CHARACTERIZATION OF 3' 5' CYCLIC-NUCLEOTIDE PHOSPHODIESTERASE ACTIVITIES OF MOUSE NEUROBLASTOMA N18TG2 CELLS/

Characterization of 'low K(m)' 3':5' cyclic nucleotide phosphodiestera se activities (PDE) expressed in mouse N18TG2 neuroblastoma cells is r eported. At least 3 peaks of activity were isolated by DEAE chromatogr aphy, none of which was calcium-calmodulin stimulated and CGMP stimula ted or inhibited. A first peak elutes at 200 mM sodium acetate; it spe cifically hydrolyzes cGMP with a K(m) of 4.7 muM and shows sensitivity to zaprinast [M&B 22948] (1.8 muM). A second peak eluting at 410 mM s odium acetate hydrolyzes both cyclic nucleotides. A third peak, specif ic for cAMP hydrolysis, elutes at 580 mM sodium acetate, has a K(m) of 3.2 muM and is sensitive to RO 20 1724 (7.6 muM) and rolipram (2 muM) . Hydrodynamic analysis showed for the first peak a Stokes radius of 5 .3 nm with a sedimentation coefficient of 8.1 S, a frictional ratio (f /f(o)) of 1.41 and a native molecular mass of 182 kDa. The same analys is for peak 3 showed a Stokes radius of 4.1 nm with a sedimentation co efficient of 3.2 S, a frictional ratio of 1.63 and a native molecular mass of 56 kDa. The biochemical features reported for the enzyme eluti ng in the first peak, and its cGMP-binding activity stimulated by inhi bitors of phosphodiesterase activity, demonstrate that it belongs to t he PDE V subfamily; on the other hand the cAMP specific enzyme eluting in the third peak can be assigned to the 'RO 20 1724 inhibited' form. The significance of these findings is discussed in relation to the fu nctional characteristics of the N18TG2 cell line.