FACTORS AFFECTING THE OLIGOMERIC STRUCTURE OF YEAST EXTERNAL INVERTASE
FACTORS AFFECTING THE OLIGOMERIC STRUCTURE OF YEAST EXTERNAL INVERTASE
复制标题
DOI：
10.1016/0003-9861(83)90619-7
复制
发表时间：
1983-01-01
影响因子：
3.9
通讯作者：
MALEY, F
中科院分区：
文献类型：
--
作者：
CHU, FK;WATOREK, W;MALEY, F
酵母外转化酶被认为是一个二聚体，每个亚基由一个60 kDa的多肽链组成。现在有证据表明，在其最适pH为5.0时，外源转化酶的主要形式是平均大小为8倍的八聚体。105Da。在超速离心过程中，八聚体解离为低分子量形式，包括六聚体、四聚体和二聚体。所有形式的酶都具有相同的比活性，并且含有相似的碳水化合物与蛋白质的比例。虽然单体亚基(1.×.105Da)的碳水化合物含量不均一，每个亚基含有9条寡糖链。十二烷基硫酸钠-聚丙烯酰胺凝胶电泳法对蛋白质和酶活性进行染色时，只有低聚形式的酶表现出活性。在用4M盐酸胍部分失活转化酶时，凝胶上有明显的八聚体和单体，但只有前者有活性。在pH 2.5的条件下，在十二烷基硫酸钠存在下孵育，只能产生失活的单体。与活性低聚物不同的是，十二烷基硫酸钠-聚丙烯酰胺凝胶电泳后，该单体不能对蔗糖进行水解。与体外研究一致的是，新鲜制备的酵母裂解物中含有该酶的主要活性形式--外源转化酶的八聚体。从这些研究和其他使用脱糖转化酶的研究中得出结论，外源转化酶的碳水化合物成分不仅有助于稳定酶的活性，而且有助于维持其寡聚体结构。
It has been assumed that yeast external invertase is a dimer with each subunit composed of a 60-kDa [dalton] polypeptide chain. Evidence is now presented that at its optimal pH of 5.0, the predominant form of external invertase is an octamer with an average size of 8 .times. 105 Da. During ultracentrifugation the octamer dissociated to lower MW forms, including a hexamer, tetramer and dimer. All forms of the enzyme possess identical specific activities and contain a similar carbohydrate to protein ratio. Although the monomer subunits (1 .times. 105 Da) were hetereogeneous in carbohydrate content, each subunit possessed 9 oligosaccharide chains. When stained for protein and enzyme activity following sodium dodecyl sulfate-polyacrylamide gel electrophoresis, only the oligomeric form of the enzyme appeared to be active. On partially inactivating invertase with 4 M guanidine hydrochloride both octamer and monomer were evident on the gels but only the former was active. Incubating at pH 2.5 in the presence of sodium dodecyl sulfate yielded only inactive monomer. The monomer, unlike the active oligomeric aggregate, was unable to hydrolyze sucrose after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Consistent with the in vitro studies, freshly prepared yeast lysate was shown to contain the octameric species of external invertase as the major active form of this enzyme. From these studies and others which employed deglycosylated invertase, it is concluded that the carbohydrate component of external invertase contributes not only to stabilizing enzyme activity, but also to maintaining its oligomeric structure.