Characterisation of taurochenodeoxycholic acid 6 alpha-hydroxylase from pig liver microsomes.
A fraction of cytochrome P-450 catalysing an efficient 6 alpha-hydroxylation of taurine-conjugated 3 alpha,7 alpha-dihydroxy-5 beta- cholanoic acid (taurochenodeoxycholic acid) was partially purified from pig liver microsomes. The specific content of cytochrome P-450 was 6 nmol/mg protein and the preparation showed two major protein bands upon SDS/PAGE. These two bands were isolated after SDS/PAGE and protein blotting. The protein band with a molecular mass of 53 kDa had an N-terminal amino acid sequence and internal sequences resembling that of the cytochrome P-450 4A subfamily (CYP 4A). Polyclonal antibodies raised against this protein were able to, after SDS/PAGE and immunoblotting, detect the protein in microsomal fractions as well as in the purified cytochrome P-450 fraction. Furthermore, addition of these antibodies to a reconstituted system containing the cytochrome P-450 fraction, inhibited 6 alpha-hydroxylation of taurochenodeoxycholic acid by up to 90%. Experiments with irrelevant antibodies did not show inhibition of 6 alpha-hydroxylation. The purified cytochrome P-450 fraction catalysed in addition omega- and omega-1 hydroxylation of lauric acid and 6 alpha-hydroxylation of 3 alpha-hydroxy-5 beta-cholanoic acid (lithocholic acid). However, these hydroxylase activities were rather low compared to 6 beta-hydroxylation of taurochenodexycholic acid. The enzyme fraction did not show hydroxylase activities towards cholesterol and 5 beta-cholestane-3 alpha,7 alpha-diol. These results indicate that 6 alpha-hydroxylation of taurochenodeoxycholic acid is catalysed by a specific species of cytochrome P-450 that, according to N-terminal amino acid sequence as well as catalytic properties, could be a member of the CYP 4A subfamily.
Araya
Z
Z
Department of Pharmaceutical Biosciences, University of Uppsala, Sweden.
Hellman
U
U
Hansson
R
R
D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D002851:Chromatography, High Pressure Liquid; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D008862:Microsomes, Liver / Q000201:enzymology*; D008969:Molecular Sequence Data; D011487:Protein Conformation; D013250:Steroid Hydroxylases / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D013552:Swine
D003577:Cytochrome P-450 Enzyme System; D013250:Steroid Hydroxylases; C095246:taurochenodeoxycholic acid 6-alpha-hydroxylase