Díez J, López-Ruiz A
Departamento de Bioquímica y Biología Molecular, Universidad de Córdoba, Spain.
Arch Biochem Biophys. 1989 Feb 1;268(2):707-15. doi: 10.1016/0003-9861(89)90339-1.
The effects of different culture conditions on nitrate reductase activity and nitrate reductase protein from Monoraphidium braunii have been studied, using two different immunological techniques, rocket immunoelectrophoresis and an enzyme-linked immunosorbent assay, to determine nitrate reductase protein. The nitrogen sources ammonium and glutamine repressed nitrate reductase synthesis, while nitrite, alanine, and glutamate acted as derepressors. There was a four- to eightfold increase of nitrate reductase activity and a twofold increase of nitrate reductase protein under conditions of nitrogen starvation versus growth on nitrate. Nitrate reductase synthesis was repressed in darkness. However, when Monoraphidium was grown under heterotrophic conditions with glucose as the carbon and energy source, the synthesis of nitrate reductase was maintained. With ammonium or darkness, changes in nitrate reductase activity correlated fairly well with changes in nitrate reductase protein, indicating that in both cases loss of activity was due to repression and not to inactivation of the enzyme. Experiments using methionine sulfoximine, to inhibit ammonium assimilation, showed that ammonium per se and not a product of its metabolism was the corepressor of the enzyme. The appearance of nitrate reductase activity after transferring the cells to induction media was prevented by cycloheximide and by 6-methylpurine, although in this latter case the effect was observed only in cells preincubated with the inhibitor for 1 h before the induction period.
利用火箭免疫电泳和酶联免疫吸附测定这两种不同的免疫学技术来测定蛋白核小球藻的硝酸还原酶蛋白，研究了不同培养条件对其硝酸还原酶活性和硝酸还原酶蛋白的影响。氮源铵和谷氨酰胺抑制硝酸还原酶的合成，而亚硝酸盐、丙氨酸和谷氨酸则作为去阻遏物。与在硝酸盐上生长相比，在氮饥饿条件下硝酸还原酶活性增加了4至8倍，硝酸还原酶蛋白增加了2倍。硝酸还原酶的合成在黑暗中受到抑制。然而，当蛋白核小球藻在以葡萄糖作为碳源和能源的异养条件下生长时，硝酸还原酶的合成得以维持。在铵存在或黑暗条件下，硝酸还原酶活性的变化与硝酸还原酶蛋白的变化相当吻合，这表明在这两种情况下，活性的丧失是由于抑制作用而非酶的失活。使用蛋氨酸亚砜亚胺抑制铵同化的实验表明，是铵本身而非其代谢产物是该酶的共阻遏物。用环己酰亚胺和6-甲基嘌呤可阻止细胞转移至诱导培养基后硝酸还原酶活性的出现，不过在后一种情况下，只有在诱导期前用抑制剂预孵育1小时的细胞中才观察到这种效果。
