Abstract
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.
Highlights
From the $Laboratory of Cellular Metabolism and the
Homogeneous preparations of bovineandovineenzymes(purified -5,000- and 2,000-fold,respectively)haddifferent specific activities, their substrate affinities and activationby calmodulin
A large portion of the calcineurin present in brain preparations can be removed by using Sepharose which has the dye Cibacronblue bound to i(6t,7) or by use of pH-dependent elutionfrom anion exchange gels [12]
Summary
Isoelectric point wasfound in bovine and ovine brain. Purification of Phosphodiesterase-For most preparations, whole Whether these represent isozymes, perhaps localized brains frozen a t a local abattoir were used. (three 10-s bursts at “Lo” setting with 10-s intervals between) in 4 mined The existenceof these two otherwise versyim- volumes (per weight of tissue) of mM sodium acetate, pH 6.8, ilar formsof the enzyme has apparently not been pre- containing 2 mMMgC12, 1 mM NaN3, and 0.2 mM EGTA.’. The gel waswashed with 1.6bedvolumes of bufferA followed by 0.6 bed volume of buffer A containing 10% glycerol and adjusted t o p H 5.45 with acetic acid.. With the use of these two differently substituted calmodulin-Sepharose preparations, volumes of columns I and I1 (8to 10 ml/kg of tissue) and flow rates through them were equal. Application of the enzyme solution was followed by addition of 1.2 column volumes of 25 mM BES, pH 7.0, containing 5 mM MgCl,, 1 mM EGTA, 2 mM CaCL, and 250 mM NaCl (buffer B). Purification from -1 kg of brain requires 10 to h and larger preparations canbe completed in 2 days
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