ANIMAL-CELL MUTANTS DEFECTIVE IN GLYCOSAMINOGLYCAN BIOSYNTHESIS
ANIMAL-CELL MUTANTS DEFECTIVE IN GLYCOSAMINOGLYCAN BIOSYNTHESIS
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DOI：
10.1073/pnas.82.10.3197
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发表时间：
1985-01-01
影响因子：
11.1
通讯作者：
TAYLOR, WH
中科院分区：
文献类型：
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作者：
ESKO, JD;STEWART, TE;TAYLOR, WH
通过筛选固定在聚酯布上的重复菌落，获得糖胺聚糖生物合成缺陷的中国仓鼠卵巢细胞突变体。根据菌株的不同，突变体每μ g细胞蛋白积累的35 S标记的糖胺聚糖比野生型少6-60倍。一些突变体将[6- 3 H]葡糖胺掺入糖胺聚糖中，其程度与野生型相同，表明硫酸盐加成被特异性改变。5株菌不能正常产生3 H标记的糖胺聚糖。在其中的4个中，糖胺聚糖组装的起始被特异性地改变，因为添加了对硝基苯基-β-葡聚糖。木糖苷使硫酸化恢复正常。从这些突变体制备的提取物中的木糖基转移酶的酶法测定显示，菌株之一，S745，所含的酶活性比野生型低15倍。该突变体提供了遗传证据，即体外测定的木糖基转移酶负责体内硫酸软骨素和硫酸乙酰肝素生物合成的起始。
Chinese hamster ovary cell mutants defective in the biosynthesis of glycosaminoglycans were obtained by screening replicate colonies immobilized on polyester cloth. Depending upon the strain, the mutants accumulated less 35S-labeled glycosaminoglycans per .mu.g of cell protein by a factor of 6-60 compared to the wild type. Some of the mutants incorporated [6-3H]glucosamine into glycosaminoglycans to the same extent as the wild type, suggesting that sulfate addition was specifically altered. In contrast, 5 strains failed to generate 3H-labeled glycosaminoglycans normally. In 4 of these, the initiation of glycosaminoglycan assembly was specifically altered, since the addition of p-nitrophenyl-.beta.-xyloside restored sulfation to normal. Enzymatic assay of the xylosyltransferase in extracts prepared from these mutants revealed that one of the strains, S745, contained less enzyme activity by a factor of 15 than the wild type. This mutant provides genetic evidence that the xylosyltransferase assayed in vitro is responsible for the initiation of chondroitin sulfate and heparan sulfate biosynthesis in vivo.