Giordano G, Grillet L, Pommier J, Terriere C, Haddock B A, Azoulay E
Eur J Biochem. 1980 Apr;105(2):297-306. doi: 10.1111/j.1432-1033.1980.tb04501.x.
The synthesis of nitrate reductase by a parental Escherichia coli K12 strain and its isogenic chlA and chlB mutants has been analyzed by protein double labelling with L-[4,5-3H]leucine and sulphur-35 and by immunoprecipitation using specific antiserum. The chlA and chlB mutants although defective in nitrate reductase activity retain the ability to synthesise the different polypeptides that are normally required for functional enzyme activity. In addition the data shows the following. 1. These polypeptides are present in unequal quantities in the membrane and in the cytoplasm of the cells. The chlB mutant synthesizes three times more nitrate reductase than the chlA mutant. 2. The subunit composition of the membrane-bound nitrate reductase present in the two mutants is different. 3. Membrane preparations from the chlB mutant contain the three subunits alpha, beta, gamma in a ratio which is similar to the wild type. 4. In the chlA mutant the two subunits beta and gamma are missing and the level of alpha subunit is very low. In the same membrane a 48,000-Mr subunit (polypeptide beta') precipitable by nitrate reductase antiserum has been found. The chlA and chlB mutants accumulate the three subunits alpha, beta and gamma in different proportion and concentrations in the cytoplasm unlike the parental strain. 5. The cytoplasm from the chlA mutant also contains the beta' polypeptide found in the membrane fraction of this mutant and in addition contain another polypeptide designated alpha' of molecular weight 105,000 which is precipitated by the nitrate reductase antiserum. The formation of particulate active nitrate reductase can be achieved by mixing the supernatant fractions of the chlA and chlB mutants (complementation) and procedes by two distinct but mutually dependent stages. Following reconstitution of activity the two peptides alpha' and beta' present in the supernatant fraction of the chlA mutant, disappear. Analysis of the immunoprecipitate polypeptides present in both the soluble and particulate nitrate reductase protein after reconstitution suggests that these polypeptides are precursors of the alpha and beta subunits following a process that remains to be elucidated.
通过用L-[4,5-³H]亮氨酸和³⁵S进行蛋白质双重标记以及使用特异性抗血清进行免疫沉淀，分析了亲本大肠杆菌K12菌株及其同基因chlA和chlB突变体中硝酸还原酶的合成情况。chlA和chlB突变体虽然在硝酸还原酶活性方面存在缺陷，但仍保留合成功能酶活性通常所需的不同多肽的能力。此外，数据显示如下：1. 这些多肽在细胞的膜和细胞质中的含量不等。chlB突变体合成的硝酸还原酶比chlA突变体多三倍。2. 两个突变体中存在的膜结合硝酸还原酶的亚基组成不同。3. chlB突变体的膜制剂中α、β、γ三个亚基的比例与野生型相似。4. 在chlA突变体中，β和γ两个亚基缺失，α亚基水平非常低。在同一膜中发现了一种可被硝酸还原酶抗血清沉淀的48,000-Mr亚基（多肽β'）。与亲本菌株不同，chlA和chlB突变体在细胞质中以不同比例和浓度积累α、β和γ三个亚基。5. chlA突变体的细胞质中还含有在该突变体膜部分中发现的β'多肽，此外还含有另一种分子量为105,000的多肽，称为α'，可被硝酸还原酶抗血清沉淀。通过混合chlA和chlB突变体的上清液部分（互补）可以实现颗粒状活性硝酸还原酶的形成，并且该过程通过两个不同但相互依赖的阶段进行。活性重建后，chlA突变体上清液部分中存在的α'和β'两种肽消失。对重建后可溶性和颗粒状硝酸还原酶蛋白中存在的免疫沉淀多肽的分析表明，这些多肽是α和β亚基的前体，其过程仍有待阐明。
