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.