A rapid direct assay of 3'-5' cyclic nucleotide phosphodiesterase activity using chromatography on immobilized acriflavin.
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.
D015105:3',5'-Cyclic-AMP Phosphodiesterases / Q000378:metabolism*; D015106:3',5'-Cyclic-GMP Phosphodiesterases / Q000378:metabolism*; D000166:Acridines; D000167:Acriflavine; D000818:Animals; D002147:Calmodulin / Q000502:physiology; D002850:Chromatography, Gel; D000242:Cyclic AMP / Q000032:analysis; D008297:Male; D009712:Nucleotides, Cyclic / Q000032:analysis*; D051381:Rats
D000166:Acridines; D002147:Calmodulin; D009712:Nucleotides, Cyclic; D000167:Acriflavine; D000242:Cyclic AMP; D015105:3',5'-Cyclic-AMP Phosphodiesterases; D015106:3',5'-Cyclic-GMP Phosphodiesterases