Seki-Chiba S, Ishimoto M
J Biochem. 1977 Dec;82(6):1663-71. doi: 10.1093/oxfordjournals.jbchem.a131862.
Nitrate reductase (NaR) linked to reduced methyl viologen from Clostridium perfringens was purified by ammonium sulfate precipitation. DEAE-cellulose chromatography, disc electrophoresis on polyacrylamide gel, and triple DEAE-Sephadex chromatography. The specific activity was increased 1,200-fold with a yield of 9%. The purified preparation was nearly homogeneous in disc electrophoresis. It was brown, and its spectrum showed a slight shoulder near 420 nm as well as a peak at 280 nm. The molecular weight was found to be 90,000 based on s020,w (5.8S) and 80,000 by Sephadex G-100 gel filtration. In SDS-polyacrylamide electrophoresis, it showed only a single band with a molecular weight of 90,000; it had no subunit structure. The isoelectric point was pH 5.5, and the optimum pH was 9. Mn2+, Fe2+, Mg2+, and Ca2+ stimulated the activity. Km for nitrate was 0.10 mM, and nitrate was stoichiometrically reduced to nitrite in the presence of 2 mM Mn2+. Ferredoxin fraction obtained from extracts of the bacterium was utilizable as an electron donor at pH 8. Cyanide and azide inhibited the enzyme. The formation of NaR was induced by nitrate and inhibited by 0.5 mM tungstate, but recovered in the presence of 0.1 mM molybdate; NaR of C. perfringens appears to be a molybdo-iron-sulfur protein.
通过硫酸铵沉淀、DEAE - 纤维素柱层析、聚丙烯酰胺凝胶圆盘电泳和三重DEAE - 葡聚糖凝胶柱层析，对与产气荚膜梭菌中还原型甲基紫精相关的硝酸还原酶（NaR）进行了纯化。比活性提高了1200倍，产率为9%。纯化后的制剂在圆盘电泳中几乎是均一的。它呈棕色，其光谱在420nm附近有一个轻微的肩峰，在280nm处有一个峰值。根据s020,w（5.8S）测定分子量为90,000，通过葡聚糖G - 100凝胶过滤法测定为80,000。在SDS - 聚丙烯酰胺电泳中，它仅显示一条分子量为90,000的条带；没有亚基结构。等电点为pH 5.5，最适pH为9。Mn2 +、Fe2 +、Mg2 +和Ca2 +能刺激该酶的活性。硝酸盐的Km值为0.10mM，在2mM Mn2 +存在下，硝酸盐按化学计量比还原为亚硝酸盐。从该细菌提取物中获得的铁氧化还原蛋白组分在pH 8时可作为电子供体。氰化物和叠氮化物抑制该酶。NaR的形成由硝酸盐诱导，0.5mM钨酸盐可抑制其形成，但在0.1mM钼酸盐存在下可恢复；产气荚膜梭菌的NaR似乎是一种钼铁硫蛋白。
