[Comparative study of the C27-steroid-hydroxylating system from bovine liver mitochondria].
A method for purification of C27-steroid hydroxylating cytochrome P-450 (cytochrome P-450(27)) from bovine liver mitochondria was developed. The purification procedure included enzyme extraction from submitochondrial particles with sodium cholate, ammonium sulfate fractionation and biospecific chromatography on cholate-Sepharose and adrenodoxin-Sepharose. The resulting enzyme preparation (317-fold purification, 16% yield) was not electrophoretically homogeneous but did not contain hemoprotein admixtures. The kinetic parameters of 5 beta-cholestane-3 alpha,7 alpha,12 alpha-triol 27-hydroxylation in a reconstituted system containing hepatoredoxin reductase, hepatoredoxin and cytochrome P-450(27) (Km = 23 microM, kcat = 0.3 s-1 at 25 degrees C) were determined. A reciprocal functional equivalency of hepatoredoxin reductase and adrenodoxin reductase as well as of hepatoredoxin and adrenodoxin in reconstituted systems of steroid 27-hydroxylation (liver) and cholesterol side chain cleavage (adrenal cortex) was established. This equivalency was thought to be due to the similarity in essential physico-chemical properties of reductase components which was especially well-pronounced in the case of hepatoredoxin and adrenodoxin. Estimation of the functional role of lysine, dicarboxylic acid and histidine residues in ferredoxin molecules by the chemical modification method revealed the similarity of the structural organization of their protein globules: the polar residues were shown to be essential for the maintenance of native conformation; dicarboxylic acid residues formed a binding domain for the interaction with electron transport proteins, whereas histidine residues seem to participate in electron transport. At the same time, cytochrome P-450(27) and cytochrome P-450 which split the side chain of cholesterol differ in their substrate specificity, immunochemical and catalytic properties.
D000302:Adrenal Cortex / Q000201:enzymology; D000596:Amino Acids; D000818:Animals; D002417:Cattle; D053493:Cholestanetriol 26-Monooxygenase; D002784:Cholesterol / Q000378:metabolism; D003577:Cytochrome P-450 Enzyme System / Q000302:isolation & purification / Q000378:metabolism*; D004579:Electron Transport; D005287:Ferredoxin-NADP Reductase / Q000302:isolation & purification / Q000378:metabolism; D005288:Ferredoxins / Q000378:metabolism; D007700:Kinetics; D008928:Mitochondria / Q000201:enzymology; D008930:Mitochondria, Liver / Q000201:enzymology*; D013250:Steroid Hydroxylases / Q000302:isolation & purification / Q000378:metabolism*; D013379:Substrate Specificity
D000596:Amino Acids; D005288:Ferredoxins; C041734:hepatoredoxin; D003577:Cytochrome P-450 Enzyme System; D002784:Cholesterol; D013250:Steroid Hydroxylases; D053493:Cholestanetriol 26-Monooxygenase; D005287:Ferredoxin-NADP Reductase
Sravnitel'noe izuchenie C27-steroidgidroksiliruiushcheĭ sistemy iz mitokhondriĭ pecheni byka.