Rochette-Egly C, Egly J M
J Cyclic Nucleotide Res. 1980;6(5):335-45.
A chromatographic method using immobilized acriflavin has been developed for the separation of unreacted cyclic nucleotides from their corresponding 5'-nucleotides, in a direct assay of 3'-5' cyclic nucleotide phosphodiesterase using [3H]-cyclic nucleotides as substrate. The method based on the so-called charge-transfer overlap recognition between flavin and indol rings, provides a rapid (15-20 min) and sensitive elution of [3H]-5'nucleotides with high recovery (up to 98%) and low blanks, while [3H]-cyclic nucleotides are retarded on the column. By this method, the formation of some secondary products by purine metabolizing enzymes such as 5'-nucleotidases, nucleosidases and/or deaminases is taken into account, using [14C]-5'-AMP thus allowing an accurate determination of phosphodiesterase activity in any preparations.
已开发出一种使用固定化吖啶黄素的色谱方法，用于在以[3H] - 环核苷酸为底物的3'-5'环核苷酸磷酸二酯酶直接测定中，从未反应的环核苷酸中分离出其相应的5'-核苷酸。该方法基于黄素和吲哚环之间所谓的电荷转移重叠识别，能够快速（15 - 20分钟）且灵敏地洗脱[3H] - 5'-核苷酸，回收率高（高达98%）且空白值低，而[3H] - 环核苷酸则在柱上被滞留。通过该方法，利用[14C] - 5'-AMP考虑了嘌呤代谢酶如5'-核苷酸酶、核苷酶和/或脱氨酶形成的一些次级产物，从而能够准确测定任何制剂中的磷酸二酯酶活性。
