Complex formation in vesicle-reconstituted mitochondrial cytochrome P450 systems (CYP11A1 and CYP11B1) as evidenced by rotational diffusion experiments using EPR and ST-EPR
| Autor: | Pyotr Kisselev, Dieter Schwarz, and Alexey Chernogolov |
|---|---|
| Rok vydání: | 1999 |
| Předmět: |
endocrine system
Rotation Cardiolipins Photochemistry Biochemistry Adrenodoxin reductase law.invention Diffusion chemistry.chemical_compound law Adrenodoxin Animals Cholesterol Side-Chain Cleavage Enzyme Electron paramagnetic resonance Ternary complex Octyl glucoside Chemistry Cholesterol side-chain cleavage enzyme Vesicle Phosphatidylethanolamines Electron Spin Resonance Spectroscopy Rotational diffusion Intracellular Membranes Mitochondria Ferredoxin-NADP Reductase Liposomes Biophysics Phosphatidylcholines Steroid 11-beta-Hydroxylase Cattle Spin Labels |
| Zdroj: | Biochemistry. 38(29) |
| ISSN: | 0006-2960 |
| Popis: | Rotational diffusion measurements using EPR and saturation transfer EPR were applied to analyze complex formation between the electron-transfer components of the mitochondrial steroid-hydroxylating cytochrome P450 systems (CYP11A1 and CYP11B1) in phosphatidylcholine/phosphatidylethanolamine/cardiolipin vesicles prepared by octyl glucoside dialysis/adsorption. Octyl glucoside reconstitution of P450SCC results in large vesicles, which have an advantage over small vesicles in that vesicle tumbling does not contribute to measured rotational diffusion rates. Immobilization of spin-labeled adrenodoxin by both P450SCC and adrenodoxin reductase indicates equimolar complexation between P450SCC and adrenodoxin as well as between adrenodoxin reductase and adrenodoxin. Combination of rotational diffusion and antibody cross-linking confirmed the complexation of adrenodoxin with P450SCC and for the first time provided direct evidence of a complex between P450SCC and P45011beta in the membrane. In contrast, no evidence was found for the existence of adrenodoxin reductase-P450SCC complexes or a ternary complex of all three proteins. Thus, these experiments confirm the shuttle mechanism of electron transfer to vesicle-reconstituted P450SCC and P45011beta. |
| Databáze: | OpenAIRE |
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