Isolation Of Vitamin K Dependent Carboxylase Using Antibody Overlay Techniques

Abstract

The standard vitamin K dependent carboxylase assay was maximized for use with purified carboxylase preparation. This preparation represented a 100-fold increase in specific activity relative to microsomes. The carboxylation assay was maximized with respect to temperature, detergent, salt and peptide parameters. The enzyme was found to react maximally at 27°C in 1.0 x 10⁻³ g/ml detergent (Renex 30), .75 M K₂HPO₄ and 2.0 mM Phe-leu-glu-glu-isoleu synthetic substrate. Reproducibility of the assay was achieved by correcting for the specific activity of the added H¹⁴CO₃. Carboxylase activity was inhibited by prothrombin antibody in the standard carboxylation assay. Visualization of carboxylase protein was obtained by reacting SDS electrophoresed carboxylase preparation with prothrombin antibody impregnated agarose. A low molecular weight band (≈30,000) was observed and may represent a carboxylase subunit.