Davis C W, Daly J W
J Cyclic Nucleotide Res. 1979;5(1):65-74.
A rapid, simple, and direct assay for 3',5'-cyclic nucleotide phospho-diesterase activity is based on the effective separation of cyclic AMP, cyclic GMP or cyclic CMP from their corresponding 5'-nucleotides and nucleosides by chromatography on a polyacrylamide-boronate gel. The affinity of the boronate residue for cis-diols results in the retention of 5'nucleotides and nucleosides while 3',5'-cyclic nucleotides are not retained. The coelution of all 5'-nucleotides and nucleosides allows for the accurate assessment of phosphodiesterase activity in preparations contaminated by other purine metabolizing enzymes such as 5'-nucleotidases and nucleotide and nucleoside deaminases. Phosphodiesterase activity assayed by this means yields linear reaction kinetics with respect to time and amount of enzyme protein. Low blank values obtained allow for detection of as little as 2-3% conversion of substrate to product.
一种快速、简单且直接的3',5'-环核苷酸磷酸二酯酶活性测定方法，是基于通过在聚丙烯酰胺-硼酸酯凝胶上进行色谱分析，将环磷酸腺苷（cAMP）、环磷酸鸟苷（cGMP）或环磷酸胞苷（cCMP）与其相应的5'-核苷酸和核苷有效分离。硼酸酯残基对顺式二醇的亲和力导致5'-核苷酸和核苷被保留，而3',5'-环核苷酸不被保留。所有5'-核苷酸和核苷的共洗脱使得能够准确评估被其他嘌呤代谢酶（如5'-核苷酸酶、核苷酸和核苷脱氨酶）污染的制剂中的磷酸二酯酶活性。用这种方法测定的磷酸二酯酶活性在时间和酶蛋白量方面呈现线性反应动力学。所获得的低空白值允许检测低至2-3%的底物向产物的转化。
