Reconstitution of cytochrome P-450-dependent digitoxin 12 beta-hydroxylase from cell cultures of foxglove (Digitalis lanata EHRH.).
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.
D002478:Cells, Cultured; D002846:Chromatography, Affinity; D002852:Chromatography, Ion Exchange; D003577:Cytochrome P-450 Enzyme System / Q000302:isolation & purification / Q000378:metabolism* / Q000494:pharmacology; D004070:Digitalis / Q000201:enzymology*; D007700:Kinetics; D050356:Lipid Metabolism; D008861:Microsomes / Q000378:metabolism; D009251:NADPH-Ferrihemoprotein Reductase / Q000302:isolation & purification; D010940:Plant Proteins / Q000378:metabolism; D010946:Plants, Medicinal; D010947:Plants, Toxic; D013250:Steroid Hydroxylases / Q000378:metabolism*
D010940:Plant Proteins; D003577:Cytochrome P-450 Enzyme System; D013250:Steroid Hydroxylases; C056960:digitoxin 12-beta-hydroxylase; D009251:NADPH-Ferrihemoprotein Reductase