Chu F K, Watorek W, Maley F
Arch Biochem Biophys. 1983 Jun;223(2):543-55. doi: 10.1016/0003-9861(83)90619-7.
It has been assumed that yeast external invertase is a dimer, with each subunit composed of a 60-kDa polypeptide chain. We now present evidence that at its optimal pH of 5.0, the predominant form of external invertase is an octamer with an average size of 8 X 10(5) Da. During ultracentrifugation the octamer dissociated to lower molecular weight forms, including a hexamer, tetramer, and dimer. All forms of the enzyme were shown to possess identical specific activities and to contain a similar carbohydrate to protein ratio. Although the monomer subunits (1 X 10(5) Da) were heterogenous in carbohydrate content, each subunit possessed nine 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. Thus, on partially inactivating invertase with 4 M guanidine hydrochloride both octamer and monomer were evident on the gels but only the former was active. Similarly, 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.
人们一直认为酵母外切转化酶是一种二聚体，每个亚基由一条60 kDa的多肽链组成。我们现在提供证据表明，在其最适pH值5.0时，外切转化酶的主要形式是一种八聚体，平均大小为8×10⁵ Da。在超速离心过程中，八聚体解离为较低分子量的形式，包括六聚体、四聚体和二聚体。所有形式的酶都显示具有相同的比活性，并且碳水化合物与蛋白质的比例相似。尽管单体亚基（1×10⁵ Da）的碳水化合物含量不同，但每个亚基都有九条寡糖链。在十二烷基硫酸钠-聚丙烯酰胺凝胶电泳后进行蛋白质和酶活性染色时，只有酶的寡聚形式似乎具有活性。因此，用4 M盐酸胍部分灭活转化酶后，凝胶上八聚体和单体都很明显，但只有前者具有活性。同样，在十二烷基硫酸钠存在下于pH 2.5孵育只产生无活性的单体。与活性寡聚聚集体不同，单体在十二烷基硫酸钠-聚丙烯酰胺凝胶电泳后不能水解蔗糖。与体外研究一致，新鲜制备的酵母裂解物显示含有外切转化酶的八聚体形式作为该酶的主要活性形式。从这些研究以及其他使用去糖基化转化酶的研究可以得出结论，外切转化酶的碳水化合物成分不仅有助于稳定酶活性，而且有助于维持其寡聚结构。
