A radiochemical assay for argininosuccinate synthetase with [U-14C]aspartate.
A simple and sensitive radiochemical procedure to assay argininosuccinate synthetase activity in crude tissue homogenates and lysates of cultured cells is described. The new method depends on the location of 14C, uniformly, in the four carbons of aspartate. On incubation in the presence of excess of L-[U-14C]aspartate, L-citrulline, ATP, and an ATP-generating system, argininosuccinase and arginase, the [14C]fumarate formed is measured as the sum of malate and fumarate. After acidification the latter two acids are separated from [14C]aspartate on a small Dowex-50 column by elution with a few milliliters of water; the unutilized amino acid substrates remain on the column. With a specific radioactivity of 9 X 10(4) cpm, 1 to 2 nmol of product can be accurately measured under kinetically optimum conditions.
D000818:Animals; D001124:Argininosuccinate Synthase / Q000032:analysis*; D001224:Aspartic Acid / Q000378:metabolism*; D002250:Carbon Radioisotopes; D002460:Cell Line; D002852:Chromatography, Ion Exchange; D005650:Fumarates / Q000302:isolation & purification; D006801:Humans; D008025:Ligases / Q000032:analysis*; D008099:Liver / Q000378:metabolism*; D008293:Malates / Q000302:isolation & purification; D011854:Radiochemistry / Q000379:methods*; D051381:Rats; D012588:Scintillation Counting
D002250:Carbon Radioisotopes; D005650:Fumarates; D008293:Malates; D001224:Aspartic Acid; D008025:Ligases; D001124:Argininosuccinate Synthase