Saido H, Watanabe F, Tamura Y, Miyatake K, Ito A, Yubisui T, Nakano Y
Department of Agricultural Chemistry, University of Osaka Prefecture, Japan.
J Nutr. 1994 Jul;124(7):1037-40. doi: 10.1093/jn/124.7.1037.
Rat liver mitochondrial NADH-linked aquacobalamin reductase was characterized to clarify its enzymological properties. Most of the enzyme was solubilized with 10 g/L Triton X-100 from rat liver mitochondrial membranes. The elution behavior of the solubilized enzyme was identical to that of NADH-cytochrome c reductase (b-type cytochromes/cytochrome b5 reductase complex) during DEAE-Sepharose Fast Flow column chromatography. By mixing both purified cytochrome b5-like hemoprotein (outer membrane-cytochrome b) and cytochrome b5 reductase, cob(II)alamin was formed from aquacobalamin and NADH. These results provide evidence that the outer membrane-cytochrome b/cytochrome b5 reductase complex has the activity of the NADH-linked aquacobalamin reductase in rat liver mitochondria. Some properties of the NADH-linked aquacobalamin reductase were studied using the function of rat liver mitochondrial membranes. The specific activity (109.5 +/- 14.3 nmol.min-1.mg protein-1) of the enzyme was shown under physiological conditions (pH 7.1 at 40 degrees C). The optimal pH and temperature for activity were 7.1 and 40 degrees C, respectively. The apparent Km values were 41.9 mumol/L for aquacobalamin in the presence of 0.2 mmol/L NADH and 14.4 mumol/L for NADH in the presence of 0.1 mmol/L aquacobalamin. The enzyme was specific for aquacobalamin, because cyanocobalamin could not be reduced by the enzyme.
对大鼠肝脏线粒体中与NADH相关的水钴胺素还原酶进行了表征，以阐明其酶学性质。大部分酶用10 g/L Triton X-100从大鼠肝脏线粒体膜中溶解出来。在DEAE-琼脂糖快速流动柱色谱过程中，溶解酶的洗脱行为与NADH-细胞色素c还原酶（b型细胞色素/细胞色素b5还原酶复合物）相同。通过将纯化的细胞色素b5样血红蛋白（外膜细胞色素b）和细胞色素b5还原酶混合，水钴胺素和NADH形成了钴胺素（II）。这些结果证明外膜细胞色素b/细胞色素b5还原酶复合物在大鼠肝脏线粒体中具有与NADH相关的水钴胺素还原酶活性。利用大鼠肝脏线粒体膜的功能研究了与NADH相关的水钴胺素还原酶的一些性质。在生理条件下（40℃，pH 7.1）显示该酶的比活性为（109.5±14.3 nmol·min-1·mg蛋白-1）。该酶活性的最适pH和温度分别为7.1和40℃。在0.2 mmol/L NADH存在下，水钴胺素的表观Km值为41.9 μmol/L；在0.1 mmol/L水钴胺素存在下，NADH的表观Km值为14.4 μmol/L。该酶对水钴胺素具有特异性，因为氰钴胺素不能被该酶还原。
