Purification and properties of calmodulin-stimulated phosphodiesterase from mammalian brain.
A new, rapid method for purification of calmodulin-stimulated phosphodiesterase from bovine, ovine, and porcine brain using only DEAE-agarose and calmodulin-Sepharose chromatography is described. Purified enzymes from the three species each exhibited a single polypeptide of Mr approximately 59,000 on gel electrophoresis under denaturing conditions. Proteolysis of ovine and bovine enzymes with alpha-chymotrypsin, however, yielded different peptides, indicating that these proteins differ in primary sequence. Homogeneous preparations of bovine and ovine enzymes (purified approximately 5,000- and 2,000-fold, respectively) had different specific activities, although their substrate affinities and activation by calmodulin (8- to 14-fold activation, Kact approximately 1 nM) were very similar. The total amount in ovine was almost twice that in bovine brain. The hydrodynamic properties of bovine and ovine enzymes were indistinguishable with a Stokes radius of 4.35 nm and s20,w of 5.95 S. The calculated frictional ratios of 1.30 to 1.38 suggest a slightly asymmetric molecule. Equilibrium sedimentation data yielded apparent Mr approximately 57,000 in the presence of 6 M guanidine and 124,000 and 112,000 for the native bovine and ovine enzymes, respectively. In addition to the enzyme that was purified to homogeneity (pI approximately 5.6), a major fraction of calmodulin-activated phosphodiesterase with a lower isoelectric point was found in bovine and ovine brain. Whether these represent isozymes, perhaps localized in different types of cells, or whether one is a post-translationally modified form, remains to be determined. The existence of these two otherwise very similar forms of the enzyme has apparently not been previously recognized.
D015105:3',5'-Cyclic-AMP Phosphodiesterases / Q000302:isolation & purification* / Q000378:metabolism; D000818:Animals; D001921:Brain / Q000201:enzymology*; D002148:Calmodulin-Binding Proteins; D002417:Cattle; D000242:Cyclic AMP / Q000378:metabolism; D006152:Cyclic GMP / Q000378:metabolism; D007700:Kinetics; D046911:Macromolecular Substances; D008970:Molecular Weight; D010749:Phosphoprotein Phosphatases / Q000302:isolation & purification; D012756:Sheep; D013379:Substrate Specificity; D013552:Swine
D002148:Calmodulin-Binding Proteins; D046911:Macromolecular Substances; D000242:Cyclic AMP; D010749:Phosphoprotein Phosphatases; D015105:3',5'-Cyclic-AMP Phosphodiesterases; D006152:Cyclic GMP