In Vitro Synthesis of Bovine Prothrombin in a Purified System

Coumarin treatment of the cow results in the appearance of a prothrombin precursor (de-carboxyprothrombin) in the blood. We have isolated from bovine liver the enzyme (prothrombin synthase) that is able to convert purified decarboxyprothrombin into prothrombin. Prothrombin synthase is localized in the microsomal fraction and extracted there from with a buffer containing 2% Triton X-100. Further purification of the enzyme was accomplished by column chromatography on Sepharose 4B, DEAE Sephacell and Sephadex G 100. The molecular weight of prothrombin synthase was found to be about 60,000 D. The enzyme activity is defined as the ability to increase the prothrombin concentration at standard conditions and it is measured as a decrease of the clotting time in the one-stage coagulation assay. In the presence of NaH¹⁴CO₃, radioactive label is incorporated into decarboxyprothrombin, parallel to the generation of prothrombin.

After extraction with a suitable organic solvent prothrombin synthase was separated from endogenously bound vitamin K. After extraction the water phase contained the enzyme, the activity of which was completely restored by supplementing the reaction mixtures with vitamin KH₂. The organic phase contained the vitamin K as was deduced from U.V.spectra. Prothrombin synthase is dependent on Mn²⁺ and molecular oxygen and is inhibited by warfarin, Cl-K and Cu²⁺.