Modification of the vitamin K-dependent carboxylase assay.
Methods are presented that describe alternative protocols for the isolation of rat liver microsomes containing the vitamin K-dependent carboxylase and the procedure in which the solubilized enzyme is assayed. The method for determining the rate of 14CO2 incorporation into low molecular weight, acid soluble substrates by the rat liver microsomal vitamin K-dependent carboxylase has been modified in order to optimize safety, accuracy and simplicity. For these studies the rat liver microsomes containing the vitamin K-dependent carboxylase were isolated by CaCl2 precipitation. These Triton X-100 solubilized microsomes were found to be equivalent to the microsomes obtained by high speed ultracentrifugation with regard to protein concentration, pentapeptide carboxylase activity, carboxylase activity, preprothrombin concentration and total carboxylatable endogenous protein substrate. This modified assay procedure requires fewer steps and pipetting transfers and is quantitatively equivalent to previously employed protocols. The described technique can be adapted for any assay where 14CO2 or H14CO3- is incorporated into non-volatile products. This newly developed assay procedure was employed to assess conditions necessary for optimal vitamin K-dependent carboxylation of the less expensive substrate, N-t-Boc-L-glutamic acid alpha-benzyl ester. The optimal conditions for the carboxylation of N-t-Boc-L-glutamic acid alpha-benzyl ester by the carboxylase were found to be 10 mM N-t-Boc-L-glutamic acid alpha-benzyl ester, 10 mM MgCl2 at 15-18 degrees C. The rate of N-t-Boc-L-glutamic acid alpha-benzyl ester carboxylation under these optimized conditions was found to be higher (1.5-fold) than the rate of carboxylation of 1 mM Phe-Leu-Glu-Glu-Ile in the presence of the cation activator, MgCl2.
D000818:Animals; D002122:Calcium Chloride; D019735:Carbon-Carbon Ligases; D005971:Glutamates; D007700:Kinetics; D008025:Ligases / Q000032:analysis*; D008274:Magnesium / Q000494:pharmacology; D015636:Magnesium Chloride; D008862:Microsomes, Liver / Q000201:enzymology*; D011732:Pyridoxal Phosphate / Q000494:pharmacology; D051381:Rats; D012995:Solubility; D013696:Temperature
D005971:Glutamates; D015636:Magnesium Chloride; C046043:tert-butyloxycarbonyl-glutamic acid 1-benzyl ester; D011732:Pyridoxal Phosphate; D008025:Ligases; D019735:Carbon-Carbon Ligases; C020660:glutamyl carboxylase; D008274:Magnesium; D002122:Calcium Chloride