Vitamin K-Dependent Carboxylase: Increased Efficiency of the Carboxylation Reaction

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Abstract
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 Km of which had been shown to be low (10 pM) and it was added in 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.