Reconstitution of cytochrome P-450-dependent digitoxin 12β-hydroxylase from cell cultures of foxglove (Digitalis lanata EHRH.)
- 1 June 1988
- journal article
- research article
- Published by Portland Press Ltd. in Biochemical Journal
- Vol. 252 (2) , 537-543
- https://doi.org/10.1042/bj2520537
Abstract
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)dimethylammoniol]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 chromatogrpahy 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.This publication has 16 references indexed in Scilit:
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