Characterization of the liver mitochondrial cytochrome P-450 catalyzing the 26-hydroxylation of 5 beta-cholestane-3 alpha, 7 alpha, 12 alpha-triol.
The cytochrome P-450 catalyzing 26-hydroxylation of C27-steroids (cytochrome P-450(26] was purified from rabbit liver mitochondria. The specific content of the cytochrome P-450 was 13.6 nmol per mg of protein and the 26-hydroxylase activity towards 5 beta-cholestane-3 alpha, 7 alpha, 12 alpha-triol was 31,300 pmol/nmol of cytochrome P-450 x min-1. The preparation also catalyzed 25-hydroxylation of vitamin D3 at a rate of 350 pmol/nmol of cytochrome P-450 x min-1. A monospecific monoclonal antibody raised against the 26-hydroxylating cytochrome P-450 was prepared. Experiments with the monoclonal antibody showed that cytochrome P-450(26) is susceptible to proteolytic degradation during purification unless the protease inhibitor TPCK is included in the buffers. After coupling to Sepharose the antibody was able to bind to cytochrome P-450(26) and to decrease the 26-hydroxylation of 5 beta-cholestane-3 alpha, 7 alpha, 12 alpha-triol. The 25-hydroxylation of vitamin D was not inhibited by the antibody. The results indicate that there are different species of cytochrome P-450 in rabbit liver mitochondria catalyzing 26-hydroxylation of 5 beta-cholestane-3 alpha, 7 alpha, 12 alpha-triol and 25-hydroxylation of vitamin D3. The N-terminal amino acid sequence of the cytochrome P-450(26) differed from those of hitherto isolated mammalian cytochromes P-450.
Dahlbäck
H
H
Department of Pharmaceutical Biochemistry, University of Uppsala, Sweden.
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D000937:Antigen-Antibody Reactions; D015153:Blotting, Western; D002777:Cholestanols; D003577:Cytochrome P-450 Enzyme System / Q000032:analysis* / Q000276:immunology; D008930:Mitochondria, Liver / Q000201:enzymology*; D011817:Rabbits; D013250:Steroid Hydroxylases / Q000032:analysis* / Q000276:immunology
D000911:Antibodies, Monoclonal; D002777:Cholestanols; D003577:Cytochrome P-450 Enzyme System; D013250:Steroid Hydroxylases