Kato H, Oyamada I, Mizutani-Funahashi M, Nakagawa H
J Biochem. 1976 May;79(5):945-53. doi: 10.1093/oxfordjournals.jbchem.a131162.
Methods were developed for the radioisotopic assay of argininosuccinate synthetase [L-citrulline: L-aspartate ligase (AMP-forming), EC 6.3.4.5] and argininosuccinase [L-argininosuccinate arginine-lyase, EC 4.3.2.1]. The assay of argininosuccinate synthetase was based on the separation of [14C]argininosuccinate formed from aspartate and [carbamoyl-14C]citrulline in the presence of ATP from the substrate citrulline. For this, the product was converted to its anhydride form by boiling for 30 min at pH 2.0 followed by application on a column of Dowex 50W (pyridine form). Argininosuccinic anhydride was eluted with 0.3 M pyridine acetate buffer, pH 4.25, while citrulline was eluted with 0.1 M pyridine acetate buffer, pH 3.80. The assay of argininosuccinase was based on the separation of [14C]argininosuccinic acid formed from arginine and [U-14C]fumaric acid from the substrate fumarate on a column of Dowex 50W(H+ form). The argininosuccinic acid was adsorbed on the column and eluted with 1 M pyridine solution, while fumarate was not adsorbed. The distributions of these two enzymes in various organs and cell fractions were reinvestigated using these methods.
已开发出用于精氨琥珀酸合成酶[L-瓜氨酸：L-天冬氨酸连接酶（AMP形成），EC 6.3.4.5]和精氨琥珀酸酶[L-精氨琥珀酸精氨酸裂解酶，EC 4.3.2.1]放射性同位素测定的方法。精氨琥珀酸合成酶的测定基于在ATP存在下，由天冬氨酸和[氨甲酰-14C]瓜氨酸形成的[14C]精氨琥珀酸与底物瓜氨酸的分离。为此，将产物在pH 2.0下煮沸30分钟转化为其酸酐形式，然后应用于Dowex 50W（吡啶型）柱上。精氨琥珀酸酸酐用pH 4.25的0.3 M吡啶乙酸缓冲液洗脱，而瓜氨酸用pH 3.80的0.1 M吡啶乙酸缓冲液洗脱。精氨琥珀酸酶的测定基于在Dowex 50W（H+型）柱上，由精氨酸形成的[14C]精氨琥珀酸与底物延胡索酸中的[U-14C]延胡索酸的分离。精氨琥珀酸被吸附在柱上并用1 M吡啶溶液洗脱，而延胡索酸不被吸附。使用这些方法重新研究了这两种酶在各种器官和细胞组分中的分布。
