Cross-linking of iodine-125-labeled, calcium-dependent regulatory protein to the Ca2+-sensitive phosphodiesterase purified from bovine heart.
The calcium-dependent regulatory protein (CDR).Ca2+ sensitive cyclic nucleotide phosphodiesterase was purified to apparent homogeneity from bovine heart by using ammonium sulfate fractionation, DEAE-ceelulose chromatography, and CDR-Sepharose affinity chromatography. The enzyme was purifed 13 750-fold with a 10% yield and a specific activity of 275 mumol of cAMP min-1 mg-1. The purified enzyme ran as a single band during sodium dodecyl sulfate-polyacrylamide gel electrophoresis with an apparent molecular weight of 57 000. Phosphodiesterase activity was stimulated 10-fold by Ca2+ and CDR with half-maximal activation occurring at 9 ng/assay. [125I]CDR was cross-linked to the purified phosphodiesterase by using dimethyl suberimidate Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the cross-linked products revealed a number of discrete 125I-labeled bands. The molecular weights of the cross-linked products indicate that the stoichiometry of the phosphodiesterase complex is A2C2, where A is the phosphodiesterase catalytic subunit and C is the calcium-dependent regulatory protein.
D000818:Animals; D002118:Calcium / Q000494:pharmacology*; D002135:Calcium-Binding Proteins / Q000378:metabolism*; D002147:Calmodulin / Q000302:isolation & purification / Q000378:metabolism*; D002417:Cattle; D003432:Cross-Linking Reagents; D004120:Dimethyl Suberimidate; D046911:Macromolecular Substances; D008970:Molecular Weight; D009206:Myocardium / Q000201:enzymology*; D010727:Phosphoric Diester Hydrolases / Q000302:isolation & purification / Q000378:metabolism*
D002135:Calcium-Binding Proteins; D002147:Calmodulin; D003432:Cross-Linking Reagents; D046911:Macromolecular Substances; D004120:Dimethyl Suberimidate; D010727:Phosphoric Diester Hydrolases; D002118:Calcium