title: Purification and characterization of 7 alpha-hydroxy-4-cholesten-3-one 12 alpha-hydroxylase.
abstract: The isoform of cytochrome P450 that catalyzes the 12 alpha-hydroxylation of 7 alpha-hydroxy-4-cholesten-3-one, an intermediate in the conversion of cholesterol to cholic acid, was purified to homogeneity from rabbit liver microsomes. The extent of purification in the various steps was judged by an assay involving high performance liquid chromatography. The purified enzyme showed a single band on SDS-polyacrylamide gel electrophoresis (M(r) = 50,000). The NH2-terminal amino acid sequence is as follows: Val-Leu-Trp-Gly-Leu-Leu-Gly-Ala-Leu-Leu-Met-Val-Met-Val-Gly-, which is different from that of any other P450s so far reported. The specific content of the enzyme was 13.3 nmol of cytochrome P450/mg of protein. Upon reconstitution with NADPH-cytochrome P450 reductase and cytochrome b5, the P450 enzyme showed a high activity of 12 alpha-hydroxylation with a turnover number of 36.6 min-1 at 37 degrees C. The omission of either cytochrome P450 or NADPH-cytochrome P450 reductase resulted in complete loss of activity, and the omission of cytochrome b5 resulted in 40% loss of activity. Antibodies prepared from mouse inhibited the 12 alpha-hydroxylase activity of rabbit liver microsomes about 90% and that of the rat liver microsomes 50%. The enzyme activity was not inhibited by other antibodies raised against other forms of P450 that catalyze different monooxygenation reactions toward xenobiotics or endogenous substrates. Anti-cytochrome b5 antibody inhibited the activity 40%, suggesting the functional role of this protein, and anti-reductase inhibited the activity almost completely. The microsomal enzyme activity was markedly elevated by starvation or streptozotocin administration to the animals. However, an immunoblotting experiment showed no correlation between the enzyme activity and the amount of protein, suggesting that post-translational modification may occur.
authors.affiliation: Department of Biochemistry, Hiroshima University School of Dentistry, Japan.
authors.forename: H
authors.initials: H
authors.lastname: Ishida
authors.forename: M
authors.initials: M
authors.lastname: Noshiro
authors.forename: K
authors.initials: K
authors.lastname: Okuda
authors.forename: M J
authors.initials: MJ
authors.lastname: Coon
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies / Q000276:immunology; D002783:Cholestenones / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid; D003577:Cytochrome P-450 Enzyme System / Q000276:immunology / Q000302:isolation & purification* / Q000378:metabolism; D015786:Cytochromes b5 / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D007527:Isoenzymes / Q000276:immunology / Q000302:isolation & purification* / Q000378:metabolism; D008862:Microsomes, Liver / Q000201:enzymology; D008969:Molecular Sequence Data; D009251:NADPH-Ferrihemoprotein Reductase / Q000378:metabolism; D011817:Rabbits; D051381:Rats; D017208:Rats, Wistar; D013253:Steroid 12-alpha-Hydroxylase / Q000302:isolation & purification* / Q000378:metabolism
chemical_list: D000906:Antibodies; D002783:Cholestenones; D007527:Isoenzymes; C002656:7 alpha-hydroxy-4-cholesten-3-one; D015786:Cytochromes b5; D003577:Cytochrome P-450 Enzyme System; C035248:7 alpha-hydroxy-4-cholesten-3-one-12 alpha monooxygenase; D013253:Steroid 12-alpha-Hydroxylase; D009251:NADPH-Ferrihemoprotein Reductase