The in vitro vitamin K-dependent carboxylation of peptide- or protein-bound glutamate residues is generally studied in detergent-solubilized microsomes from rat or cow liver. Under the conditions usually employed, the efficiency of the carboxylation reaction is low (less than 1% of the carboxylatable residues is converted into gammacarboxyglutamate). Here we describe that this efficiency may be raised to 30% by carrying out the following adaptations: 1) carboxylase was purified about 100-fold from the solubilized microsomes, so that the enzyme was obtained in a highly concentrated form and could be added in excess; 2) the HCO- 3 concentration in the reaction mixtures was raised to 50 mM and 3) a substrate was selected (decarboxylated osteocalcin from bovine bone) the K(m) of which had been shown to be low (10 μM) and it was added to rate-limiting amounts. Besides the fact that under these conditions the carboxylation reaction occurred with a higher efficiency than before, the adaptations also enabled us to express the carboxylation activity in terms of moles CO2 incorporated per mole of substrate.
|Number of pages||5|
|Journal||Thrombosis and Haemostasis|
|Publication status||Published - 1 Dec 1987|