Inoue S B, Takewaki N, Takasuka T, Mio T, Adachi M, Fujii Y, Miyamoto C, Arisawa M, Furuichi Y, Watanabe T
Department of Mycology, Nippon Roche Research Center, Kamakura, Japan.
Eur J Biochem. 1995 Aug 1;231(3):845-54. doi: 10.1111/j.1432-1033.1995.tb20770.x.
1,3-beta-D-Glucan synthase of Saccharomyces cerevisiae was solubilized and purified up to 700-fold by product entrapment. The specific activity of the partially purified enzyme was around 4 mumol glucose incorporated.min-1.mg protein-1. In SDS/PAGE, enrichment of a 200-kDa protein was clearly observed in parallel with the increase in specific activity. mAbs that could immunoprecipitate the 1,3-beta-D-glucan synthase activity were isolated, and some of them also recognized this 200-kDa protein in the Western blot. Internal amino acid sequences of this 200-kDa protein were determined after lysyl endopeptidase digestion. With the information of these amino acid sequences, we cloned two genes, GSC1 and GSC2 (glucan synthase of S. cerevisiae 1 and 2), which are very similar to each other (88% at the amino acid level); hydropathy profiles of both proteins suggest that these genes encode integral membrane proteins which can be assumed to have approximately 16 transmembrane domains. Disruption of each gene was not lethal, but disruption of both genes was lethal. The 1,3-beta-D-glucan synthase activities of membrane and partially purified enzyme of gsc1::URA3 cells were significantly lower than those of the wild-type and gsc2::LEU2 cells.
酿酒酵母的1,3-β-D-葡聚糖合酶通过产物截留法进行溶解和纯化，纯化倍数高达700倍。部分纯化酶的比活性约为4 μmol葡萄糖掺入·分钟⁻¹·毫克蛋白⁻¹。在SDS/PAGE中，随着比活性的增加，明显观察到一种200 kDa蛋白的富集。分离出了能够免疫沉淀1,3-β-D-葡聚糖合酶活性的单克隆抗体，其中一些在蛋白质印迹中也识别这种200 kDa蛋白。该200 kDa蛋白经赖氨酰内肽酶消化后测定其内部氨基酸序列。根据这些氨基酸序列信息，我们克隆了两个基因，GSC1和GSC2（酿酒酵母葡聚糖合酶1和2），它们彼此非常相似（氨基酸水平上为88%）；两种蛋白质的亲水性图谱表明，这些基因编码的是整合膜蛋白，推测大约有16个跨膜结构域。每个基因的破坏都不是致命的，但两个基因都破坏是致命的。gsc1::URA3细胞的膜和部分纯化酶的1,3-β-D-葡聚糖合酶活性明显低于野生型和gsc2::LEU2细胞。
