparation.
Solubilization of mannosyltransferase activities for the biosynthesis of mammary glycoproteins. Elongation of tetrasaccharide-lipid to heptasaccharide-lipid by a solubilized enzyme preparation.
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乳腺糖蛋白生物合成中甘露糖基转移酶活性的溶解。
DOI：
10.1111/j.1432-1033.1984.tb07980.x
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发表时间：
1984
期刊：
影响因子：
--
通讯作者：
Vijay,IK
中科院分区：
文献类型：
--
作者：
Prakash,C;Katial,A;Kang,MS;Vijay,IK
采用非离子型去污剂NP-40处理泌乳牛乳腺组织的微粒体制备物，蛋白/去污剂比例为1.5：1，去污剂浓度为0.5%。以147000 × g离心120 min后，将上清液部分与标记的糖核苷酸、GDP-Man和UDP-GlcNAc孵育。发现其可合成一系列脂质连接的双链，最高可达（Man）5-（GlcNAc）2。溶解的糖基转移酶在4 ° C下储存两周后保留高达约60%的活性。通过用CHCl3/CH3OH（2：1）提取乳腺微粒体获得的脂质混合物刺激糖脂的生物合成。通过在葡萄糖饥饿条件下用[2 - 3H]甘露糖标记幼仓鼠肾细胞，然后用CHCl3/CH3OH（2：1）提取细胞，并通过高效液相色谱法分离脂质，分离结构为Man α 1 → 3Man β → GlcNAc β → GlcNAc的标记脂质连接四糖。当将该脂联四糖与溶解的牛乳腺微粒体和GDP-Man孵育时，其被延长为具有结构Man α 1 → 2Man α 1 → 2Man α 1 → 3（Man α 1 → 6）Man β → GlcNAc β → GlcNAc的脂联七糖。延伸反应的动力学还揭示了较少量的脂质连接的五糖和六糖的中间形成。延伸反应不需要任何二价金属离子，并且具有在6.8和7.6之间的宽pH最适值。EDTA或两性霉素对延伸反应的抑制作用的缺乏支持了早期的研究，即GDP-Man而不是甘露糖基磷酸多醇是用于糖脂生物合成高达（Man）5（GlcNAc）2的甘露糖基残基的直接供体。甘露糖基磷酰基视黄醇作为延伸反应的甘露糖基供体是无效的。
The microsomal preparation from the lactating bovine mammary tissue was solubilized by treatment with nonionic detergent, NP‐40, at a protein/detergent ratio of 1.5:1 and a detergent concentration of 0.5%. Following centrifugation at 147000 ×gfor 120 min, the supernatant fraction was incubated with labeled sugar nucleotides, GDP‐Man and UDP‐GlcNAc. It was found to synthesize a series of lipid‐linked saccharides up to (Man)5‐(GlcNAc)2. The solubilized glycosyltransferases retained up to about 60% of the activity after two weeks of storage at 4°C. The biosynthesis of glycolipids was stimulated by a mixture of lipids obtained by extracting the mammary microsomes with CHCl3/CH3OH (2:1).A labeled lipid‐linked tetrasaccharide of the structure Manα1 → 3Manβ→ GlcNAcβ→ GlcNAc was isolated by labeling baby hamster kidney cells with [2‐3H]mannose under conditions of glucose starvation followed by extraction of the cells with CHCl3/CH3OH (2:1) and separation of the lipids by high‐performance liquid chromatography. When this lipid‐linked tetrasaccharide was incubated with the solubilized bovine mammary microsomes and GDP‐Man, it was elongated to a lipid‐linked heptasaccharide having the structure Manα1 → 2Manα1 → 2Manα1 → 3(Manα1 → 6)Manβ→ GlcNAcβ→ GlcNAc. The kinetics of the elongation reaction also revealed the intermediary formation of smaller amounts of lipid‐linked pentasaccharide and hexasaccharide. The elongation reaction did not require any divalent metal ion and had a broad pH optimum between 6.8 and 7.6. The lack of inhibition of the elongation reaction by EDTA or amphomycin support earlier studies that GDP‐Man rather than mannosylphosphoryldolichol, is the direct donor of mannosyl residues for the biosynthesis of glycolipids up to (Man)5(GlcNAc)2. Mannosylphosphorylretinol was ineffective as mannosyl donor for the elongation reaction.