The 50-kDa glucose 6-phosphate-sensitive hexokinase of Schistosoma mansoni.
The 50-kDa glucose 6-phosphate-sensitive hexokinase of Schistosoma mansoni.
复制标题
DOI：
--
复制
发表时间：
1994-10
期刊：
影响因子：
--
通讯作者：
A. Tielens;J. M. V. D. Heuvel;H. J. V. Mazijk;J. Wilson;C. Shoemaker
中科院分区：
文献类型：
--
作者：
A. Tielens;J. M. V. D. Heuvel;H. J. V. Mazijk;J. Wilson;C. Shoemaker
已从曼氏血吸虫成虫中提纯了己糖激酶，其活性与一种M(R)约为50,000的单一蛋白物种有关。用抗鼠I型己糖激酶的抗血清或用先前克隆的cDNA在大肠杆菌中表达的重组曼氏葡萄球菌己糖激酶的抗血清探测的Western blotting上可以识别该蛋白。纯化的曼氏葡萄球菌己糖激酶的18个残基的N末端序列与从该基因的核苷酸序列推导出的序列完全相同，这与所克隆的cDNA编码本研究中所描述的己糖激酶的观点一致。曼氏葡萄球菌酶对葡萄糖的Km相对较低(约60微米)，对其产物葡萄糖6-磷酸(GLC-6-P)的抑制(竞争性对ATP，Ki约为50微米)敏感。具有这些动力学性质和50 kDa分子量的曼氏葡萄球菌己糖激酶类似于祖先通过基因复制和融合而产生的100 kDa GLC-6-P敏感的哺乳动物己糖激酶。血吸虫己糖激酶是第一个已获得序列的50 kDa Glc-6-P敏感己糖激酶。血吸虫己糖激酶不与线粒体结合，这与它在N端缺乏疏水片段一致，这是哺乳动物I和II同工酶与线粒体结合所必需的。曼氏尾蚴表现出显著的克拉布特里效应至少部分归因于对葡萄糖有很高亲和力但对GLC-6-P的产物抑制只有中等敏感性的己糖激酶的高水平表达。
Hexokinase has been purified from adult Schistosoma mansoni worms and the activity shown to be associated with a single protein species having an M(r) about 50,000. This protein is recognized on Western blots probed with antisera against rat Type I hexokinase or against a recombinant S. mansoni hexokinase that had been expressed in Escherichia coli using a previously cloned cDNA. An 18-residue N-terminal sequence determined for the purified S. mansoni hexokinase is identical to that deduced from the nucleotide sequence of the cDNA, consistent with the view that the cloned cDNA encodes the hexokinase characterized in the present study. The S. mansoni enzyme has a relatively low Km (approximately 60 microM) for glucose and is sensitive to inhibition (competitive versus ATP, Ki approximately 50 microM) by its product, glucose 6-phosphate (Glc-6-P). With these kinetic properties and 50 kDa molecular mass, S. mansoni hexokinase resembles the ancestral hexokinase predicted to have given rise, by gene duplication and fusion, to the present day 100-kDa Glc-6-P-sensitive mammalian hexokinases. The schistosomal hexokinase represents the first 50-kDa Glc-6-P-sensitive hexokinase whose sequence has been obtained. The schistosomal hexokinase does not bind to mitochondria, consistent with its lack of a hydrophobic segment at the N terminus which is required for binding of the mammalian Type I and II isoenzymes to mitochondria. The marked Crabtree effect exhibited by S. mansoni cercariae may be at least partly attributed to the expression of rather high levels of a hexokinase having a high affinity for glucose but only a moderate sensitivity to product inhibition by Glc-6-P.