Purification and characterization of bovine lung calmodulin-dependent cyclic nucleotide phosphodiesterase. An enzyme containing calmodulin as a subunit.
A rabbit lung cyclic nucleotide phosphodiesterase (PDE) prepared by successive chromatography on DEAE-cellulose and G-200 Sephadex columns in the presence of EGTA was activated by Ca2+ and contained calmodulin (CaM), suggesting that the enzyme exists as a stable CaM X PDE complex (Sharma, R. K., and Wirch, E. (1979) Biochem. Biophys. Res. Commun. 91, 338-344). An enzyme with similar properties was demonstrated to exist in bovine lung extract. C1, a monoclonal antibody previously shown to react with the 60-kDa subunit of bovine brain PDE isozymes (Sharma, R. K., Adachi, A.-M., Adachi, K., and Wang, J. H.) (1984) J. Biol. Chem. 259, 9248-9254), cross-reacted with the lung enzyme. Purification of the lung enzyme by C1 antibody immunoaffinity chromatography rendered the enzyme dependent on exogenous CaM for Ca2+ stimulation. Further purification was achieved by CaM affinity chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of the purified enzyme showed a predominant polypeptide of Mr 58,000 and a minor band of about 50,000. The purified enzyme could be reconstituted into a PDE X CaM complex upon incubation with CaM in the presence of either Ca2+ or EGTA. The reconstituted protein complex did not dissociate in buffers containing 0.1 mM EGTA. Analysis of the purified and reconstituted lung phosphodiesterase by Sephacryl S-300 gel filtration indicated that the lung enzyme is a dimeric protein and that the reconstituted enzyme contained two molecules of calmodulin. Analysis of the reconstituted phosphodiesterase by sodium dodecyl sulfate-polyacrylamide gel electrophoresis also showed it to contain equimolar calmodulin and the enzyme subunit. The CaM antagonists, fluphenazine, compound 48/80, and calcineurin at concentrations abolishing CaM stimulation of bovine brain PDE had little effect on the activity of reconstituted bovine lung phosphodiesterase.
D015087:2',3'-Cyclic-Nucleotide Phosphodiesterases / Q000378:metabolism*; D000818:Animals; D000911:Antibodies, Monoclonal; D002118:Calcium / Q000378:metabolism; D002147:Calmodulin / Q000378:metabolism*; D002148:Calmodulin-Binding Proteins / Q000494:pharmacology; D002417:Cattle; D002848:Chromatography, DEAE-Cellulose; D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D005476:Fluphenazine / Q000494:pharmacology; D007700:Kinetics; D008168:Lung / Q000201:enzymology*; D003189:p-Methoxy-N-methylphenethylamine / Q000494:pharmacology
D000911:Antibodies, Monoclonal; D002147:Calmodulin; D002148:Calmodulin-Binding Proteins; D003189:p-Methoxy-N-methylphenethylamine; D015087:2',3'-Cyclic-Nucleotide Phosphodiesterases; D005476:Fluphenazine; D002118:Calcium