Vitamin K-dependent gamma-glutamyl carboxylase activity in the chick embryonic chorioallantoic membrane.
During embryonic development of the chick, the onset of calcium transport by the chorioallantoic membrane (CAM) is concomitant with the appearance of a calcium-binding protein (CaBP). The development-specific expression of the CaBP in the CAM is inhibited by vitamin K antagonism in ovo with the anticoagulant, warfarin. However, the CaBP remains immunologically detectable in the CAM of warfarin-treated embryos, suggesting the presence of a precursor form of the CaBP. Previously, we have demonstrated that CaBP expression in CAM organ cultures is inducible by vitamin K. Furthermore, the CaBP contains several residues of the modified amino acid, gamma-carboxyglutamic acid (gamma-CGlu), which has been shown to be formed by vitamin K-dependent carboxylation of glutamic acid in several plasma clotting proteins. This study reports the presence of a post-translational, vitamin K-dependent gamma-glutamyl carboxylase activity in the CAM. Our results show that explants of CAM incorporate H14CO3 in an age-specific and vitamin K-dependent manner. Incorporation of H14CO3 by the CAM is further potentiated by warfarin treatment of the embryos, presumably owing to an elevation of the amount of endogenous uncarboxylated protein precursor(s). Among the subcellular (nuclear, mitochondrial, microsomal, and soluble) fractions of the CAM, only microsomes exhibit specific incorporation of of H14CO3 into gamma-CGlu. The CAM microsomal carboxylation activity is post-translational, vitamin K-dependent, specific for prenylated homologs of vitamin K, sensitive to warfarin, and appears to be unrelated to the activities of biotin-dependent carboxylases or phosphoenolpyruvate carboxykinase. Optimal carboxylation activity occurs after incubation of the microsomes with H14CO3 for 60 min at 37 degrees C in the presence of over 100 microgram of vitamin K1/ml.
D000818:Animals; D019735:Carbon-Carbon Ligases; D002642:Chick Embryo; D002823:Chorion / Q000201:enzymology* / Q000502:physiology; D005971:Glutamates; D005779:Immunodiffusion; D007700:Kinetics; D008025:Ligases / Q000378:metabolism*; D008099:Liver / Q000201:enzymology; D008861:Microsomes / Q000201:enzymology / Q000648:ultrastructure; D011516:Prothrombin; D064030:S100 Calcium Binding Protein G / Q000378:metabolism; D014812:Vitamin K / Q000494:pharmacology*; D014859:Warfarin / Q000494:pharmacology
D005971:Glutamates; D064030:S100 Calcium Binding Protein G; D014812:Vitamin K; D014859:Warfarin; D011516:Prothrombin; D008025:Ligases; D019735:Carbon-Carbon Ligases; C020660:glutamyl carboxylase