Purification and characterization of a low-Km 3':5'-cyclic adenosine phosphodiesterase from post-meiotic male mouse germ cells.
We describe the purification and the study of the kinetic and hydrodynamic properties of a 'low Km' cAMP phosphodiesterase specifically expressed in haploid male germ cells of the mouse. The enzyme has been purified approx. 13,000-fold with respect to the activity in total cell homogenate. The purified enzyme hydrolyzed specifically cAMP with a Km of 3.3 microM and with a Vmax of 10.5 mumol of cAMP hydrolyzed/min per mg of protein. The hydrolytic activity was neither stimulated nor inhibited by cGMP, whereas it was inhibited by RO 20-1724 and Rolipram. The enzyme showed a Stokes radius of 3.8 nm and a sedimentation coefficient of 3.1 S, corresponding to a native molecular mass of 50 kDa and a frictional ratio of 1.53. Sodium dodecyl sulphate polyacrylamide gel electrophoresis analysis of sucrose gradient fractions of the purified enzyme showed a major band of 43 kDa copeaking with enzyme activity.
Giorgi
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M
Dipartimento di Sanità Pubblica e Biologia Cellulare, Università degli studi di Roma Tor Vergata, Italy.
Piscitelli
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Rossi
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Geremia
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D015105:3',5'-Cyclic-AMP Phosphodiesterases / Q000302:isolation & purification* / Q000378:metabolism*; D012368:4-(3-Butoxy-4-methoxybenzyl)-2-imidazolidinone / Q000494:pharmacology; D000818:Animals; D002499:Centrifugation, Density Gradient; D002846:Chromatography, Affinity; D002848:Chromatography, DEAE-Cellulose; D002850:Chromatography, Gel; D003600:Cytosol / Q000201:enzymology; D007700:Kinetics; D008297:Male; D051379:Mice; D008815:Mice, Inbred Strains; D008970:Molecular Weight; D010726:Phosphodiesterase Inhibitors / Q000494:pharmacology; D011487:Protein Conformation; D011760:Pyrrolidinones / Q000494:pharmacology; D020889:Rolipram; D013737:Testis / Q000201:enzymology*
D010726:Phosphodiesterase Inhibitors; D011760:Pyrrolidinones; D012368:4-(3-Butoxy-4-methoxybenzyl)-2-imidazolidinone; D015105:3',5'-Cyclic-AMP Phosphodiesterases; D020889:Rolipram