Cross-linking studies of the cholesterol hydroxylation system from bovine adrenocortical mitochondria.

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TitleCross-linking studies of the cholesterol hydroxylation system from bovine adrenocortical mitochondria.
Publication TypeJournal Article
Year of Publication1989
AuthorsTurko, IV, Adamovich, TB, Kirillova, NM, Usanov, SA, Chashchin, VL
JournalBiochim Biophys Acta
Date Published1989 Jun 13
KeywordsAdrenal Cortex, Adrenodoxin, Animals, Binding Sites, Cattle, Cholesterol, Cholesterol Side-Chain Cleavage Enzyme, Cross-Linking Reagents, Ferredoxin-NADP Reductase, Hydroxylation, Macromolecular Substances, Mitochondria, Molecular Weight, Spectrum Analysis

Cytochrome P-450SCC and adrenodoxin were cross-linked with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide. The sample containing 94% of cross-linked complex and 6% of free cytochrome P-450SCC was obtained after purification on cholate-Sepharose. Cytochrome P-450SCC in cross-linked complex completely preserves its high-spin form in the presence of Tween 20 or pregnenolone. Utilization of radioactively labelled adrenodoxin, chemical cleavage of cytochrome P-450SCC from cross-linked complex with o-iodosobenzoic acid and HPLC for separation of peptides allow us to conclude that the complex of cytochrome P-450SCC with adrenodoxin was cross-linked through two amino acid sequences of cytochrome P-450SCC-Leu-88-Thr-107 and Leu-368-Gly-416. The cross-linked complex of adrenodoxin reductase, adrenodoxin and cytochrome P-450SCC with an apparent molecular mass of 114 kDa was obtained with N-succinimidyl-6-(4'-azido-2'-nitrophenylamino)hexanoate. The composition of cross-linked complex was determined by immunoblotting and by evaluation of radioactivity using preliminary N-ethyl[2,3-14C]maleimide-modified adrenodoxin. From this data it appears that the ternary complex may exist in solution.

Alternate JournalBiochim. Biophys. Acta
PubMed ID2736257