Biosynthesis of prolyl hydroxylase: evidence for two separate dolichol-media pathways of glycosylation.
Biosynthesis of prolyl hydroxylase: evidence for two separate dolichol-media pathways of glycosylation.
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DOI：
10.1021/bi00342a041
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发表时间：
1985-10
期刊：
影响因子：
2.9
通讯作者：
N. Kedersha;J. Tkacz;R. Berg
中科院分区：
文献类型：
--
作者：
N. Kedersha;J. Tkacz;R. Berg
脯氨酸羟基酶是一种含有两个不同亚基的糖蛋白，即α和β。从13日龄鸡胚胎中分离出的Pro羟基酶的α亚基含有一个高甘露糖低聚糖，含有7个甘露糖基残基。已证明从17日龄鸡胚胎肌腱细胞纯化的酶中存在两种形式的α亚基，其中一种在分子量和碳水化合物含量上似乎与13日龄鸡胚胎的单一α酶相同，另一种形式(α‘)包含两个寡糖，每个低聚糖包含八个甘露糖单元[参见KederashN.L.，Tkacz，J.S.，&Berg，R.A.(1985)生物化学]。用[2-~3H]甘露糖、[6-~3H]氨基葡萄糖、[14C(U)]甘露糖和[14C(U)]葡萄糖对鸡腱细胞进行生物合成标记研究。在十二烷基硫酸钠中用聚丙烯酰胺凝胶电泳法对标记产物进行分析表明，只有α‘上的寡糖结合了可测量的甘露糖或氨基葡萄糖同位素；然而，在相似的条件下，两个α亚基都含有14C氨基酸混合物和[14C(U)]葡萄糖[代谢转化为[14C(U)]甘露糖]。用14C氨基酸混合物进行的脉冲追逐标记研究表明，糖基化多肽链α和α‘是同时合成的，α和α’之间没有明显的前体产物关系。在衣霉素存在的情况下，既没有检测到阿尔法，也没有检测到阿尔法‘；相反，出现了一个流动性更大的单一多肽。用抑制浓度的氨基葡萄糖孵育细胞，部分抑制了α‘的糖基化，但允许α的糖基化。
Prolyl hydroxylase is a glycoprotein containing two nonidentical subunits, alpha and beta. The alpha subunit of prolyl hydroxylase isolated from 13-day-old chick embryos contains a single high mannose oligosaccharide having seven mannosyl residues. Two forms of alpha subunit have been shown to exist in enzyme purified from tendon cells of 17-day-old chick embryos, one of which (alpha) appears to be identical in molecular weight and carbohydrate content with the single alpha of enzyme from 13-day-old chick embryos, as well as another form (alpha') that contains two oligosaccharides, each containing eight mannosyl units [see Kedersha, N. L., Tkacz, J. S., & Berg, R. A. (1985) Biochemistry (preceding paper in this issue)]. Biosynthetic labeling studies were performed with chick tendon cells using [2-3H]mannose, [6-3H]glucosamine, [14C(U)]mannose, and [14C(U)]glucose. Analysis of the labeled products using polyacrylamide gel electrophoresis in sodium dodecyl sulfate showed that only the oligosaccharides on alpha' incorporated measurable mannose or glucosamine isotopes; however, both alpha subunits incorporated 14C amino acid mix and [14C(U)]glucose [metabolically converted to [14C(U)]mannose] under similar conditions. Pulse-chase labeling studies using 14C amino acid mix demonstrated that both glycosylated polypeptide chains alpha and alpha' were synthesized simultaneously and that no precursor product relationship between alpha and alpha' was apparent. In the presence of tunicamycin, neither alpha nor alpha' was detected; a single polypeptide of greater mobility appeared instead. Incubation of the cells with inhibitory concentrations of glucosamine partially depressed the glycosylation of alpha' but allowed the glycosylation of alpha.(ABSTRACT TRUNCATED AT 250 WORDS)