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Purification and characterization of a vitamin D3 25-hydroxylase from pig liver microsomes.

ABSTRACT: A cytochrome P-450 which catalyses 25-hydroxylation of vitamin D3 has been purified to apparent homogeneity from pig liver microsomes. The specific content of cytochrome P-450 was 12 nmol.mg of protein-1, and the preparation showed a single band with an apparent M(r) of 50,500 upon SDS/PAGE. A monoclonal antibody raised against the vitamin D3 25-hydroxylase reacted strongly with the purified 25-hydroxylating cytochrome P-450 from pig kidney microsomes [Bergman & Postlind (1990) Biochem. J. 270, 345-350]. The liver enzyme showed structural and functional properties very similar to those of the kidney enzyme. The two enzymes differed with respect to only one of the first 16 N-terminal amino acids. The vitamin D3 25-hydroxylase in pig liver microsomes exhibited a turnover and an apparent Km for 25-hydroxylation of vitamin D3 which were of the same order of magnitude as those of a well-characterized male-specific 25-hydroxylating cytochrome P-450 in rat liver microsomes. The two enzymes differed structurally. The pig liver enzyme was, in contrast to the rat liver enzyme, not sex-specific, and did not catalyse 16 alpha-hydroxylation of testosterone. These properties of the 25-hydroxylase in rat liver microsomes have led to questions on the role of microsomal 25-hydroxylation of vitamin D3. It is concluded that studies on microsomal 25-hydroxylation with the rat may be misleading. The results of the present study show that the pig appears to be a representative species for evaluation of vitamin D3 hydroxylases in other mammals, including man.

SUBMITTER: Axen E

PROVIDER: S-EPMC1133068 | biostudies-other | 1992 Nov

REPOSITORIES: biostudies-other

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Project description:The cytochrome P450 mitochondrial enzyme 25-hydroxyvitamin D3 1alpha-hydroxylase (1alpha-hydroxylase) of renal tubule cells hydroxylates the major circulating form of vitamin D (25(OH)D3) to the active systemic hormone 1,25(OH)2D3. Local production of 1,25(OH)2D3 appears to occur also at other sites where 1alpha-hydroxylase is expressed for autocrine/paracrine regulation. To reduce risks of hypercalcemia during treatment with vitamin D, we have previously suggested use of non-1alpha-hydroxylated vitamin D analogues to target tissues where 1alpha-hydroxylase is expressed, including the parathyroid glands in secondary hyperparathyroidism. The present study was undertaken to examine expression of 1alpha-hydroxylase in breast cancer and to investigate whether a non-1alpha-hydroxylated vitamin D analogue displayed biological function. In addition, expression of the 25-hydroxyvitamin D3 24-hydroxylase (24-hydroxylase) and the vitamin D receptor (VDR) was investigated.The expression of 1alpha-hydroxylase, 24-hydroxylase and VDR was investigated in breast cancer specimens (n = 19) and normal breast tissues (n = 10) by immunohistochemistry and/or RT-PCR. Consecutive cryosections of 6 mum essentially free of immune cells were used in the analyses. The effect of vitamin D analogues on transcriptional activation was analyzed in transiently transfected MCF-7 breast cancer cells.1alpha-hydroxylase protein was demonstrated in 79% and 100% of breast cancer specimens and normal breast, respectively. The overall relative mRNA levels of 1alpha-hydroxylase and 24-hydroxylase in normal breast compared to breast tumors were: 1alpha-hydroxylase, 1 +/- 0.07 versus 0.7 +/- 0.05, respectively (p < 0.001); 24-hydroxylase, 1 +/- 0.08 verus 2.1 +/- 0.2, respectively (p < 0.001). The VDR was expressed in 95% of the tumors as expected, with mRNA levels of 1 +/- 0.09 and 1.4 +/- 0.12 (p < 0.05) in breast cancer and normal breast, respectively. The ketoconazole-sensitive transcription activation potential of the non-1alpha-hydroxylated vitamin D analogue prodrug of EB1089 (EB1285) was demonstrated in MCF-7 cells, which express 1alpha-hydroxylase. The activity of EB1285 was about 20% of 1,25(OH)2D3.These results demonstrate nearly normal expression levels of 1alpha-hydroxylase, 24-hydroxylase and VDR in the majority of investigated breast cancer specimens. A non-1alpha-hydroxylated vitamin D analogue displayed activity in breast cancer cells. Such analogues may present future therapeutic options for proliferative disorders where 1alpha-hydroxylase is expressed.

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Purification and characterization of a vitamin D3 25-hydroxylase from pig liver microsomes.

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