Vitamin K-dependent carboxylase: increased efficiency of the carboxylation reaction.
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 microM) 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.
D000818:Animals; D002135:Calcium-Binding Proteins / Q000378:metabolism; D019735:Carbon-Carbon Ligases; D002417:Cattle; D004796:Clinical Enzyme Tests / Q000379:methods; D004789:Enzyme Activation; D006863:Hydrogen-Ion Concentration; D008025:Ligases / Q000302:isolation & purification / Q000502:physiology*; D008099:Liver / Q000201:enzymology; D009994:Osmolar Concentration; D015675:Osteocalcin; D013379:Substrate Specificity; D013696:Temperature
D002135:Calcium-Binding Proteins; D015675:Osteocalcin; D008025:Ligases; D019735:Carbon-Carbon Ligases; C020660:glutamyl carboxylase