Phosphorylation and characterization of bovine heart calmodulin-dependent phosphodiesterase.
Calmodulin-dependent phosphodiesterase was purified to apparent homogeneity from the total calmodulin-binding fraction of bovine heart in a single step by immunoaffinity chromatography. The isolated enzyme had significantly higher affinity for calmodulin than the bovine brain 60-kDa phosphodiesterase isozyme. The cAMP-dependent protein kinase was found to catalyze the phosphorylation of the purified cardiac calmodulin-dependent phosphodiesterase with the incorporation of 1 mol of phosphate/mol of subunit. The phosphodiesterase phosphorylation rate was increased severalfold by histidine without affecting phosphate incorporation into the enzyme. Phosphorylation of phosphodiesterase lowered its affinity for calmodulin and Ca2+. At constant saturating concentrations of calmodulin (650 nM), the phosphorylated calmodulin-dependent phosphodiesterase required a higher concentration of Ca2+ (20 microM) than the nonphosphorylated phosphodiesterase (0.8 microM) for 50% activity. Phosphorylation could be reversed by the calmodulin-dependent phosphatase (calcineurin), and dephosphorylation was accompanied by an increase in the affinity of phosphodiesterase for calmodulin.
Sharma
R K
RK
Department of Medical Biochemistry, University of Calgary, Alberta, Canada.
D015105:3',5'-Cyclic-AMP Phosphodiesterases / Q000302:isolation & purification / Q000378:metabolism*; D000818:Animals; D001921:Brain / Q000201:enzymology; D002021:Buffers; D002147:Calmodulin / Q000378:metabolism / Q000494:pharmacology; D002417:Cattle; D002846:Chromatography, Affinity; D054677:Cyclic Nucleotide Phosphodiesterases, Type 1; D007527:Isoenzymes / Q000378:metabolism; D007700:Kinetics; D008970:Molecular Weight; D009206:Myocardium / Q000201:enzymology*; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism
D002021:Buffers; D002147:Calmodulin; D007527:Isoenzymes; D011494:Protein Kinases; D015105:3',5'-Cyclic-AMP Phosphodiesterases; D054677:Cyclic Nucleotide Phosphodiesterases, Type 1