从高等植物长春花中纯化并部分鉴定微粒体NADH-细胞色素b5还原酶

Purification and partial characterization of microsomal NADH-cytochrome b5 reductase from higher plant Catharanthus roseus.

作者信息

Madyastha K M, Chary N K, Holla R, Karegowdar T B

机构信息

Department of Organic Chemistry, Indian Institute of Science, Bangalore.

出版信息

Biochem Biophys Res Commun. 1993 Dec 15;197(2):518-22. doi: 10.1006/bbrc.1993.2509.

DOI:10.1006/bbrc.1993.2509

PMID:8267585

Abstract

A simple three step procedure was used to purify microsomal NADH-cytochrome b5 (ferricyanide) reductase to homogeneity from the higher plant C. roseus. The microsomal bound reductase was solubilized using zwitterionic detergent-CHAPS. The solubilized reductase was subjected to affinity chromatography on octylamino Sepharose 4B, blue 2-Sepharose CL-6B and NAD(+)-Agarose. The homogeneous enzyme has an apparent molecular weight of 33,000 as estimated by SDS-PAGE. The purified enzyme catalyzes the reduction of purified cytochrome b5 from C. roseus in the presence of NADH. The reductase also readily transfers electrons from NADH to ferricyanide (Km 56 microM), 2, 6-dichlorophenolindophenol (Km 65 microM) and cytochrome c via cytochrome b5 but not to menadione.

摘要

采用一种简单的三步程序从高等植物长春花中纯化微粒体NADH - 细胞色素b5（铁氰化物）还原酶至同质状态。使用两性离子去污剂CHAPS使微粒体结合的还原酶溶解。将溶解的还原酶进行辛基氨基琼脂糖4B、蓝色2 - 琼脂糖CL - 6B和NAD(+) - 琼脂糖亲和层析。通过SDS - PAGE估计，该同质酶的表观分子量为33,000。纯化后的酶在NADH存在下催化长春花纯化细胞色素b5的还原反应。该还原酶还能轻易地通过细胞色素b5将电子从NADH转移至铁氰化物（Km为56微摩尔）、2,6 - 二氯酚靛酚（Km为65微摩尔）和细胞色素c，但不能转移至甲萘醌。