Purification of a Ca2+-activatable cyclic nucleotide phosphodiesterase from bovine heart by specific interaction with its Ca2+-dependent modulator protein.
A Ca2+-activatable cyclic nucleotide phosphodiesterase from bovine heart can be eluted from a DEAE-cellulose column either in the free form by buffers containing 0.1 mM ethylene glycol bis(beta-aminoethyl ether)N-N,N'N'-tetraacetic acid (EGTA) or as a complex of the enzyme with its protein modulator by buffers containing 0.01 mM CaCl2. A purification procedure based primarily on the significantly different affinity of the two forms of the enzyme for DEAE-cellulose was developed for the purification of the enzyme from bovine heart. The procedure involves ammonium sulfate fractionation, three chromatographic steps on DEAE-cellulose, and gel filtration on Sephadex G-200 with a 5000-fold purification over the crude extract. The purified enzyme has a specific activity of 120 mumol of cAMP/mg/min, can be activated 5-fold by Ca2+, but is only 80% pure as judged by analytical disc gel electrophoresis. The purified enzyme is unstable but can be stabilized by addition of Ca2+ and the protein modulator; this is in contrast to the less pure preparations of Ca2+-activatable phosphodiesterase which are destabilized by the protein modulator in the presence of Ca2+.
D015087:2',3'-Cyclic-Nucleotide Phosphodiesterases / Q000302:isolation & purification* / Q000378:metabolism; D015105:3',5'-Cyclic-AMP Phosphodiesterases / Q000302:isolation & purification / Q000378:metabolism; D000818:Animals; D002118:Calcium / Q000494:pharmacology*; D002352:Carrier Proteins / Q000378:metabolism*; D002417:Cattle; D004355:Drug Stability; D004789:Enzyme Activation; D007700:Kinetics; D008970:Molecular Weight; D009124:Muscle Proteins / Q000378:metabolism*; D009206:Myocardium / Q000201:enzymology*; D010727:Phosphoric Diester Hydrolases / Q000302:isolation & purification*
D002352:Carrier Proteins; D009124:Muscle Proteins; D015087:2',3'-Cyclic-Nucleotide Phosphodiesterases; D010727:Phosphoric Diester Hydrolases; D015105:3',5'-Cyclic-AMP Phosphodiesterases; D002118:Calcium