A radiometric technique for the measurement of adenylosuccinate lyase

When radioactive adenylosuccinic acid (AMP-S) is metabolized to AMP and fumaric acid by the enzyme adenylosuccinate lyase (EC 4.3.2.2), a proton is released to the solvent as 3H2O. This removal is believed to be stereospecifically identical to that catalyzed by the enzyme, L-aspartase [1-5], and therefore entails the loss of a proton from C-3 of the dicarboxylic acid moiety of the nucleotide. Advantage has been taken of this fact in the design of a facile assay for this enzyme. Adenylosuccinic acid, tritiated on C-2 and C-3 of the L-aspartate moiety, is prepared by chemical synthesis. This product is purified, lyophilized to dryness and reconstituted in a solution of unlabelled AMP-S, bringing the final concentration to 5 X 10(-3) M, and the final specific activity to 8.0 microCi/mumol. 5-microliter aliquots of this substrate are then incubated at 37 degrees C with 5-microliter aliquots of tissue extract; after an appropriate period, any tritium released to the solvent water is distilled at room temperature overnight into a 5 microliter droplet of saturated aqueous KOH adherent to the lid of the sealed reaction vessel. The lid is removed and tritium thereon is measured by scintillation spectrometry. The assay, performed as prescribed, is facile, in that it permits the simultaneous estimation of the lyase activity in a large battery of samples, is not interfered with by opalescent or proteinaceous suspensions, is accurate and outstandingly sensitive.