Scott R H, DeMoss J A
J Bacteriol. 1976 Apr;126(1):478-86. doi: 10.1128/jb.126.1.478-486.1976.
When Escherichia coli was grown on medium containing 10 mM tungstate the formation of active formate dehydrogenase, nitrate reductase, and the complete formate-nitrate electron transport pathway was inhibited. Incubation of the tungstate-grown cells with 1 mM molybdate in the presence of chloramphenicol led to the rapid activation of both formate dehydrogenase and nitrate reductase, and, after a considerable lag, the complete electron transport pathway. Protein bands which corresponded to formate dehydrogenase and nitrate reductase were identified on polyacrylamide gels containing Triton X-100 after the activities were released from the membrane fraction and partially purified Cytochrome b1 was associated with the protein band corresponding to formate dehydrogenase but was not found elsewhere on the gels. When a similar fraction was prepared from cells grown on 10 mM tungstate, an inactive band corresponding to formate dehydrogenase was not observed on polyacrylamide gels; rather, a new faster migrating band was present. Cytochrome b1 was not associated with this band nor was it found anywhere else on the gels. This new band disappeared when the tungstate-grown cells were incubated with molybdate in the presence of chloramphenicol. The formate dehydrogenase activity which was formed, as well as a corresponding protein band, appeared at the original position on the gels. Cytochrome b1 was again associated with this band. The protein band which corresponded to nitrate reductase also was severely depressed in the tungstate-grown cells and a new faster migrating band appeared on the polyacrylamide gels. Upon activation of the nitrate reductase by incubation of the cells with molybdate, the new band diminished and protein reappeared at the original position. Most of the nitrate reductase activity which was formed appeared at the original position of nitrate reductase on gels although some was present at the position of the inactive band formed by tungstate-grown cells. Apparently, inactive forms of both formate dehydrogenase and nitrate reductase accumulate during growth on tungstate which are electrophoretically distinct from the active enzymes. Activation by molybdate results in molecular changes which include the reassociation of cytochrome b1 with formate dehydrogenase and restoration of both enzymes to their original electrophoretic mobilities.
当大肠杆菌在含有10 mM钨酸盐的培养基上生长时，活性甲酸脱氢酶、硝酸还原酶的形成以及完整的甲酸 - 硝酸盐电子传递途径均受到抑制。在氯霉素存在的情况下，将在钨酸盐培养基上生长的细胞与1 mM钼酸盐一起孵育，会导致甲酸脱氢酶和硝酸还原酶迅速激活，并且在相当长的延迟后，完整的电子传递途径也被激活。在从膜部分释放活性并进行部分纯化后，在含有 Triton X - 100的聚丙烯酰胺凝胶上鉴定出了与甲酸脱氢酶和硝酸还原酶相对应的蛋白条带。细胞色素b1与对应于甲酸脱氢酶的蛋白条带相关，但在凝胶的其他位置未发现。当从在10 mM钨酸盐上生长的细胞制备类似部分时，在聚丙烯酰胺凝胶上未观察到与甲酸脱氢酶相对应的无活性条带；相反，出现了一条新的迁移速度更快的条带。细胞色素b1既不与这条带相关，在凝胶的其他任何位置也未发现。当在氯霉素存在的情况下将在钨酸盐培养基上生长的细胞与钼酸盐一起孵育时，这条新带消失了。形成的甲酸脱氢酶活性以及相应的蛋白条带出现在凝胶上的原始位置。细胞色素b1再次与这条带相关。在钨酸盐培养基上生长的细胞中，与硝酸还原酶相对应的蛋白条带也严重减少，并且在聚丙烯酰胺凝胶上出现了一条新的迁移速度更快的条带。通过将细胞与钼酸盐一起孵育来激活硝酸还原酶后，新带减少，蛋白在原始位置重新出现。形成的大部分硝酸还原酶活性出现在凝胶上硝酸还原酶的原始位置，尽管在由在钨酸盐培养基上生长的细胞形成的无活性条带位置也有一些活性。显然，在钨酸盐上生长期间，甲酸脱氢酶和硝酸还原酶的无活性形式会积累，它们在电泳上与活性酶不同。钼酸盐激活会导致分子变化，包括细胞色素b1与甲酸脱氢酶重新结合以及两种酶恢复到其原始电泳迁移率。
