1.Modification of the vitamin K-dependent carboxylase assay.
Romiti S, Kappel WK. J Biochem Biophys Methods. 1985 May;11(1):59-68.
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.
2.An approach to trapping gamma-glutamyl radical intermediates proposed for vitamin K dependent carboxylase: alpha,beta-methyleneglutamic acid.
Slama JT1, Satsangi RK, Simmons A, Lynch V, Bolger RE, Suttie J. J Med Chem. 1990 Feb;33(2):824-32.
The vitamin K dependent carboxylase activates the glutamyl gamma-CH of substrate peptides for carboxylation by producing a gamma-glutamyl free radical, a gamma-glutamyl carbanion, or through a concerted carboxylation. We propose to intercept the putative gamma-glutamyl free radical by the intramolecular rearrangement of a substrate containing the alpha,beta-cyclopropane analogue of glutamic acid. The rearrangement of cyclopropylcarbinyl radicals into 2-butenyl radicals is rapid, exothermic, and considered diagnostic of free-radical formation. 1-Amino-2-(carboxymethyl)cyclopropane-1-carboxylate, the beta-cyclopropane analogue of glutamic acid, was synthesized starting from diethyl alpha-ketoglutarate. The alpha-keto ester was first treated with benzonitrile in sulfuric acid, to yield diethyl alpha,alpha-dibenzamidoglutarate. The alpha,alpha-dibenzamido acid was cleaved to produce the alpha,beta-dehydroamino acid and benzamide on treatment with p-toluenesulfonic acid in hot benzene.