Vitamin K-dependent carboxylase. Development of a peptide substrate

Rat liver microsomes contain a vitamin K-dependent carboxylase activity that converts specific glutamyl residues of microsomal prothrombin precursor to gamma-carboxyglutamic acid residues. This activity has now been solubilized by treatment with Triton X-100. The pentapeptide, Phe-Leu-Glu-Glu-Val, has been synthesized; and it has been demonstrated that, in the presence of this peptide, the solubilized microsomes catalyze a vitamin K-dependent incorporation of added H14CO3- into a low molecular weight trichloroacetic acid-soluble compound. The carboxylated product has been identified as peptide-bound gamma-carboxyglutamic acid by its chemical stability during acidic and alkaline hydrolysis and by co-chromatography of an alkaline hydrolysate of the product with authentic gamma-carboxyglutamic acid. The conditions for peptide carboxylation appear to be identical with those demonstrated for precursor carboxylation.