Mutant analysis of glyceraldehyde 3-phosphate dehydrogenase in Escherichia coli.
Mutant analysis of glyceraldehyde 3-phosphate dehydrogenase in Escherichia coli.
复制标题
大肠杆菌中甘油醛3-磷酸脱氢酶的突变分析。
DOI：
10.1042/bj1790099
复制
发表时间：
1979
期刊：
影响因子：
--
通讯作者：
J. Hillman
中科院分区：
文献类型：
--
作者：
J. Hillman
通过琼脂糖己烷NAD(+)亲和层析和重复结晶的相对简单的步骤，从大肠杆菌中纯化了NAD(+)专一性的3-磷酸甘油醛脱氢酶(EC 1.2.1.12)。针对该蛋白的兔抗血清在对纯酶的双扩散研究中产生了一条沉淀线，对野生型大肠杆菌菌株的粗提物产生了两条线。这两条沉淀线代表抗体与甘油醛3-磷酸脱氢酶特异性决定簇的相互作用。9株缺乏3-磷酸甘油醛脱氢酶活性的独立突变体均对野生型酶有一定的抗原性交叉反应物质。根据双向扩散实验中观察到的沉淀线的类型和数量，突变体可分为三组；一组几乎没有形成交叉反应的物质。还测试了几个突变菌株粗提液中的交叉反应物质对NAD(+)的亲和力和磷酸化活性的变化。累积的数据表明，几个突变菌株的蛋白质发生了严重的改变，因此甘油醛3-磷酸脱氢酶不太可能具有必要的非催化功能，如缓冲烟酰胺核苷酸或糖酵解中间浓度。其他接受测试的突变体在所研究的几个参数上具有与野生型酶相似的交叉反应物质；这些菌株的蛋白质一旦纯化，可能会成为野生型酶的有用类似物。
NAD(+)-specific glyceraldehyde 3-phosphate dehydrogenase (EC 1.2.1.12) from Escherichia coli was purified to homogeneity by a relatively simple procedure involving affinity chromatography on agarose-hexane-NAD(+) and repeated crystallization. Rabbit antiserum directed against this protein produced one precipitin line in double-diffusion studies against the pure enzyme, and two lines against crude extracts of wild-type E. coli strains. Both precipitin lines represent the interaction of antibody with determinants specific for glyceraldehyde 3-phosphate dehydrogenase. Nine independent mutants of E. coli lacking glyceraldehyde 3-phosphate dehydrogenase activity all possessed some antigenic cross-reacting material to the wild-type enzyme. The mutants could be divided into three groups on the basis of the types and amounts of precipitin lines observed in double-diffusion experiments; one group formed little cross-reacting material. The cross-reacting material in crude cell-free extracts of several of the mutant strains were also tested for alterations in their affinity for NAD(+) and their phosphorylative activity. The cumulative data indicate that the protein in several of the mutant strains is severely altered, and thus that glyceraldehyde 3-phosphate dehydrogenase is unlikely to have an essential, non-catalytic function such as buffering nicotinamide nucleotide or glycolytic-intermediate concentrations. Others of the mutants tested have cross-reacting material which behaved like the wild-type enzyme for the several parameters studied; the proteins from these strains, once purified, might serve as useful analogues of the wild-type enzyme.