Cytochrome P450 enzymes in the biosynthesis and metabolism of bile acids and active forms of vitamin D

University dissertation from Uppsala : Acta Universitatis Upsaliensis

Abstract: Cytochrome P450 enzymes catalyzing hydroxylations in the biosynthesis and metabolism of bile acids and active forms of vitamin D3 were studied.A microsomal cytochrome P450 enzyme with a molecular mass of 53 kDa, catalyzing the 6α-hydroxylation of taurochenodeoxycholic acid was purfied from pig liver microsomes and characterized with respect to catalytic and electrophoretic properties as well as polyclonal antibodies. The enzyme was also characterized by N-terminal and some internal amino acid sequencings. These sequences showed similarities with those of mammalian cytochromes P450 (CYP) in the CYP4A subfamily. .CYP3A4, a major drug-metabolizing enzyme in human liver, was found to be active in and responsible for a major part of the 6α-hydroxylation of taurochenodeoxycholic acid and lithocholic acid in human liver microsomes.Purified CYP27A from pig kidney mitochondria was shown to catalyze 1α-and 27-hydroxylaton but not 24-hydroxylation of 25-hydroxyvitamin D3. The same enzyme was found to convert 25-hydroxyvitamin D3 into another hydroxylated major metabolite. Recombinantly expressed human CYP27A was able to catalyze the same hydroxylations.Regulation of rabbit liver CYP7A and CYP27A enzymes by cholic acid and cholestyramine was studied. There was no coordinate regulation of CYP7A and CYP27A at a transcriptional level. In contrast to CYP7A, CYP27A was not subject to a negative feedback control by bile acids neither at a transcriptional nor at a post-transcriptional level.A 5 kb DNA fragment of the human CYP27A gene, upstream from the translation initiation codon, was studied using luciferase reporter constructs in HepG2 cells. The DNA fragment was found to contain a functional promotor. Three fragments of the 5 kb DNA obtained after Sma I digestion were analyzed for promotor activity. DNA sequence analysis of two of the fragments revealed the presence of possible promotor sequences (TATA) in both fragments. The response of the human CYP27A-luciferase plasmid to dexamethasone, growth hormone, 1α,25-dihydroxyvitamin D3 and bile acids was studied. The transcriptional activity was induced two-fold by growth hormone and three-fold by dexamethasone. 1α,25-Dihydroxyvitamin D3 or bile acids had no significant effects on the expression of the human CYP27A gene.

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