Detection of a calmodulin-sensitive cyclic nucleotide phosphodiesterase in rat parotid gland.
Calmodulin coupled to Sepharose has provided a rapid and sensitive means of isolating a cyclic nucleotide phosphodiesterase activity which is stimulated by the calmodulin-Ca2+ complex, from rat parotid gland. Initial experiments established that phosphodiesterase activity sensitive to calmodulin and Ca2+ could not be demonstrated in crude extracts of rat parotid gland or after partial purification of rat parotid phosphodiesterase over DEAE-cellulose. However, it was possible to readily demonstrate the presence of a cyclic nucleotide phosphodiesterase activity regulated by calmodulin if the extracts were first purified by batch ion-exchange chromatography over DEAE-cellulose followed by affinity chromatography with calmodulin coupled to Sepharose. The batch ion-exchange chromatography step removed the major portion of free parotid calmodulin which could compete with calmodulin-coupled Sepharose for the proteins regulated by calmodulin. Thus, by employing an initial chromatography step over DEAE-cellulose to separate phosphodiesterase activity from calmodulin, it was possible to increase the recovery of calmodulin-sensitive phosphodiesterase after affinity chromatrography with calmodulin coupled to Sepharose. This approach should be useful for demonstrating the presence of and for purifying other parotid proteins regulated by calmodulin.
D015087:2',3'-Cyclic-Nucleotide Phosphodiesterases / Q000302:isolation & purification / Q000378:metabolism*; D000818:Animals; D002118:Calcium / Q000378:metabolism; D002135:Calcium-Binding Proteins / Q000378:metabolism*; D002147:Calmodulin / Q000302:isolation & purification / Q000378:metabolism*; D002846:Chromatography, Affinity; D002848:Chromatography, DEAE-Cellulose; D002852:Chromatography, Ion Exchange; D010306:Parotid Gland / Q000201:enzymology* / Q000378:metabolism; D010727:Phosphoric Diester Hydrolases / Q000378:metabolism*; D051381:Rats
D002135:Calcium-Binding Proteins; D002147:Calmodulin; D015087:2',3'-Cyclic-Nucleotide Phosphodiesterases; D010727:Phosphoric Diester Hydrolases; D002118:Calcium