Substrate specificity of N-acetylglucosamine 1-phosphate transferase activity in Chinese hamster ovary cells.
Substrate specificity of N-acetylglucosamine 1-phosphate transferase activity in Chinese hamster ovary cells.
复制标题
中国仓鼠卵巢细胞中N-乙酰氨基葡萄糖1-磷酸转移酶活性的底物特异性。
DOI：
10.1093/glycob/2.4.313
复制
发表时间：
1992
期刊：
影响因子：
4.3
通讯作者：
Krag,SS
中科院分区：
文献类型：
--
作者：
McLachlan,KR;Krag,SS
在哺乳动物细胞中，天冬酰胺连接的糖蛋白的寡糖链的组装途径始于在N-乙酰氨基葡萄糖1-磷酸转移酶催化的反应中形成GlcNAc-PP-DOLICHOL。以中国仓鼠卵巢(CHO)细胞膜为酶源，研究了两种脂类底物对Anin体外测定转移酶活性的影响。实验是用不同浓度的多氯乙酯或其前体聚戊烯基磷酸酯进行的。我们确定该酶的最适pH为9，此时该酶对乙醇底物的Vmax提高了3倍，Km降低了2倍。在pH 7.4时，两种脂类的KmandVmax相差10倍。在所有检测条件下，我们发现GlcNAc-PP-Lid是唯一形成的产物。根据这些结果，我们得出结论，在CHO细胞中，多氯乙酯磷酸而不是聚戊烯基磷酸是转移酶的首选底物。这一观察结果具有重要意义，因为我们以前分离到的CHO糖基化突变体无法将聚戊烯醇转化为多元醇，因此利用聚戊烯基衍生物进行糖基化反应。因此，这些结果有助于我们理解突变细胞系中的糖基化缺陷。
The assembly pathway of the oligosaccharide chains of asparagine-linked glycoproteins in mammalian cells begins with the formation of GlcNAc-PP-dolichol in a reaction catalysed by the enzymeN-acetylglucosamine 1-phosphate transferase. We have investigated the efficiency of two lipid substrates for the transferase activity in anin vitroassay using Chinese hamster ovary (CHO) cell membranes as an enzyme source. Experiments were carried out with varying concentrations of dolichyl phosphate or its precursor, polyprenyl phosphate. We determined that enzyme activity was optimal at pH 9, where the enzyme exhibited a 3-fold higher Vmaxand a 2-fold lowerKmfor the dolichol substrate. At pH 7.4, theKmandVmaxdifferences between the two lipids were 10-fold. Under all assay conditions tested, we found that GlcNAc-PP-lipid was the only product formed. We conclude from these results that dolichyl phosphate rather than polyprenyl phosphate is the preferred substrate for the transferase enzyme in CHO cells. This observation is significant in light of the fact that we have previously isolated CHO glycosylation mutants which fail to convert polyprenol into dolichol, and hence utilize polyprenyl derivatives for glycosylation reactions. Thus, these results contribute to our understanding of the glycosylation defects in the mutant cell lines.