Cyclic 3′ : 5′-Nucleotide Phosphodiesterase: PURIFICATION, CHARACTERIZATION, AND ACTIVE FORM OF THE PROTEIN ACTIVATOR FROM BOVINE BRAIN

Abstract

Abstract 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: sedimentation coefficient (s20,w), 1.85 S; diffusion coefficient (D20,w), 1.09 x 10-6 cm2 per s; partial specific volume (v), 0.72 cm3 per g; frictional ratio (f:f0), 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 pI of 4.3. Sulfhydryl groups, cystine, and tryptophan were not detected. The NH2-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 Ca2+ binding sites per mole of activator; their dissociation constants ranged from 4 to 18 µm. Stimulation of phosphodiesterase by the activator required a low concentration of Ca2+, and chelation of Ca2+ by ethylene glycol bis(β-amino-ethyl ether)-N ,N'-tetraacetic acid rendered the activator inactive. The concentration of Ca2+ needed to give half-maximum activation of phosphodiesterase was 3 µm. 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) J. 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 mm Mg2+ or 1 mm EDTA. Staining of the activator with periodate-Schiff reagent or pyronine Y after electrophoresis on acrylamide gel was negative. Bovine brain phosphodiesterase(s) hydrolyzes adenosine 3' : 5'-monophosphate (cyclic AMP) and guanosine 3' : 5'-monophosphate (cyclic GMP). At millimolar substrate concentration the rate of hydrolysis of cyclic AMP was greater than that of cyclic GMP, whereas at micromolar concentration, the reverse was found. Although the activator stimulated the hydrolysis of both nucleotides, it stimulated cyclic AMP hydrolysis more, regardless of the concentration used. The stimulation of the hydrolysis of both substrates was greater at micromolar concentrations than at millimolar. Caffeine inhibited phosphodiesterase both in the presence and absence of the activator; this inhibition was not reversed by increased concentrations of the activator.