Research PapersCo-purification of Microsomal Epoxide Hydrolase with the Warfarin-Sensitive Vitamin K1 Oxide Reductase of the Vitamin K Cycle
Section snippets
Materials
CHAPS, DIFP, Tris, benzamidine, ammonium sulfate, glycerol, protein A-agarose, purified rabbit IgG, and vitamin K1 were obtained from the Sigma Chemical Co. Asolectin phospholipid preparation was obtained from the Fluka Chemical Corp. Sepharose 6B and Sephadex G25 were obtained from Pharmacia Biotech Inc. Hydroxyapatite and reagents used for SDS–PAGE were obtained from Bio-Rad Laboratories. Antiserum to purified homogeneous rat liver HYL1 was raised in adult male rabbits, as described. [25].
Substrate Synthesis
Results
Table 1 shows the partial purification of VKOR activity from rat liver microsomes. The specific activity of the enzyme was enhanced 118-fold by this procedure. The VKOR activity of this preparation was inhibited 85% in the presence of 10 μM warfarin, and 92% in the presence of 25 μM warfarin, indicating that it indeed represents the VKOR activity of the vitamin K cycle. SDS–PAGE analysis of the eluted proteins shows a complex mixture, comprising a major protein component with apparent molecular
Discussion
The data presented clearly indicate a copurification of HYL1 activity with the VKOR complex. That HYL1, or a very similar protein, is a major component of the isolated mixture of proteins is evident from the relatively high abundance of the 50 kDa band on the SDS-polyacrylamide gel electrophoretogram, as well as from the high immunoreactivity of the 50 kDa protein with an anti-HYL1 antibody, as shown by the immunoblots in Fig. 1, Fig. 2. The high relative abundance of HYL1 in this mixture of
Acknowledgements
This work was supported, in part, by USDA Grant NCR-9403041 to R. W.
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Polymorphisms of vitamin K-related genes (EPHX1 and VKORC1L1) and stable warfarin doses
2018, GeneCitation Excerpt :Because the rate of conversion of K to KH2 by VKOR was considerably slower than the rate of conversion of KO to K, it has been suggested that other enzymes may exist in the reduction pathway of vitamin K (Limdi, 2012). VKOR protein resides in the endoplasmic reticulum, and may be complexed with microsomal epoxide hydrolase, encoded by EPHX1, to produce a multiprotein complex that catalyzes hydrolysis of a large number of epoxides, and is responsible for vitamin K epoxide reduction (Guenthner et al., 1998; Morisseau and Hammock, 2005). The polymorphism of rs3738042 showed that patients carrying variant homozygotes (AA) required lower doses than those with the wild G allele although it did not reach statistical significance.
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2012, Toxicology and Applied PharmacologyCitation Excerpt :For example, in cerebral tissues, mEH is primarily localized in glial cells (Teissier et al., 1998) and its activity is particularly high in tissues which function as blood- and cerebrospinal fluid-brain barriers such as the choroid plexus (Ghersi-Egea et al., 1994). In addition to the role in xenobiotic metabolism, mEH is implicated as a participant in endogenous steroid metabolism (Fandrich et al., 1995), and in the vitamin K reductase complex (Guenthner et al., 1998). mEH is known to be expressed on the plasma membrane and has been reported to act as a Na+-dependent bile acid transporter (von Dippe et al., 1993).
Structure and Function of Vitamin K Epoxide Reductase
2008, Vitamins and HormonesCitation Excerpt :These GSTs were further purified by an agarose–glutathione affinity column and they were proposed to be the warfarin‐sensitive component of the VKOR enzyme complex in the ER membrane (Cain et al., 1997). Further results from this group suggested that microsomal epoxide hydrolase, an integral membrane protein, is the second component of VKOR complex (Guenthner et al., 1998; Wallin and Guenthner, 1997). Based on their results, these authors proposed that dimeric GST provided the warfarin‐sensitive thiol redox center while the microsomal epoxide hydrolase provided the KO binding site for the catalytic reduction reaction (Guenthner et al., 1998; Wallin and Guenthner, 1997).