[Chemical modification of steroid-hydroxylating monooxygenases with fluorescein isothiocyanate].
Chemical modifications of cytochrome P-450scc and cytochrome P-450(11) beta with fluorescein-, diiodofluorescein-, eosine- and rhodamine isothiocyanate have been carried out. At a low reagent/protein ratio and neutral pH, a selective chemical modification was known to take place which did not affect the spectral properties of cytochrome P-450scc. Covalent chromatography was found useful to discriminate between covalent modification of cytochrome P-450scc and non-specific binding of FITC with cytochrome P-450scc. Proteolytic modification of cytochrome P-450scc and structural analysis indicate that a lysine residue of the C-terminal sequence of cytochrome P-450scc is accessible to FITC. The residue was shown, by the analysis of the chymotryptic hydrolysate of the fragment F2, to be Lys338. Effect of modification with FITC on the interaction of cytochrome P-450scc with cholesterol or adrenodoxin, on the reduction kinetics and on the conversion of cholesterol to pregnenolone was also studied.
Lapko
A G
AG
Smettan
G
G
Rukpaul'
K
K
Usanov
S A
SA
D000595:Amino Acid Sequence; D002786:Cholesterol Side-Chain Cleavage Enzyme / Q000737:chemistry*; D002851:Chromatography, High Pressure Liquid; D016650:Fluorescein-5-isothiocyanate / Q000737:chemistry*; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D013056:Spectrophotometry, Ultraviolet; D013252:Steroid 11-beta-Hydroxylase / Q000737:chemistry*
D013252:Steroid 11-beta-Hydroxylase; D002786:Cholesterol Side-Chain Cleavage Enzyme; D016650:Fluorescein-5-isothiocyanate
Khimicheskaia modifikatsiia steroidgidroksiliruiushchikh monooksigenaz fluorestseinizotiotsianatom.