Cyclic 3':5'-nucleotide phosphodiesterase. Purification, characterization, and active form of the protein activator from bovine brain.

A protein activator of cyclic 3’:5’-nucleotide phosphodiesterase from bovine brain has been purified to homogeneity by the criteria of analytical polyacrylamide gel electrophoresis, polyacrylamide gel isoelectric focusing, Sephadex G-100 column chromatography, and analytical ultracentrifugation. The over-all purification was about 1700-fold with a yield of 7%. The molecular weight of the activator is 15,000 as determined by several methods. Other physical properties are: sedrmentatron coefficient (s~,,,~ ) 1.85 S; diffusion coefficient , (D20,w), 1.09 X 1OV cm* per s; partial specific volume (LJ), 0.72 cm3 per g; frictional ratio (f:fo), 1.20; and isoelectric point (PI), 4.3. Amino acid analysis of the acid hydrolyzate of the activator showed high content of aspartic and glutamic acids compared to basic amino acids, in agreement with a low p1 of 4.3. Sulfhydryl groups, cystine, and tryptophan were not detected. The NHz-terminal residue was identified as valine. This, together with the fact that the molecular weight of 15,000 remained unchanged in 6 N guanidine hydrochloride, indicates that the activator is a single polypeptide. The activator binds Ca2+ specifically. A Scatchard plot of data obtained from equilibrium binding revealed four Ca*f binding sites per mole of activator; their dissociation constants ranged from 4 to 18 PM. Stimulation of phosphodiesterase by the activator required a low concentration of Ca*+, and chelation of Ca2+ by ethylene glycol bis(@-aminoethyl ether)-N, N’-tetraacetic acid rendered the activator inactive. The concentration of Ca*+ needed to give halfmaximum activation of phosphodiesterase was 3 pM. Ca2+ alone did not activate phosphodiestersae. These results suggest that the active form of the activator is a Ca2+-activator complex, in agreement with the findings of Teo and Wang (TEo, T. S., AND WANG, J. H. (1973) /. Biol. Chem. 248, 5950). In contrast to the heart activator (TEo, T. S., WANG, T. H., AND WANG, J. H. (1973) J. Biol. Chem. 248, 588), the brain activator was stable in the presence of either 1 mu Mg*+ or 1