Two complementary radiometric methods for the measurement of 5-amino-4-imidazole-N-succinocarboxamide ribonucleotide synthetase (SAICAR synthetase).
Two complementary methods have been devised for measuring the activity of 5-amino-4-imidazole-N-succinocarboxamide ribonucleotide synthetase (SAICAR synthetase, EC 6.3.2.6), a critical enzyme in the pathway of purine biosynthesis. In the first method, L-[4-14C]aspartic acid is condensed with 5-amino-4-imidazolecarboxylic acid ribonucleotide (AICOR) via the action of SAICAR synthetase. Unreacted L-[4-14C]aspartic acid is quantitatively dissipated by enzymatic decarboxylation, and the residual radioactivity is measured by scintillation spectrometry. In the second method, the reverse reaction of SAICAR synthetase is measured; radioactive 5-amino-4-imidazole-N-succinocarboxamide ribonucleotide (SAICAR) is synthesized enzymatically, using a partially purified preparation of SAICAR synthetase from chicken liver. To the purified [14C]SAICAR is added: sodium arsenate, Tris-HCl buffer containing ADP--MgCl2 or buffer alone, and to initiate the reaction, a 12 000 x g supernatant or other suitable source of enzyme. As a consequence of the arsenolytic cleavage of [14C]SAICAR, L-[4-14C]aspartic acid is generated in stoichiometric amounts. The fourth carbon of this amino acid is then detached by selective enzymatic decarboxylation, trapped in 40% KOH and quantitated by scintillation spectrometry. The assays, performed as prescribed, are facile and notably sensitive; using them, the specific activity of SAICAR synthetase has been measured in acetone powders of the livers of representative members of Vertebrata, and also in the principal viscera of the mouse. Of the livers examined, pigeon liver was the richest source of the investigated enzyme.
D000818:Animals; D001224:Aspartic Acid; D002250:Carbon Radioisotopes; D002645:Chickens; D002855:Chromatography, Thin Layer; D008099:Liver / Q000201:enzymology; D008297:Male; D051379:Mice; D010453:Peptide Synthases / Q000032:analysis*; D011865:Radioisotope Dilution Technique; D014018:Tissue Distribution
D002250:Carbon Radioisotopes; D001224:Aspartic Acid; D010453:Peptide Synthases; C022379:phosphoribosylaminoimidazole-succinocarboxamide synthetase