Bérczi A, Fredlund K M, Møller I M
Institute of Biophysics, Biological Research Center, Hungarian Academy of Sciences, Szeged.
Arch Biochem Biophys. 1995 Jun 20;320(1):65-72. doi: 10.1006/abbi.1995.1343.
Plasma membranes were purified from spinach (Spinacea oleracea L.) leaves by aqueous two-phase partitioning. The NADH-hexacyanoferrate(III) reductase was released from the membrane by Chaps solubilization and purified 360-fold by ion-exchange chromatography followed by affinity chromatography and size-exclusion chromatography on FPLC. A major band of 45 kDa and a minor contaminant of 66 kDa were detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The band at 45 kDa cross-reacted with antibodies raised against an NADH-hexacyanoferrate(III) reductase from potato tuber microsomes. The native size of the enzyme was 160 kDa as determined by size-exclusion chromatography indicating that it is a tetramer. Two-dimensional gel electrophoresis, isoelectric focusing, followed by SDS-PAGE revealed three main bands of identical molecular weight with pI of 5.3-5.6. The enzyme contained about one flavin adenine dinucleotide (FAD) per 45-kDa subunit as determined by fluorescence spectroscopy, was specific for the beta-hydrogen of NADH, preferred NADH over NADPH as electron donor, and preferred hexacyanoferrate(III) as electron acceptor, e.g., it reduced Fe3+-EDTA, cytochrome c, oxygen, and duroquinone at < 10% of the rate with hexacyanoferrate(III). p-Chloromercurobenzoate, mersalyl, and dicumarol inhibited the activity by > 70% whereas FAD, flavin mononucleotide, duroquinone, and ubiquinone0 did not affect the activity.
通过双水相分配法从菠菜（Spinacea oleracea L.）叶片中纯化质膜。用Chaps增溶法从膜上释放NADH-高铁氰化物（III）还原酶，并通过离子交换色谱法，随后在FPLC上进行亲和色谱和尺寸排阻色谱法纯化360倍。通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）检测到一条45 kDa的主要条带和一条66 kDa的次要污染物条带。45 kDa处的条带与针对马铃薯块茎微粒体中的NADH-高铁氰化物（III）还原酶产生的抗体发生交叉反应。通过尺寸排阻色谱法测定该酶的天然大小为160 kDa，表明它是一种四聚体。二维凝胶电泳、等电聚焦，随后进行SDS-PAGE显示出三条分子量相同、pI为5.3 - 5.6的主要条带。通过荧光光谱法测定，该酶每个45 kDa亚基含有约一个黄素腺嘌呤二核苷酸（FAD），对NADH的β-氢具有特异性，作为电子供体时优先选择NADH而非NADPH，作为电子受体时优先选择高铁氰化物（III），例如，它还原Fe3+-EDTA、细胞色素c、氧气和硬脂醌的速率不到还原高铁氰化物（III）速率的10%。对氯汞苯甲酸、汞撒利和双香豆素抑制活性超过70%，而FAD、黄素单核苷酸、硬脂醌和泛醌0不影响活性。
