Site-Directed Mutagenesis of VKORC1, the Target Protein of Coumarin-Type Anticoagulants

Novel mutations in the VKORC1 gene of wild rats and mice – a response to 50 years of selection pressure by warfarin?

“…Blue shaded amino acid residues refer to the CXXC motif which is supposed to be the active site of the VKORC1 protein. The TYA motif which is highlighted in pink is the proposed warfarin binding site [ 25 ].…”

“…Therefore, rats bearing either of the Tyr139 mutations should have an enormous selective advantage in warfarin-exposed areas. We have speculated earlier that Tyr139 may be part of the warfarin binding site of VKORC1 [ 25 ].…”

“…For the recombinant expression of rat and mouse VKORC1 variants HEK293-EBNA cells (Invitrogen, Karlsruhe, Germany) were used as described earlier [ 25 ]. The VKOR enzymatic activity of the various VKORC1 proteins was measured in whole-cell extracts before and after addition of different warfarin concentrations as described by Rost et al, 2005 [ 25 ].…”

“…For the recombinant expression of rat and mouse VKORC1 variants HEK293-EBNA cells (Invitrogen, Karlsruhe, Germany) were used as described earlier [ 25 ]. The VKOR enzymatic activity of the various VKORC1 proteins was measured in whole-cell extracts before and after addition of different warfarin concentrations as described by Rost et al, 2005 [ 25 ]. Aliquots of whole-cell protein extracts were separated on 4–12% NuPAGE gels (Invitrogen, Karlsruhe, Germany) according to the manufacturer's instructions and blotted onto nitrocellulose membranes (Whatman Schleicher & Schuell, Dassel, Germany).…”