Elsevier

Biochemical Pharmacology

Volume 55, Issue 2, 15 January 1998, Pages 169-175
Biochemical Pharmacology

Research Papers
Co-purification of Microsomal Epoxide Hydrolase with the Warfarin-Sensitive Vitamin K1 Oxide Reductase of the Vitamin K Cycle

https://doi.org/10.1016/S0006-2952(97)00431-0Get rights and content

Abstract

Vitamin K1 oxide reductase activity has been partially purified from rat liver microsomes. A three-step procedure produced a preparation in which warfarin-sensitive vitamin K1 oxide reductase activity was 118-fold enriched over the activity in intact rat liver microsomes. A major component of the multi-protein mixture was identified as a 50 kDa protein that strongly cross-reacts with antiserum prepared against homogeneous rat liver microsomal epoxide hydrolase. The reductase preparation also had a high level of epoxide hydrolase activity against two xenobiotic epoxide substrates. The Km values for hydrolysis by the reductase preparation were similar to those for homogeneous microsomal epoxide hydrolase itself, and the specific hydrolase activities of the reductase preparation were 25–35% of the specific activities measured for the homogeneous hydrolase preparation. Antibodies prepared against homogeneous microsomal epoxide hydrolase inhibited up to 80% of reductase activity of the reductase preparation. Homogeneous microsomal epoxide hydrolase had no vitamin K1 oxide reductase activity. This evidence suggests that microsomal epoxide hydrolase, or a protein that is very similar to it, is a major functional component of a multi-protein complex that is responsible for vitamin K1 oxide reduction in rat liver microsomes.

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.

References (35)

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