Abstract
The reductase activity mediating the ketone reduction of the acetonyl side-chain of warfarin and analogs has been partially purified from rabbit liver cytosol. The reductase activity is resolved in two different fractions (A and B) by DEAE-Sephacel chromatography. Both fractions reduce the acetonyl group of warfarin analogs with marked substrate (R-enantiomers), as well as product stereoselectivity (alcohols of the S-configuration). The reductases are NADPH-dependent, which is absolute for fraction B. The enzyme kinetics of the R-enantiomers of warfarin and three 4'-derivatives (4'-nitro-, 4'-chloro-, and 4'-methoxywarfarin) has been investigated. In contrast to fraction B, fraction A is sensitive in its KM for 4' substitution: the KM values of 4'-nitro- and chloro-analogs are approximately 6 times lower than the KM values of the 4'-methoxy analog or warfarin itself. On the other hand, the Vmax values of fraction A are all in the range of about 1 to 2 nmol/mg x min, whereas the Vmax values of fraction B vary from about 1(4'-methoxywarfarin) to 12 (the 4'-nitro analog). The intrinsic activities (Vmax/KM) of both enzymes show the same rank order: 4'-nitro greater than 4'-chloro greater than 4'-methoxy = warfarin. Warfarin reductase activity of both enzymes is not inhibited by pyrazole, sodium barbitone, or dicoumarol, but is strongly inhibited by quercetin, indomethacin, furosemide, and prostaglandin E2 (PGE2). In addition, fraction A is inhibited by menadione and androsterone; fraction B is inhibited by estrone. Various compounds were tested as substrates for these enzyme fractions.(ABSTRACT TRUNCATED AT 250 WORDS)
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