The V255M mutant shows higher levels of modified MGP than wild-type (WT) carboxylase. (A) The overall reaction for MGP carboxylation comprises binding, catalysis, and release, which produces MGP containing 5 Glas (indicated by Ys). (B) MGP_1-64 and FIX_−18/+41 were incubated with the same amount of WT carboxylase, followed by SDS-PAGE and PhosphorImager analysis to quantitate [¹⁴C]-CO₂ incorporation into the peptides. (C) The same assay was used to measure MGP_1-64 carboxylation by WT and mutant carboxylases, using equivalent amounts of enzyme as determined by western blot analysis with an anti-carboxylase antibody. Higher carboxylase concentrations were used than in panel B, and the duplicate samples that were assayed gave almost identical results and consequent small error bars. The overall reaction for WT carboxylase took 60 minutes, as determined by comparing pmol of Gla in MGP_1-64 with the amount of carboxylase. (D-F) The specific catalytic step was monitored using preformed MGP_FLAG-carboxylase complexes generated in cells, which were adsorbed to anti-FLAG resin to remove free carboxylase. The amount of carboxylase was quantitated by an epoxidase assay,²⁰ and equivalent amounts of complex were then incubated in reactions containing KH₂ and [¹⁴C]-CO₂, followed by gel electrophoresis and PhosphorImager analysis to monitor [¹⁴C]-CO₂ incorporation into MGP_FLAG.