Rapid separation and quantitation of 3',5'-cyclic nucleotides and 5'-nucleotides in phosphodiesterase reaction mixtures using high-performance liquid chromatography.
A simple, rapid high-performance liquid-chromatography system for the fractionation and direct quantitation of substrates and products in crude phosphodiesterase reaction mixtures is described. Phosphate buffers and a pellicular anion exchange resin are used at ambient temperature. The method is sensitive, measuring picomoles of products with ultraviolet detection and femtomoles with isotopic measurement, and offers several advantages over the more popular batch sorption and manual methods for measuring phosphodiesterase activity. The time required for analysis, less than 8 min for single substrate reaction mixtures, is a fraction of that required with other chromatographic systems, and precision is +/- 5%. Results of studies with an activatable form of phosphodiesterase demonstrate the accuracy, precision and utility of the procedure for biochemical analyses.
D000241:Adenosine / Q000302:isolation & purification; D000249:Adenosine Monophosphate / Q000032:analysis; D000818:Animals; D001921:Brain / Q000201:enzymology; D002147:Calmodulin; D002417:Cattle; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D008832:Microchemistry; D009712:Nucleotides, Cyclic / Q000302:isolation & purification*; D010727:Phosphoric Diester Hydrolases; D012265:Ribonucleotides / Q000302:isolation & purification*; D013806:Theophylline / Q000302:isolation & purification
D002147:Calmodulin; D009712:Nucleotides, Cyclic; D012265:Ribonucleotides; D000249:Adenosine Monophosphate; D013806:Theophylline; D010727:Phosphoric Diester Hydrolases; D000241:Adenosine