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. 1992 Nov 1;287(Pt 3):725–731. doi: 10.1042/bj2870725

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

E Axén 1, T Bergman 1, K Wikvall 1
PMCID: PMC1133068  PMID: 1445236

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.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Addya S., Zheng Y. M., Shayiq R. M., Fan J. Y., Avadhani N. G. Characterization of a female-specific hepatic mitochondrial cytochrome P-450 whose steady-state level is modulated by testosterone. Biochemistry. 1991 Aug 27;30(34):8323–8330. doi: 10.1021/bi00098a007. [DOI] [PubMed] [Google Scholar]
  2. Andersson S., Boström H., Danielsson H., Wikvall K. Purification from rabbit and rat liver of cytochromes P-450 involved in bile acid biosynthesis. Methods Enzymol. 1985;111:364–377. doi: 10.1016/s0076-6879(85)11023-2. [DOI] [PubMed] [Google Scholar]
  3. Andersson S., Holmberg I., Wikvall K. 25-hydroxylation of C27-steroids and vitamin D3 by a constitutive cytochrome P-450 from rat liver microsomes. J Biol Chem. 1983 Jun 10;258(11):6777–6781. [PubMed] [Google Scholar]
  4. Andersson S., Jörnvall H. Sex differences in cytochrome P-450-dependent 25-hydroxylation of C27-steroids and vitamin D3 in rat liver microsomes. J Biol Chem. 1986 Dec 25;261(36):16932–16936. [PubMed] [Google Scholar]
  5. Bergman T., Postlind H. Characterization of pig kidney microsomal cytochrome P-450 catalysing 25-hydroxylation of vitamin D3 and C27 steroids. Biochem J. 1990 Sep 1;270(2):345–350. doi: 10.1042/bj2700345. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Bhattacharyya M. H., DeLuca H. F. Subcellular location of rat liver calciferol-25-hydroxylase. Arch Biochem Biophys. 1974 Jan;160(1):58–62. doi: 10.1016/s0003-9861(74)80008-1. [DOI] [PubMed] [Google Scholar]
  7. Björkhem I., Holmberg I., Oftebro H., Pedersen J. I. Properties of a reconstituted vitamin D3 25-hydroxylase from rat liver mitochondria. J Biol Chem. 1980 Jun 10;255(11):5244–5249. [PubMed] [Google Scholar]
  8. Dahlbäck H., Wikvall K. 25-Hydroxylation of vitamin D3 by a cytochrome P-450 from rabbit liver mitochondria. Biochem J. 1988 May 15;252(1):207–213. doi: 10.1042/bj2520207. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Dahlbäck H., Wikvall K. 25-hydroxylation of vitamin D3 in rat liver: roles of mitochondrial and microsomal cytochrome P-450. Biochem Biophys Res Commun. 1987 Feb 13;142(3):999–1005. doi: 10.1016/0006-291x(87)91513-0. [DOI] [PubMed] [Google Scholar]
  10. DeLuca H. F. The transformation of a vitamin into a hormone: the vitamin D story. Harvey Lect. 1979 1980;75:333–379. [PubMed] [Google Scholar]
  11. Hansson R., Wikvall K. Properties of reconstituted cholesterol 7 alpha-hydroxylase system from rat and rabbit liver microsomes. Eur J Biochem. 1979 Jan 15;93(2):419–426. doi: 10.1111/j.1432-1033.1979.tb12838.x. [DOI] [PubMed] [Google Scholar]
  12. Haugen D. A., Coon M. J. Properties of electrophoretically homogeneous phenobarbital-inducible and beta-naphthoflavone-inducible forms of liver microsomal cytochrome P-450. J Biol Chem. 1976 Dec 25;251(24):7929–7939. [PubMed] [Google Scholar]
  13. Hayashi S., Morohashi K., Yoshioka H., Okuda K., Omura T. Expression of a rat liver microsomal cytochrome P-450 catalyzing testosterone 16 alpha-hydroxylation in Saccharomyces cerevisiae: vitamin D3 25-hydroxylase and testosterone 16 alpha-hydroxylase are distinct forms of cytochrome P-450. J Biochem. 1988 May;103(5):858–862. doi: 10.1093/oxfordjournals.jbchem.a122361. [DOI] [PubMed] [Google Scholar]
  14. Hayashi S., Noshiro M., Okuda K. Isolation of a cytochrome P-450 that catalyzes the 25-hydroxylation of vitamin D3 from rat liver microsomes. J Biochem. 1986 Jun;99(6):1753–1763. doi: 10.1093/oxfordjournals.jbchem.a135653. [DOI] [PubMed] [Google Scholar]
  15. Hayashi S., Omura T., Watanabe T., Okuda K. Immunochemical evidence for the catalysis of vitamin D3 25-hydroxylation and testosterone 16 alpha-hydroxylation by homologous forms of cytochrome P-450 in rat liver microsomes. J Biochem. 1988 May;103(5):853–857. doi: 10.1093/oxfordjournals.jbchem.a122360. [DOI] [PubMed] [Google Scholar]
  16. Holmberg I., Berlin T., Ewerth S., Björkhem I. 25-Hydroxylase activity in subcellular fractions from human liver. Evidence for different rates of mitochondrial hydroxylation of vitamin D2 and D3. Scand J Clin Lab Invest. 1986 Dec;46(8):785–790. doi: 10.3109/00365518609084051. [DOI] [PubMed] [Google Scholar]
  17. Holmberg I. Inhibition of reconstituted vitamin D3 25-hydroxylase by a protein fraction from rat liver microsomes. Biochem Biophys Res Commun. 1984 Sep 28;123(3):1209–1214. doi: 10.1016/s0006-291x(84)80261-2. [DOI] [PubMed] [Google Scholar]
  18. Kaiser R., Holmquist B., Hempel J., Vallee B. L., Jörnvall H. Class III human liver alcohol dehydrogenase: a novel structural type equidistantly related to the class I and class II enzymes. Biochemistry. 1988 Feb 23;27(4):1132–1140. doi: 10.1021/bi00404a009. [DOI] [PubMed] [Google Scholar]
  19. LOWRY O. H., ROSEBROUGH N. J., FARR A. L., RANDALL R. J. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951 Nov;193(1):265–275. [PubMed] [Google Scholar]
  20. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  21. Madhok T. C., DeLuca H. F. Characteristics of the rat liver microsomal enzyme system converting cholecalciferol into 25-hydroxycholecalciferol. Evidence for the participation of cytochrome p-450. Biochem J. 1979 Dec 15;184(3):491–499. doi: 10.1042/bj1840491. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Masumoto O., Ohyama Y., Okuda K. Purification and characterization of vitamin D 25-hydroxylase from rat liver mitochondria. J Biol Chem. 1988 Oct 5;263(28):14256–14260. [PubMed] [Google Scholar]
  23. Morgan E. T., Gustafsson J. A. Sex-specific isozymes of cytochrome P-450. Steroids. 1987 Apr-May;49(4-5):215–245. doi: 10.1016/0039-128x(87)90001-8. [DOI] [PubMed] [Google Scholar]
  24. Nebert D. W., Nelson D. R., Coon M. J., Estabrook R. W., Feyereisen R., Fujii-Kuriyama Y., Gonzalez F. J., Guengerich F. P., Gunsalus I. C., Johnson E. F. The P450 superfamily: update on new sequences, gene mapping, and recommended nomenclature. DNA Cell Biol. 1991 Jan-Feb;10(1):1–14. doi: 10.1089/dna.1991.10.1. [DOI] [PubMed] [Google Scholar]
  25. OMURA T., SATO R. THE CARBON MONOXIDE-BINDING PIGMENT OF LIVER MICROSOMES. II. SOLUBILIZATION, PURIFICATION, AND PROPERTIES. J Biol Chem. 1964 Jul;239:2379–2385. [PubMed] [Google Scholar]
  26. Ohyama Y., Masumoto O., Usui E., Okuda K. Multi-functional property of rat liver mitochondrial cytochrome P-450. J Biochem. 1991 Mar;109(3):389–393. doi: 10.1093/oxfordjournals.jbchem.a123391. [DOI] [PubMed] [Google Scholar]
  27. Saarem K., Pedersen J. I. 25-Hydroxylation of 1 alpha-hydroxyvitamin D-3 in rat and human liver. Biochim Biophys Acta. 1985 May 29;840(1):117–126. doi: 10.1016/0304-4165(85)90168-0. [DOI] [PubMed] [Google Scholar]
  28. Wray W., Boulikas T., Wray V. P., Hancock R. Silver staining of proteins in polyacrylamide gels. Anal Biochem. 1981 Nov 15;118(1):197–203. doi: 10.1016/0003-2697(81)90179-2. [DOI] [PubMed] [Google Scholar]
  29. Yasukochi Y., Masters B. S. Some properties of a detergent-solubilized NADPH-cytochrome c(cytochrome P-450) reductase purified by biospecific affinity chromatography. J Biol Chem. 1976 Sep 10;251(17):5337–5344. [PubMed] [Google Scholar]

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