Identification of a warfarin-sensitive protein component in a 200S rat liver microsomal fraction catalyzing vitamin K and vitamin K 2,3-epoxide reduction.
A partially purified, 200S submicrosomal fraction exhibiting thiol-dependent vitamin K1 (vitamin K) and epoxide reductase activities has been isolated by partial solubilization of rat hepatic microsomes with sodium cholate and separation by centrifugation at 105 000 g into a discontinuous sucrose gradient. At pH 7.4, the rates of vitamin K and vitamin K 2,3-epoxide reduction per milligram of 200S fraction protein were equivalent and were 2.5-3.0 times faster than in microsomes. Reduction of vitamin K 2,3-epoxide occurred in a tightly coupled, two-step reaction initially to vitamin K and subsequently to vitamin K hydroquinone (vitamin KH2). Incorporation of glycerol or sucrose and of sodium cholate into reaction mixtures equivalently affected the rates of both vitamin K and vitamin K 2,3-epoxide reduction, but in the case of epoxide metabolism, the ratios of vitamin KH2/vitamin K were much lower, suggesting that the second reaction has been partially uncoupled from the first. A 14 000-17 000-dalton warfarin-sensitive protein (WSP) that participates in vitamin K and vitamin K 2,3-epoxide reduction in the 200S fraction was identified by incorporation of N-[3H]ethylmaleimide ([3H]NEM) into the catalytically active reduced form of one or more attached disulfides. Reduction of WSP with dithiothreitol was required for reaction with [3H]NEM, and the substrates vitamin K and vitamin K 2,3-epoxide and the inhibitor warfarin all effectively blocked the reaction. 2-Mercaptoethanol could not substitute for dithiothreitol.(ABSTRACT TRUNCATED AT 250 WORDS)
D000818:Animals; D002458:Cell Fractionation; D002499:Centrifugation, Density Gradient; D002851:Chromatography, High Pressure Liquid; D005033:Ethylmaleimide / Q000494:pharmacology; D007700:Kinetics; D008862:Microsomes, Liver / Q000201:enzymology*; D006899:Mixed Function Oxygenases / Q000378:metabolism*; D008970:Molecular Weight; D051381:Rats; D014812:Vitamin K / Q000378:metabolism*; D064417:Vitamin K Epoxide Reductases; D014859:Warfarin / Q000494:pharmacology*
D014812:Vitamin K; D014859:Warfarin; D006899:Mixed Function Oxygenases; D064417:Vitamin K Epoxide Reductases; D005033:Ethylmaleimide