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, M., Piscitelli, D., Rossi, P., Geremia, R. (1992). Purification and characterization of a low-Km 3':5'-cyclic adenosine phosphodiesterase from post-meiotic male mouse germ cells. BIOCHIMICA ET BIOPHYSICA ACTA-PROTEIN STRUCTURE AND MOLECULAR ENZYMOLOGY, 1121(1-2), 178-182 [10.1016/0167-4838(92)90352-E].

Purification and characterization of a low-Km 3':5'-cyclic adenosine phosphodiesterase from post-meiotic male mouse germ cells

ROSSI, PELLEGRINO;GEREMIA, RAFFAELE
1992-05-22

Abstract

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.
Pubblicato
Rilevanza internazionale
Articolo
Esperti anonimi
Settore BIO/16
English
Con Impact Factor ISI
Rolipram; Centrifugation, Density Gradient; Animals; Testis; Pyrrolidinones; 3',5'-Cyclic-AMP Phosphodiesterases; Mice; Molecular Weight; Chromatography, Affinity; Mice, Inbred Strains; Phosphodiesterase Inhibitors; Chromatography, DEAE-Cellulose; Chromatography, Gel; Kinetics; Cytosol; 4-(3-Butoxy-4-methoxybenzyl)-2-imidazolidinone; Male; Protein Conformation
Giorgi, M., Piscitelli, D., Rossi, P., Geremia, R. (1992). Purification and characterization of a low-Km 3':5'-cyclic adenosine phosphodiesterase from post-meiotic male mouse germ cells. BIOCHIMICA ET BIOPHYSICA ACTA-PROTEIN STRUCTURE AND MOLECULAR ENZYMOLOGY, 1121(1-2), 178-182 [10.1016/0167-4838(92)90352-E].
Giorgi, M; Piscitelli, D; Rossi, P; Geremia, R
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/2108/65813
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