从毛花洋地黄（Digitalis lanata EHRH.）细胞培养物中重组细胞色素P-450依赖性洋地黄毒苷12β-羟化酶。
Reconstitution of cytochrome P-450-dependent digitoxin 12 beta-hydroxylase from cell cultures of foxglove (Digitalis lanata EHRH.).
作者信息
Petersen M, Seitz H U
机构信息
Institut für Entwicklungs- und Molekularbiologie der Pflanzen, Universität Düsseldorf, Federal Republic of Germany.
出版信息
Biochem J. 1988 Jun 1;252(2):537-43. doi: 10.1042/bj2520537.
Cytochrome P-450-dependent digitoxin 12 beta-hydroxylase from cell cultures of foxglove (Digitalis lanata) was solubilized from microsomal membranes with CHAPS (3-[(3-cholamidopropyl)dimethylammonio]propane-1-sulphonic acid). Cytochrome P-450 was separated from NADPH: cytochrome c (P-450) reductase by ion-exchange chromatography on DEAE-Sephacel. NADPH:cytochrome c (P-450) reductase was further purified by affinity chromatography on 2',5'-ADP-Sepharose 4B. This procedure resulted in a 248-fold purification of the enzyme; on SDS/polyacrylamide-gel electrophoresis after silver staining, only one band, corresponding to a molecular mass of 80 kDa, was present. The digitoxin 12 beta-hydroxylase activity could be reconstituted by incubating partially purified cytochrome P-450 and NADPH:cytochrome c (P-450) reductase together with naturally occurring microsomal lipids and flavin nucleotides. This procedure yielded about 10% of the original amount of digitoxin 12 beta-hydroxylase.
从毛地黄（Digitalis lanata）细胞培养物中提取的细胞色素P - 450依赖性洋地黄毒苷12β - 羟化酶，用CHAPS（3 - [(3 - 胆酰胺丙基)二甲基铵基]丙烷 - 1 - 磺酸）从微粒体膜中溶解出来。通过在DEAE - Sephacel上进行离子交换色谱，将细胞色素P - 450与NADPH：细胞色素c（P - 450）还原酶分离。NADPH：细胞色素c（P - 450）还原酶通过在2',5'-ADP - Sepharose 4B上进行亲和色谱进一步纯化。该方法使酶得到了248倍的纯化；银染后进行SDS/聚丙烯酰胺凝胶电泳，仅出现一条带，对应分子量为80 kDa。通过将部分纯化的细胞色素P - 450和NADPH：细胞色素c（P - 450）还原酶与天然存在的微粒体脂质和黄素核苷酸一起孵育，可以重建洋地黄毒苷12β - 羟化酶活性。此方法产生的洋地黄毒苷12β - 羟化酶约为原始量的10%。
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