Reconstitution of cytochrome P-450-dependent digitoxin 12 beta-hydroxylase from cell cultures of foxglove (Digitalis lanata EHRH.)
| Autor: | H U Seitz, Maike Petersen |
|---|---|
| Rok vydání: | 1988 |
| Předmět: |
Cytochrome
Digitoxin Reductase Biochemistry Chromatography Affinity Affinity chromatography Digitalis lanata Cytochrome P-450 Enzyme System Microsomes medicine Molecular Biology Cells Cultured NADPH-Ferrihemoprotein Reductase Plant Proteins Digitalis Plants Medicinal biology Chemistry Cytochrome c Cytochrome P450 Cell Biology biology.organism_classification Chromatography Ion Exchange Lipid Metabolism Kinetics Plants Toxic Steroid Hydroxylases biology.protein Microsome medicine.drug Research Article |
| Zdroj: | The Biochemical journal. 252(2) |
| ISSN: | 0264-6021 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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