Solomonson L P, Howard W D, Yamaya T, Oaks A
Arch Biochem Biophys. 1984 Sep;233(2):469-74. doi: 10.1016/0003-9861(84)90469-7.
The molecular basis for the action of two natural inactivator proteins, isolated from rice and corn, on a purified assimilatory nitrate reductase has been examined by several physical techniques. Incubation of purified Chlorella nitrate reductase with either rice inactivator protein or corn inactivator protein results in a loss of NADH:nitrate reductase and the associated partial activity, NADH:cytochrome c reductase, but no loss in nitrate-reducing activity with reduced methyl viologen as the electron donor. The molecular weight of the reduced methyl viologen:nitrate reductase species, determined by sedimentation equilibrium in the Beckman airfuge after complete inactivation with rice inactivator protein or with corn inactivator protein, was 595,000 and 283,000, respectively, compared to a molecular weight of 376,000 for the untreated control determined under the same conditions. Two protein peaks were observed after molecular-sieve chromatography on Sephacryl S-300 of nitrate reductase inactivated by corn inactivator protein. The Stokes radii of these fragments were 68 and 24 A, compared to a value of 81 A for untreated nitrate reductase. The large fragment contained molybdenum and heme but no flavin, and had nitrate-reducing activity with reduced methyl viologen as electron donor. The small fragment contained FAD but had no NADH:cytochrome c reductase or nitrate-reducing activities. Molecular weights determined by sodium dodecyl sulfate-gel electrophoresis were 67,000 and 28,000 for the large and small fragments, respectively, compared to a subunit molecular weight of 99,000 determined for the untreated control. No change in subunit molecular weight of nitrate reductase after inactivation by rice inactivator protein was observed. These results indicate that rice inactivator protein acts by binding to nitrate reductase. The stoichiometry of binding is 1-2 molecules of rice inactivator protein to one tetrameric molecule of nitrate reductase. Corn inactivator protein, in contrast, acts by cleavage of a Mr 30,000 fragment from nitrate reductase which is associated with FAD. The remaining fragment is a tetramer of Mr 70,000 subunits which retains nitrate-reducing activity and contains molybdenum and heme but has no NADH:dehydrogenase activity. The action of rice inactivator protein was partially prevented by NADH and completely prevented by a combination of NADH and cyanide, while the action of corn inactivator protein was not significantly affected by these effectors.
通过多种物理技术，研究了从水稻和玉米中分离出的两种天然失活蛋白对纯化的同化型硝酸还原酶的作用分子基础。用水稻失活蛋白或玉米失活蛋白孵育纯化的小球藻硝酸还原酶，会导致NADH：硝酸还原酶及相关的部分活性NADH：细胞色素c还原酶丧失，但以还原型甲基紫精作为电子供体时，硝酸还原活性并未丧失。在用水稻失活蛋白或玉米失活蛋白完全失活后，通过贝克曼空气离心机中的沉降平衡测定，还原型甲基紫精：硝酸还原酶物种的分子量分别为595,000和283,000，而在相同条件下测定的未处理对照的分子量为376,000。在用玉米失活蛋白失活的硝酸还原酶在Sephacryl S - 300上进行分子筛层析后，观察到两个蛋白峰。这些片段的斯托克斯半径分别为68和24 Å，而未处理的硝酸还原酶的值为81 Å。大的片段含有钼和血红素，但不含黄素，以还原型甲基紫精作为电子供体时具有硝酸还原活性。小的片段含有FAD，但没有NADH：细胞色素c还原酶或硝酸还原活性。通过十二烷基硫酸钠 - 凝胶电泳测定，大、小片段的分子量分别为67,000和28,000，而未处理对照的亚基分子量为99,000。在用水稻失活蛋白失活后，未观察到硝酸还原酶亚基分子量的变化。这些结果表明，水稻失活蛋白通过与硝酸还原酶结合起作用。结合化学计量为每一个硝酸还原酶四聚体分子结合1 - 2个水稻失活蛋白分子。相比之下，玉米失活蛋白通过从硝酸还原酶上切割下一个与FAD相关的30,000 Mr片段起作用。剩余的片段是一个由70,000 Mr亚基组成的四聚体，保留硝酸还原活性，含有钼和血红素，但没有NADH：脱氢酶活性。NADH部分抑制水稻失活蛋白的作用，NADH和氰化物的组合完全抑制其作用，而玉米失活蛋白的作用不受这些效应物的显著影响。
