Cross-linking studies of the cholesterol hydroxylation system from bovine adrenocortical mitochondria.
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.
Turko
I V
IV
Institute of Bioorganic Chemistry, B.S.S.R. Academy of Sciences, Minsk, U.S.S.R.
Adamovich
T B
TB
Kirillova
N M
NM
Usanov
S A
SA
Chashchin
V L
VL
D000302:Adrenal Cortex / Q000201:enzymology*; D000325:Adrenodoxin / Q000302:isolation & purification / Q000378:metabolism*; D000818:Animals; D001665:Binding Sites; D002417:Cattle; D002784:Cholesterol / Q000378:metabolism*; D002786:Cholesterol Side-Chain Cleavage Enzyme / Q000302:isolation & purification / Q000378:metabolism*; D003432:Cross-Linking Reagents; D005287:Ferredoxin-NADP Reductase / Q000302:isolation & purification / Q000378:metabolism; D006900:Hydroxylation; D046911:Macromolecular Substances; D008928:Mitochondria / Q000201:enzymology*; D008970:Molecular Weight; D013057:Spectrum Analysis
D003432:Cross-Linking Reagents; D046911:Macromolecular Substances; D000325:Adrenodoxin; D002784:Cholesterol; D002786:Cholesterol Side-Chain Cleavage Enzyme; D005287:Ferredoxin-NADP Reductase