Cross-linking studies of steroidogenic electron transfer: covalent complex of adrenodoxin reductase with adrenodoxin.
Bifunctional reagents 3,3'-dithiobis(succinimidyl propionate), 1-ethyl 3-(3-dimethylaminopropyl)carbodiimide and N-succinimidyl 3-(2-pyridyldithio)propionate have been used in an attempt to study molecular organization and covalent cross-linking of adrenodoxin reductase with adrenodoxin, the components of steroidogenic electron transfer system in bovine adrenocortical mitochondria. There was no cross-linking of individual proteins by the bifunctional reagents used, except for adrenodoxin cross-linking with water-soluble carbodiimide. Substantial cross-linking of adrenodoxin reductase with adrenodoxin was observed when water-soluble carbodiimide was used as cross-linking reagent. However, the cross-linked complex failed to transfer electrons. Significant amounts of the functional cross-linked complex (up to 42%) were observed when the proteins were cross-linked with N-succinimidyl 3-(2-pyridyldithio)propionate. Using gel filtration, ion-exchange chromatography and affinity chromatography on adrenodoxin-Sepharose, the complex was obtained in a highly purified form. In the presence of cytochrome P-450scc or cytochrome c, the cross-linked complex of adrenodoxin reductase with adrenodoxin was active in electron transfer from NADPH to heme proteins. The data obtained indicate that there are distinct binding sites on the adrenodoxin molecule responsible for the adrenodoxin reductase and cytochrome P-450scc binding, which suggests that steroidogenic electron transfer may be realized in an organized complex.
D000302:Adrenal Cortex / Q000378:metabolism; D000325:Adrenodoxin / Q000378:metabolism*; D000818:Animals; D002417:Cattle; D003432:Cross-Linking Reagents / Q000494:pharmacology*; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism; D004579:Electron Transport; D005287:Ferredoxin-NADP Reductase / Q000378:metabolism*; D007700:Kinetics; D008928:Mitochondria / Q000378:metabolism; D009247:NADH, NADPH Oxidoreductases / Q000378:metabolism*; D013329:Structure-Activity Relationship
D003432:Cross-Linking Reagents; D000325:Adrenodoxin; D003577:Cytochrome P-450 Enzyme System; D005287:Ferredoxin-NADP Reductase; D009247:NADH, NADPH Oxidoreductases