Characterization of 3':5' cyclic nucleotide phosphodiesterase activities of mouse neuroblastoma N18TG2 cells.
Characterization of 'low Km' 3':5' cyclic nucleotide phosphodiesterase activities (PDE) expressed in mouse N18TG2 neuroblastoma cells is reported. At least 3 peaks of activity were isolated by DEAE chromatography, none of which was calcium-calmodulin stimulated and cGMP stimulated or inhibited. A first peak elutes at 200 mM sodium acetate; it specifically hydrolyzes cGMP with a Km of 4.7 microM and shows sensitivity to zaprinast [M&B 22948] (1.8 microM). A second peak eluting at 410 mM sodium acetate hydrolyzes both cyclic nucleotides. A third peak, specific for cAMP hydrolysis, elutes at 580 mM sodium acetate, has a Km of 3.2 microM and is sensitive to RO 20 1724 (7.6 microM) and rolipram (2 microM). 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/fo) of 1.41 and a native molecular mass of 182 kDa. The same analysis for peak 3 showed a Stokes radius of 4.1 nm with a sedimentation coefficient 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 eluting in the first peak, and its cGMP-binding activity stimulated by inhibitors of phosphodiesterase activity, demonstrate that it belongs to the 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 functional characteristics of the N18TG2 cell line.
Dipartimento di Scienze e Tecnologie Biomediche e Biometria, Università dell'Aquila, Italy.
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Giorgi
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Caniglia
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Scarsella
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Augusti-Tocco
D015087:2',3'-Cyclic-Nucleotide Phosphodiesterases / Q000037:antagonists & inhibitors / Q000302:isolation & purification / Q000378:metabolism*; D000818:Animals; D001792:Blood Platelets / Q000201:enzymology; D002499:Centrifugation, Density Gradient; D002848:Chromatography, DEAE-Cellulose; D002850:Chromatography, Gel; D006152:Cyclic GMP / Q000378:metabolism; D003600:Cytosol / Q000201:enzymology; D004591:Electrophoresis, Polyacrylamide Gel; D007700:Kinetics; D008168:Lung / Q000201:enzymology; D051379:Mice; D008970:Molecular Weight; D009447:Neuroblastoma / Q000201:enzymology*; D011688:Purinones / Q000494:pharmacology; D051381:Rats; D014407:Tumor Cells, Cultured
D011688:Purinones; D015087:2',3'-Cyclic-Nucleotide Phosphodiesterases; C011145:zaprinast; D006152:Cyclic GMP