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. 1985 May;82(9):2713–2717. doi: 10.1073/pnas.82.9.2713

Purification of a vitamin K epoxide reductase that catalyzes conversion of vitamin K 2,3-epoxide to 3-hydroxy-2-methyl-3-phytyl-2,3-dihydronaphthoquinone.

I Mukharji, R B Silverman
PMCID: PMC397635  PMID: 3857611

Abstract

An enzyme from bovine liver microsomes that catalyzes the reduction of vitamin K 2,3-epoxide to 2- and 3-hydroxy-2-methyl-3-phytyl-2,3-dihydronaphthoquinone was purified 1152-fold to apparent homogeneity. Microsomes were solubilized with 3-[3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), and the enzyme was purified by chromatography on PBE-94 ion exchanger, hydroxylapatite, and DEAE-cellulose, and then gel filtration on Sephacryl S-200. The homogeneity of the final preparation was established by polyacrylamide slab gel electrophoresis in the presence of sodium dodecyl sulfate. The molecular weight of the native enzyme is 25,000 and that of denatured enzyme is 12,400, which suggests that the enzyme is a dimer with identical subunits. No chromophoric cofactors are associated with the enzyme. Dithiothreitol and CHAPS are essential for activity, but high concentrations of glycerol reduces the activity. The enzyme is not inhibited by warfarin, a potent inhibitor of the vitamin K epoxide reductase, which catalyzes the conversion of vitamin K 2,3-epoxide to vitamin K. Evidence is presented indicating that the purified enzyme is not simply a fragment of the warfarin-sensitive vitamin K epoxide reductase.

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Selected References

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  1. BOYLAND E., WILLIAMS K. AN ENZYME CATALYSING THE CONJUGATION OF EPOXIDES WITH GLUTATHIONE. Biochem J. 1965 Jan;94:190–197. doi: 10.1042/bj0940190. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bell R. G. Metabolism of vitamin K and prothrombin synthesis: anticoagulants and the vitamin K--epoxide cycle. Fed Proc. 1978 Oct;37(12):2599–2604. [PubMed] [Google Scholar]
  3. Fasco M. J., Hildebrandt E. F., Suttie J. W. Evidence that warfarin anticoagulant action involves two distinct reductase activities. J Biol Chem. 1982 Oct 10;257(19):11210–11212. [PubMed] [Google Scholar]
  4. Fasco M. J., Preusch P. C., Hildebrandt E., Suttie J. W. Formation of hydroxyvitamin K by vitamin K epoxide reductase of warfarin-resistant rats. J Biol Chem. 1983 Apr 10;258(7):4372–4380. [PubMed] [Google Scholar]
  5. Fasco M. J., Principe L. M. R- and S-Warfarin inhibition of vitamin K and vitamin K 2,3-epoxide reductase activities in the rat. J Biol Chem. 1982 May 10;257(9):4894–4901. [PubMed] [Google Scholar]
  6. Fasco M. J., Principe L. M. Vitamin K1 hydroquinone formation catalyzed by a microsomal reductase system. Biochem Biophys Res Commun. 1980 Dec 31;97(4):1487–1492. doi: 10.1016/s0006-291x(80)80033-7. [DOI] [PubMed] [Google Scholar]
  7. Hildebrandt E. F., Preusch P. C., Patterson J. L., Suttie J. W. Solubilization and characterization of vitamin K epoxide reductase from normal and warfarin-resistant rat liver microsomes. Arch Biochem Biophys. 1984 Feb 1;228(2):480–492. doi: 10.1016/0003-9861(84)90014-6. [DOI] [PubMed] [Google Scholar]
  8. Jones J. P., Gardner E. J., Cooper T. G., Olson R. E. Vitamin K-dependent carboxylation of peptide-bound glutamate. The active species of "CO2" utilized by the membrane-bound preprothrombin carboxylase. J Biol Chem. 1977 Nov 10;252(21):7738–7742. [PubMed] [Google Scholar]
  9. Kumagai H., Miles E. W. The B protein of Escherichia coli tryptophan synthetase. II. New -elimination and -replacement reactions. Biochem Biophys Res Commun. 1971 Sep;44(5):1271–1278. doi: 10.1016/s0006-291x(71)80223-1. [DOI] [PubMed] [Google Scholar]
  10. 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]
  11. Larson A. E., Friedman P. A., Suttie J. W. Vitamin K-dependent carboxylase. Stoichiometry of carboxylation and vitamin K 2,3-epoxide formation. J Biol Chem. 1981 Nov 10;256(21):11032–11035. [PubMed] [Google Scholar]
  12. Lee J. J., Fasco M. J. Metabolism of vitamin K and vitamin K 2,3-epoxide via interaction with a common disulfide. Biochemistry. 1984 May 8;23(10):2246–2252. doi: 10.1021/bi00305a024. [DOI] [PubMed] [Google Scholar]
  13. Lu A. Y., Jerina D. M., Levin W. Liver microsomal epoxide hydrase. J Biol Chem. 1977 Jun 10;252(11):3715–3723. [PubMed] [Google Scholar]
  14. Lu A. Y., Ryan D., Jerina D. M., Daly J. W., Levin W. Liver microsomal expoxide hydrase. Solubilization, purification, and characterization. J Biol Chem. 1975 Oct 25;250(20):8283–8288. [PubMed] [Google Scholar]
  15. Matschiner J. T., Bell R. G., Amelotti J. M., Knauer T. E. Isolation and characterization of a new metabolite of phylloquinone in the rat. Biochim Biophys Acta. 1970 Feb 24;201(2):309–315. doi: 10.1016/0304-4165(70)90305-3. [DOI] [PubMed] [Google Scholar]
  16. Morrissey J. H. Silver stain for proteins in polyacrylamide gels: a modified procedure with enhanced uniform sensitivity. Anal Biochem. 1981 Nov 1;117(2):307–310. doi: 10.1016/0003-2697(81)90783-1. [DOI] [PubMed] [Google Scholar]
  17. Olson R. E., Suttie J. W. Vitamin K and gamma-carboxyglutamate biosynthesis. Vitam Horm. 1977;35:59–108. doi: 10.1016/s0083-6729(08)60521-x. [DOI] [PubMed] [Google Scholar]
  18. Preusch P. C., Suttie J. W. Formation of 3-hydroxy-2,3-dihydrovitamin K1 in vivo: relationship to vitamin K epoxide reductase and warfarin resistance. J Nutr. 1984 May;114(5):902–910. doi: 10.1093/jn/114.5.902. [DOI] [PubMed] [Google Scholar]
  19. Sadowski J. A., Suttie J. W. Mechanism of action of coumarins. Significance of vitamin K epoxide. Biochemistry. 1974 Aug 27;13(18):3696–3699. doi: 10.1021/bi00715a012. [DOI] [PubMed] [Google Scholar]
  20. Suttie J. W., Geweke L. O., Martin S. L., Willingham A. K. Vitamin K epoxidase: dependence of epoxidase activity on substrates of the vitamin K-dependent carboxylation reaction. FEBS Lett. 1980 Jan 14;109(2):267–270. doi: 10.1016/0014-5793(80)81102-1. [DOI] [PubMed] [Google Scholar]
  21. Suttie J. W., Jackson C. M. Prothrombin structure, activation, and biosynthesis. Physiol Rev. 1977 Jan;57(1):1–70. doi: 10.1152/physrev.1977.57.1.1. [DOI] [PubMed] [Google Scholar]
  22. Suttie J. W. Mechanism of action of vitamin K: synthesis of gamma-carboxyglutamic acid. CRC Crit Rev Biochem. 1980;8(2):191–223. doi: 10.3109/10409238009105469. [DOI] [PubMed] [Google Scholar]
  23. Wallin R., Gebhardt O., Prydz H. NAD(P)H dehydrogenase and its role in the vitamin K (2-methyl-3-phytyl-1,4-naphthaquinone)-dependent carboxylation reaction. Biochem J. 1978 Jan 1;169(1):95–101. doi: 10.1042/bj1690095. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Wallin R., Suttie J. W. Vitamin K-dependent carboxylation and vitamin K epoxidation. Evidence that the warfarin-sensitive microsomal NAD(P)H dehydrogenase reduces vitamin K1 in these reactions. Biochem J. 1981 Mar 15;194(3):983–988. doi: 10.1042/bj1940983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Whitlon D. S., Sadowski J. A., Suttie J. W. Mechanism of coumarin action: significance of vitamin K epoxide reductase inhibition. Biochemistry. 1978 Apr 18;17(8):1371–1377. doi: 10.1021/bi00601a003. [DOI] [PubMed] [Google Scholar]
  26. Zimmermann A., Matschiner J. T. Biochemical basis of hereditary resistance to warfarin in the rat. Biochem Pharmacol. 1974 Mar 15;23(6):1033–1040. doi: 10.1016/0006-2952(74)90002-1. [DOI] [PubMed] [Google Scholar]
  27. de Metz M., Soute B. A., Hemker H. C., Fokkens R., Lugtenburg J., Vermeer C. Studies on the mechanism of the vitamin K-dependent carboxylation reaction. Carboxylation without the concurrent formation of vitamin K 2,3-epoxide. J Biol Chem. 1982 May 25;257(10):5326–5329. [PubMed] [Google Scholar]

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