Processive MGP carboxylation is impaired in the V255M and S300F mutants. (A) Complexes between MGP_FLAG and variant carboxylases were reacted in the presence of a challenge peptide (MGP_1-64) that contained both the EBD and the entire Gla domain. (B-D) MGP_FLAG-carboxylase complexes were isolated on anti-FLAG resin, and the amount of complex was determined by an epoxidase assay, as before.²⁰ Equivalent amounts of complex were then analyzed in the challenge assay. Carboxylation of MGP_FLAG in the complexes and MGP_1-64 was quantitated by monitoring [¹⁴C]-CO₂ incorporation into each form, using gel electrophoresis and PhosphorImager analysis. (E-G) MGP_1-64 carboxylation occurred after MGP_FLAG in the complex with wild-type carboxylase. Both MGP forms were carboxylated at the same time by mutant carboxylases, which produced higher levels of MGP_1-64 than MGP_FLAG in the complex.