A cytochrome P450 expressed in pig liver was cloned by polymerase chain reaction using oligonucleotide primers based on amino acid sequences of the purified taurochenodeoxycholic acid 6␣-hydroxylase. This enzyme catalyzes a 6␣-hydroxylation of chenodeoxycholic acid, and the product hyocholic acid is considered to be a primary bile acid specific for the pig. The cDNA encodes a protein of 504 amino acids. The primary structure of the porcine taurochenodeoxycholic acid 6␣-hydroxylase, designated CYP4A21, shows about 75% identity with known members of the CYP4A subfamily in rabbit and man. Transfection of the cDNA for CYP4A21 into COS cells resulted in the synthesis of an enzyme that was recognized by antibodies raised against the purified pig liver enzyme and catalyzed 6␣-hydroxylation of taurochenodeoxycholic acid. The hitherto known CYP4A enzymes catalyze hydroxylation of fatty acids and prostaglandins and have frequently been referred to as fatty acid hydroxylases. A change in substrate specificity from fatty acids or prostaglandins to a steroid nucleus among CYP4A enzymes is notable. The results of mutagenesis experiments indicate that three amino acid substitutions in a region around position 315 which is highly conserved in all previously known CYP4A and CYP4B enzymes could be involved in the altered catalytic activity of CYP4A21.
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...
7a-Hydroxylation of cholesterol was studied in reconstituted systems containing cytochrome P-450 and NADPH-cytochrome P-450 reductase from rat and rabbit liver microsomes. NADPHcytochrome P-450 reductase was prepared with a biospecific affinity chromatographic method. Cytochrome P-450 was prepared by solubilization with non-ionic detergents and various chromatographic procedures. After the last purification step two fractions of cytochrome P-450 from rat liver were analyzed for cholesterol 7a-hydroxylase activity. One fraction had low or no detectable 7a-hydroxylase activity but was able to catalyze 7a-hydroxylation of taurodeoxycholic acid, and 1 1-hydroxylation and 12-hydroxylation of lauric acid. The other fraction catalyzed an efficient 7a-hydroxylation of cholesterol as well as 7a-hydroxylation of cholestanol. This fraction also catalyzed 12a-hydroxylation and 26-hydroxylation of 5j?-cholestane-3a,7a-diol, 7a-hydroxylation of taurodeoxycholic acid and, 11-hydroxylation and 12-hydroxylation of lauric acid. Three cytochrome P-450 fractions from rabbit liver, obtained after the final purification step, were analyzed for cholesterol 7a-hydroxylase activity. Two fractions had no detectable 7a-hydroxylase activity but were able to catalyze 12a-hydroxylation and 25-hydroxylation of 5P-cholestane-3a,7a-diol, 25-hydroxylation of .5~-cholestane-3a,7a,l2~-triol and 1 1-hydroxylation and 12-hydroxylation of lauric acid. The third fraction catalyzed in addition 7a-hydroxylation of cholesterol and cholestanol.The cholesterol 7a-hydroxylase activity showed an absolute requirement for cytochrome P-450 1 and NADPH-cytochrome P-450 reductase 7a-hydroxylation.Phospholipid had no -significant effect on cholesterolThe first and rate-limiting step in the conversion of cholesterol into bile acids is the 7a-hydroxylation of cholesterol [l-41. The reaction is catalyzed by a microsomal monooxygenase system and is inhibited by carbon monoxide, indicating the participation of cytochrome P-450 [5 -71. The reaction has been shown to be inhibited by an antibody against NADPHcytochrome c(P-450) reductase [ 5 ] . In 1974, it was reported that 7a-hydroxylation of cholesterol could be shown in reconstituted systems from rat liver microsomes containing partially purified cytochrome P-450 and NADPH-cytochrome P-450 reductase [8]. Since then, a number of attempts have been made to purify further the cytochrome P-450 fraction containing Enzymes. NADPH-cytochrome c(P-450) reductase (EC 1.6.2.4) ; cholesterol 7cr-monooxygenase or cholesterol 7a-hydroxylase (EC 1.14.13.17).Trivial Name.?. Cholestanol, 5B-cholestan-3/3-01; cholic acid, 3a,7a,l2a-trihydroxy-5P-cholanoic acid; deoxycholic acid, 3a, 12a-dihydroxy-5/3-cholanoic acid. cholesterol 7a-hydroxylase activity. Various chromatographic procedures described by Coon, Lu and their associates [9,10] have been tested but in most cases little or no cholesterol 7a-hydroxylase activity has been recovered. In a recent preliminary communication it was reported that cytochrome P-450 could be fractionated w...
Cytochromes P-450 LM3b and LM4 were prepared from untreated and cholestyramine-treated rabbits. The catalytic properties of these cytochrome P-450 fractions towards substrates in bile acid biosynthesis were studied in reconstituted systems containing NADPH -cytochrome P-450 reductase and phospholipid. Cytochrome P-450 LM3b showed no hydroxylase activity towards cholesterol and only low activity towards some other Cz7-steroids whereas it catalyzed efficient hydroxylation of testosterone and demethylation of ethylmorphine. Preparations of cytochrome P-450 LM4 catalyzed hydroxylation of cholesterol and other Cz7-steroids more efficiently than microsomes. Cytochrome hs had no stimulatory effect on the Cz,-steroid hydroxylase activities.
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