An improved assay of cyclic 3',5'-nucleotide phosphodiesterases with QAE-Sephadex columns.
A method for the assay of cyclic nucleotide phosphodiesterases is described which employs chromatographical separation of cyclic nucleotides and nucleosides on QAE A-25 Sephadex columns and 5'-nucleotidase as an auxiliary enzyme. The assay allows quantitative recovery of adenosine, guanosine and their metabolites from the anion exchanger and thus is suitable for use in crude phosphodiesterase preparations containing purine catabolizing enzymes. In comparative studies, this method was found to be considerably more sensitive than previous reported methods because of lower assay blanks and higher recoveries of the nucleoside catabolites. The method is suitable for kinetic analysis of crude enzyme preparations from micromolar to millimolar substrate concentrations.
D015105:3',5'-Cyclic-AMP Phosphodiesterases / Q000032:analysis*; D015106:3',5'-Cyclic-GMP Phosphodiesterases / Q000032:analysis*; D000818:Animals; D001921:Brain / Q000201:enzymology; D002852:Chromatography, Ion Exchange / Q000379:methods; D007700:Kinetics; D008297:Male; D051381:Rats
D015105:3',5'-Cyclic-AMP Phosphodiesterases; D015106:3',5'-Cyclic-GMP Phosphodiesterases