ABSTRACT The formation of 1α,25-dihydroxyvitamin D3 requires a 25-hydroxylation followed by a 1α-hydroxylation catalyzed by cytochrome P450 (CYP) enzymes in liver and kidney. Two hepatic P450 enzymes catalyzing 25-hydroxylation of vitamin D3 exist in mammalian liver – one mitochondrial and one microsomal. The mitochchondrial vitamin D3 25-hydrooxylase is apparently identical with CYP27A, an obligatory enzyme in bile acid biosynthesis in liver. The microsomal 25-hydroxylase has been purified to apparent homogeneity from pig liver. The enzyme catalyzed 25-hydroxylation of vitamin D3, 1α-hydroxyvitamin D3, vitamin D2 and 1α-hydroxyvitamin D2. A cDNA encoding pig liver microsomal vitamin D3 25-hydroxylase has recently been isolated in this laboratory. The structure of vitamin D3 25-hydroxylase shows 70-80% identity with members of the CYP2D subfamily and has been assigned the name CYP2D25. In this laboratory, a mitochondrial P450 fraction catalyzing 1α-hydroxylation but not 24-hydroxylation of 25-hydroxyvitamin D3 was purified from pig kidney. The enzyme preparation catalyzed also 27-hydroxylation. A monoclonal antibody directed against liver CYP27A immunoprecipitated more than 90% of the 1α-hydroxylase activity in a crude P450 extract from kidney mitochondria. Treatment of rats with a single i.v. dose of 1α,25-dihydroxyvitamin D3 resulted in a marked suppression of CYP27A mRNA levels in kidney. The results provide evidence for a role of CYP27A as a renal mitochondrial 1α-hydroxylase. Recently, the isolation of cDNA encoding mouse, rat and human kidney 25-hydroxyvitamin D3 1α-hydroxylase was reported. The amino acid sequences deduced from these cDNA clones were similar but different from that of CYP27A. This 1α-hydroxylase has been reported to constitute a new CYP27 subfamily, CYP27B. A microsomal P450 catalyzing 1α-hydroxylation of 25-hydroxyvitamin D3 has been purified to apparent homogeneity from pig kidney. This finding demonstrate the presence in kidney of a previously unknown microsomal 1α-hydroxylase in addition to the mitochondrial 1α-hydroxylases. It may be conclude that multiple 1α-hydroxylating cytochromes P450 in kidney. i.e. CYP27A, CYP27B and a microsomal 1α-hydroxylase, have been described. The relative importance and regulation of the different renal 1α-hydroxylases in the bioactivation of vitamin D3 will be subject for future studies.
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