A new carboxylation reaction. The vitamin K-dependent incorporation of H-14-CO3- into prothrombin.

TLDR

Prothrombin contains gamma‑carboxyglutamic acid residues that are formed post‑translationally in a vitamin K‑dependent reaction, with incorporation of H‑14‑CO₃⁻ into microsomal proteins requiring the prothrombin precursor and factors from the post‑microsomal supernatant. Radioactive prothrombin isolated from microsomal pellets was found to carry all its H‑14‑CO₃⁻ radioactivity in the amino‑terminal fragment, and hydrolysis showed that half the activity was lost while the remaining radioactivity resided in glutamic acid, confirming that the label was incorporated into the carboxyl groups of gamma‑carboxyglutamic acid residues.

Abstract

The bovine plasma zymogen prothrombin contains a number of gamma-carboxyglutamic acid residues which are not found in an abnormal prothrombin produced when cattle are given the vitamin K antagonist dicoumarol. These modified glutamic acid residues appear to be formed post-translationally by a reaction which requires vitamin K. It has been shown that postmitochondrial supernates from vitamin K-deficient rats incorporate added H-14-CO3- minus into microsomal proteins upon the addition of vitamin K. This incorporation is dependent upon the presence of the prothrombin precursor in the microsomal preparations, and upon factors which are present in the postmicrosomal supernatant. Most of the radioactive protein which can be obtained from the microsomal pellet by extraction with 0.25% Triton X-100 has been identified as prothrombin and it can be shown that all of the radioactivity is in the amino-terminal activation fragment of prothrombin. This portion of the protein has previously been shown to contain the gamma-carboxyglutamic acid residues. Hydrolysis of the purified radioactive prothrombin resulted in a loss of 50% of the radioactivity and subsequent chromatography of the amino acid hydrolyzate demonstrated that the remaining radioactivity was entirely in glutamic acid. These results are consistent with the hypothesis that all of the H-14-CO3- minus was incorporated into the carboxyl groups of gamma-carboxyglutamic acid residues.