Cyclic 3':5'-nucleotide phosphodiesterase. Ca2+ confers more helical conformation to the protein activator.
The ultraviolet spectrum of a protein activator of cyclic nucleotide phosphodiesterase and adenylate cyclase purified to homogeneity from bovine brain displayed absorption peaks at 252, 259, 265, 269, and 277 nm. The activator contained no phosphate and did not serve as a substrate for cyclic adenosine 3':5'-monophosphate- or cyclic guanosine 3':5'-monophosphate-dependent protein kinases. The activator binds Ca2+, and the active form appears to be a Ca2+ activator complex (Lin, Y.M., Liu, Y.P., and Cheung, W.Y. (1974) J. Biol. Chem. 249, 4943-4954). Optical rotatory dispersion measurement showed that the Ca2+-free activator exhibited a reduced mean residue rotation ([m']231) of -5700, corresponding to 39% of helical content. In the presence of Ca2+, the [m']231 was increased to -7500, corresponding to 57% of helical content. The Ca2+ -induced conformational change was corroborated by a chemical method. In the presence of Ca2+, the activator was more resistant to trypsin inactivation, presumably because proteins with more helical structures are more resistant to tryptic attack. The activator is rich in aspartate and glutamate. Chemical block of some of the carboxyl groups with glycine ethyl ester or methoxyamine diminished the [m']231 of the activator and its activity, suggesting that blockade of some of the carboxyl groups in the activator unfolded the molecule, leading to a loss of activity. We conclude that Ca2+, which confers more helical structure to the activator, converts the inactive, less helical structure to the active, more helical structure, and that chemical modification of the activator leading to unfolding of the molecule abolishes its biological activity.
D015105:3',5'-Cyclic-AMP Phosphodiesterases / Q000378:metabolism*; D000818:Animals; D001921:Brain / Q000201:enzymology; D002118:Calcium / Q000494:pharmacology*; D002417:Cattle; D000242:Cyclic AMP / Q000494:pharmacology; D006152:Cyclic GMP / Q000494:pharmacology; D004533:Egtazic Acid / Q000494:pharmacology; D004789:Enzyme Activation / Q000187:drug effects; D007700:Kinetics; D009419:Nerve Tissue Proteins / Q000502:physiology*; D009905:Optical Rotatory Dispersion; D010727:Phosphoric Diester Hydrolases / Q000378:metabolism*; D011485:Protein Binding; D011487:Protein Conformation; D011494:Protein Kinases / Q000378:metabolism*; D013056:Spectrophotometry, Ultraviolet
D009419:Nerve Tissue Proteins; D004533:Egtazic Acid; D000242:Cyclic AMP; D011494:Protein Kinases; D010727:Phosphoric Diester Hydrolases; D015105:3',5'-Cyclic-AMP Phosphodiesterases; D006152:Cyclic GMP; D002118:Calcium