Heterogeneity of the coumarin anticoagulant targeted vitamin K epoxide reduction system. Study of kinetic parameters in susceptible and resistant mice (Mus musculus domesticus).
Vitamin K epoxide reductase (VKOR) activity in liver microsomes from a susceptible and a genetically warfarin-resistant strain of mice (Mus Musculus domesticus) was analyzed to determine the mechanism of resistance to this 4-hydroxycoumarin derivative. Kinetic parameters for VKOR were calculated for each strain by incubating liver microsomes with vitamin K epoxide +/- warfarin. In susceptible mice, an Eadie-Hofstee plot of the data was not linear and suggested the involvement of at least two different components. Apparent kinetic parameters were obtained by nonlinear regression using a Michaelis--Menten model, which takes into account two enzymatic components. Component A presents a high Km and a high Vm, and as a consequence only an enzymatic efficiency Vm/Km was obtained (0.0024 mL/min/mg). Estimated warfarin Ki was 0.17 microM. Component B presented an apparent Km of 12.73 microM, an apparent Vm of 0.32 nmol/min/mg, and an apparent Ki for warfarin of 6.0 microM. In resistant mice, the enzymatic efficiency corresponding to component A was highly decreased (0.0003-0.00066 mL/min/mg) while the Ki for warfarin was not modified. The apparent Vm of component B was poorly modified between susceptible and resistant mice. The apparent Km of component B observed in resistant mice was similar to the Km observed in susceptible mice. These modifications of the catalytic properties are associated with a single nucleotide polymorphism (T175G) in the VKOR-C1 gene, which corresponds to a Trp59Gly mutation in the protein.
Lasseur
Romain
R
UMR 1233 INRA/DGER, Métabolisme des Xénobiotiques et Mycotoxines, Lyon College of Veterinary Medicine, BP 83, 69280 Marcy l'Etoile, France.
Grandemange
Agnès
A
Longin-Sauvageon
Christiane
C
Berny
Philippe
P
Benoit
Etienne
E
D000818:Animals; D000925:Anticoagulants / Q000494:pharmacology*; D001483:Base Sequence; D020134:Catalytic Domain / Q000235:genetics; D004247:DNA / Q000235:genetics; D004351:Drug Resistance / Q000235:genetics*; D007700:Kinetics; D051379:Mice; D008862:Microsomes, Liver / Q000201:enzymology*; D006899:Mixed Function Oxygenases / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D009154:Mutation; D020641:Polymorphism, Single Nucleotide; D064417:Vitamin K Epoxide Reductases; D014859:Warfarin / Q000494:pharmacology*
D000925:Anticoagulants; D014859:Warfarin; D004247:DNA; D006899:Mixed Function Oxygenases; D064417:Vitamin K Epoxide Reductases