Characterisation of Taurochenodeoxycholic Acid 6α‐Hydroxylase from Pig Liver Microsomes

Abstract

A fraction of cytochrome P-450 catalysing an efficient 6a-hydroxylation of taurine-conjugated 3a,7a-dihydroxy-5/?-cholanoic acid (taurochenodeoxycholic acid) was partially purified from pig liver microsomes. The specific content of cytochrome P-450 was 6 nmoymg protein and the preparation showed two major protein bands upon SDSPAGE. These two bands were isolated after SDSPAGE 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 SDSPAGE 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 6a-hydroxylation of taurochenodeoxycholic acid by up to 90%. Experiments with irrelevant antibodies did not show inhibition of 6a-hydroxylation. The purified cytochrome P-450 fraction catalysed in addition a-and 0-1 hydroxylation of lauric acid and 6a-hydroxylation of 3a-hydroxy-5P-cholanoic acid (lithocholic acid). However, these hydroxylase activities were rather low compared to 6a-hydroxylation of taurochenodeoxycholic acid. The enzyme fraction did not show hydroxylase activities towards cholesterol and 5P-cholestane-3a,7a-diol. These results indicate that 6a-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.Keywords. Pig microsomes ; cytochrome P-450 ; 6a-hydroxylase ; taurochenodeoxycholic acid ; hyocholic acid.The biosynthesis and metabolism of bile acids include a number of hydroxylations catalysed by the microsomal and mitochondria1 fractions of liver [l]. All these reactions have been shown to be catalysed by different species of cytochrome P-450 [2]. Some of the bile acids are unique for certain species [3]. 3a,6a,7a-Trihydroxy-5~-cholanoic acid (hyocholic acid) is considered to be a species-specific bile acid in pigs although it has been reported that hyocholic acid is excreted in small amounts by healthy humans [3, 41. The excretion was increased in cholestatic patients and in pregnant women with cholestasis [4,5]. In the pig, hyocholic acid is formed by a 6a-hydroxylation of 3a,7a-dihydroxy-5~-cholanoic acid (chenodeoxycholic acid) or of the taurine conjugate of the substrate. Hyocholic acid is the only trihydroxylated bile acid in pig bile.In a previous report from this laboratory the partial fractionation and characterisation of 6a-hydroxylase from pig liver was described [6]. That enzyme fraction showed, however, a low Correspondence to R. Hansson, Division of Biochemistry, Department of Pharmaceutical Biosciences, University of Uppsala, Box 578, S-751 23 Uppsala, Sweden F a : +46 18 558778. Abbreviations. Chenodeoxycholic acid, 3a,7a-dihydroxy-SP-cholan...

Cloning and Expression of a Pig Liver Taurochenodeoxycholic Acid 6α-Hydroxylase (CYP4A21)

“…1. Underlined sequences correspond to peptides obtained by tryptic cleavage and N-terminal sequencing of the purified protein as reported previously (5). All tryptic fragments have Arg and Lys in positions that permit trypsin cleavage, and the peptide sequences completely match those encoded by the cDNA.…”

“…In previous reports from this laboratory, the purification and characterization of the taurochenodeoxycholic acid 6␣-hydroxylase from pig liver have been described (4,5). The enzyme was shown to be a microsomal cytochrome P450 heme protein.…”

“…Incubations were performed at 37°C for 20 or 30 min and terminated with 5 ml of ethanol. The incubation mixtures were prepared for thin layer chromatography as described previously (5). The chromatoplates were analyzed by radioactivity scanning.…”

Cloning and expression of two novel pig liver and kidney fatty acid hydroxylases [cytochrome P450 (CYP)4A24 and CYP4A25]

“…52 kDa, similar to that of the purified taurochenodeoxycholic acid 6α-hydroxylase [10], which was used as control together with pig kidney microsomes. No immunoreactive pro- The complete sequence of CYP4A24 is shown together with the variable residues in CYP4A25.…”

“…A pig kidney ω-and (ωk1)-hydroxylase has previously been purified and characterized, but was not reported to be cloned [9]. CYP4A21 was cloned by PCR using primers designed from the purified taurochenodeoxycholic acid 6α-hydroxylase [8,10]. Results obtained by restriction-enzyme digest analysis indicated that the PCR product contained more than one distinct sequence.…”