Figure 2.
V255M carboxylase generates higher levels of modified FIX than wild-type carboxylase. (A) Carboxylation of Glu (E) to Gla (Y) residues in FIX occurs in 3 steps (ie, binding, catalysis, and release). The overall reaction was studied using FIX−18/+41, which contains the 18–amino acid EBD covalently linked to the 41–amino acid Gla domain with 12 Glu residues. The EBD in FIX is a propeptide that is cleaved subsequent to carboxylation. (B) FLAG-tagged wild-type (WT) and mutant carboxylases were immunopurified using anti-FLAG antibody and quantitated in western blots with anti-FLAG and anti-carboxylase antibodies. Equivalent amounts of variant carboxylases were then reacted with FIX−18/+41, followed by SDS-PAGE and PhosphorImager analysis. (C-E) The specific catalytic step was monitored using FIX-carboxylase complexes generated in cells expressing FIX and individual variant carboxylases. After isolation on anti-FIX resin, aliquots were monitored for the amount of FIX-carboxylase complex by an epoxidation assay,20 which was confirmed by western blot analysis. Equivalent amounts of complex (6 pmol) were then monitored for pmol of Gla incorporation into FIX using gel electrophoresis and PhosphorImager analysis. (F) The amounts of carboxylated FIX−18/+41 or FIX in the complex in panels B and E were compared with the amount of WT carboxylaseFLAG to determine the times to complete the reactions. The time for an individual FIX-carboxylase complex to become fully carboxylated was ∼15 minutes (lower panel), whereas FIX−18/+41 was carboxylated in ∼75 minutes (as indicated by a ratio of 1 for FIX−18/+41/carboxylase levels in the upper panel). (G) FIX−18/+41 was reacted with WT or mutant carboxylases as in panel B, and the products were precipitated with chloroform/methanol17 and resuspended in 25 mM of ammonium bicarbonate. Aliquots were quantitated by scintillation counting, and similar levels of counts were subjected to isoelectric focusing using a pH 3-10 Criterion IEF gel (Bio-Rad Laboratories) and the buffers recommended by the manufacturer. This analysis revealed a more basic FIX−18/+41 product with the V255M mutant than with WT carboxylase, indicating partial carboxylation.

V255M carboxylase generates higher levels of modified FIX than wild-type carboxylase. (A) Carboxylation of Glu (E) to Gla (Y) residues in FIX occurs in 3 steps (ie, binding, catalysis, and release). The overall reaction was studied using FIX−18/+41, which contains the 18–amino acid EBD covalently linked to the 41–amino acid Gla domain with 12 Glu residues. The EBD in FIX is a propeptide that is cleaved subsequent to carboxylation. (B) FLAG-tagged wild-type (WT) and mutant carboxylases were immunopurified using anti-FLAG antibody and quantitated in western blots with anti-FLAG and anti-carboxylase antibodies. Equivalent amounts of variant carboxylases were then reacted with FIX−18/+41, followed by SDS-PAGE and PhosphorImager analysis. (C-E) The specific catalytic step was monitored using FIX-carboxylase complexes generated in cells expressing FIX and individual variant carboxylases. After isolation on anti-FIX resin, aliquots were monitored for the amount of FIX-carboxylase complex by an epoxidation assay,20 which was confirmed by western blot analysis. Equivalent amounts of complex (6 pmol) were then monitored for pmol of Gla incorporation into FIX using gel electrophoresis and PhosphorImager analysis. (F) The amounts of carboxylated FIX−18/+41 or FIX in the complex in panels B and E were compared with the amount of WT carboxylaseFLAG to determine the times to complete the reactions. The time for an individual FIX-carboxylase complex to become fully carboxylated was ∼15 minutes (lower panel), whereas FIX−18/+41 was carboxylated in ∼75 minutes (as indicated by a ratio of 1 for FIX−18/+41/carboxylase levels in the upper panel). (G) FIX−18/+41 was reacted with WT or mutant carboxylases as in panel B, and the products were precipitated with chloroform/methanol17 and resuspended in 25 mM of ammonium bicarbonate. Aliquots were quantitated by scintillation counting, and similar levels of counts were subjected to isoelectric focusing using a pH 3-10 Criterion IEF gel (Bio-Rad Laboratories) and the buffers recommended by the manufacturer. This analysis revealed a more basic FIX−18/+41 product with the V255M mutant than with WT carboxylase, indicating partial carboxylation.

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