Isolation of similar rolipram-inhibitable cyclic-AMP-specific phosphodiesterases from rat brain and heart.
A cyclic AMP phosphodiesterase form of rat brain cytosol was purified by means of affinity chromatography on an immobilized analog of the specific inhibitor rolipram, followed by an exclusion chromatography step. The resulting preparation presented two protein bands in polyacrylamide gel electrophoresis, both with phosphodiesterase activity. Kinetics of cyclic AMP hydrolysis by the purified enzyme proved of the Michaelis type, with a Km of 3 microM, while hydrolysis of cyclic GMP displayed anomalous negatively cooperative kinetics. At micromolar concentrations, this enzyme from hydrolyzed highly specifically cyclic AMP (50-fold faster than cyclic GMP). Cyclic GMP proved a poor competitor of cyclic AMP hydrolysis (Ki 1.04 mM). The neurotropic compound, rolipram, strongly inhibited the enzyme, in a competitive manner (Ki 0.9 microM). This enzyme displayed a molecular mass of around 44 kDa as determined by exclusion chromatography, but two molecular masses of 42 kDa and 89 kDa were observable by electrophoresis on a polyacrylamide gradient gel, compatible with an equilibrium between dimeric and monomeric forms. Isoelectric focusing of the preparation gave rise to two activity peaks of pI 4.8 and 6.7, with identical properties, probably representing two charge isomers of the same protein. An enzyme prepared from rat heart cytosol by the same techniques as for brain phosphodiesterase isolation shared numerous characteristics with the enzyme of cerebral origin, suggesting identity of the rolipram-sensitive form between the two tissues. Since the rolipram-sensitive form detected in crude brain preparations markedly differs from the above-described isolated enzyme, both by its molecular mass in exclusion chromatography and by its pI, it is suggested that an alteration of the native protein, due to dissociation of putative subunits, occurs during the purification procedure.
Némoz
G
G
Institut National de la Santé et de la Recherche Médicale Unité 205, Laboratoire de Chimie Biologique, Villeurbanne, France.
Moueqqit
M
M
Prigent
A F
AF
Pacheco
H
H
D015105:3',5'-Cyclic-AMP Phosphodiesterases / Q000037:antagonists & inhibitors / Q000302:isolation & purification*; D000818:Animals; D001665:Binding Sites; D001921:Brain / Q000201:enzymology*; D007525:Isoelectric Focusing; D007700:Kinetics; D008297:Male; D008970:Molecular Weight; D009206:Myocardium / Q000201:enzymology*; D010726:Phosphodiesterase Inhibitors / Q000494:pharmacology*; D011760:Pyrrolidinones / Q000494:pharmacology*; D051381:Rats; D011919:Rats, Inbred Strains; D020889:Rolipram
D010726:Phosphodiesterase Inhibitors; D011760:Pyrrolidinones; D015105:3',5'-Cyclic-AMP Phosphodiesterases; D020889:Rolipram