Purification of cAMP-specific phosphodiesterase from rat heart by affinity chromatography on immobilized rolipram.
Affinity chromatography on a cAMP-specific phosphodiesterase inhibitor related to Rolipram, immobilized to AH Sepharose allowed to perform an efficient purification of the cAMP-specific phosphodiesterase isoenzyme from rat heart cytosol (102-fold purification with a 35% yield in a single step). This affinity chromatography involved a biospecific interaction since a 2 mM cAMP elution step at 30 degrees C was necessary for releasing the cAMP specific form tightly bound on the affinity gel. The cAMP eluate fraction exhibited a high specificity towards cAMP (cAMP/cGMP hydrolysis ratio 5-10), a marked sensitivity to Rolipram inhibition and could be resolved in two cAMP-specific, highly Rolipram-sensitive peaks of pI 6.7 and 4.8 by IEF on polyacrylamide gel plates. Protein stain of the IEF gel revealed a single band at pI 6.7.
D015105:3',5'-Cyclic-AMP Phosphodiesterases / Q000302:isolation & purification*; D000818:Animals; D002846:Chromatography, Affinity / Q000379:methods; D000242:Cyclic AMP / Q000378:metabolism; D006152:Cyclic GMP / Q000378:metabolism; D007525:Isoelectric Focusing; D009206:Myocardium / Q000201:enzymology*; D011760:Pyrrolidinones / Q000378:metabolism*; D051381:Rats; D011919:Rats, Inbred Strains; D020889:Rolipram
D011760:Pyrrolidinones; D000242:Cyclic AMP; D015105:3',5'-Cyclic-AMP Phosphodiesterases; D006152:Cyclic GMP; D020889:Rolipram