Characterization of cyclic-nucleotide phosphodiesterase activities in resting and N-formylmethionylleucylphenylalanine-stimulated human neutrophils.
Cyclic nucleotide phosphodiesterase activities in human neutrophils were characterized. Neutrophil sonicates exhibited high-affinity and low-affinity cAMP phosphodiesterase activities, with apparent Km values of 1.9 microM and 112 microM, respectively. No cGMP phosphodiesterase activity was detected. Approx. 70% of cAMP phosphodiesterase activity measured at 1 microM cAMP was present in the soluble subcellular fraction, and the remainder was localized in the particulate fraction. Chromatography of the soluble subcellular fraction on DE-52 ion-exchange resin yielded a low-affinity cAMP phosphodiesterase activity and a high-affinity cAMP phosphodiesterase activity. The soluble high-affinity cAMP phosphodiesterase activity exhibited moderate calmodulin sensitivity. After incubation of intact neutrophils with N-formylmethionylleucylphenylalanine (fMet-Leu-Phe), a 25-30% increase in the activity of the high-affinity cAMP phosphodiesterase activity was observed in the sonicate and in the soluble fraction. Maximal increases were achieved after 2 min of incubation and the increases persisted for at least 10 min. The increase in activity was independent of calmodulin and guanine nucleotide regulatory proteins. These results indicate that a soluble high-affinity cAMP phosphodiesterase comprises the majority of phosphodiesterase activity in neutrophils and that increases in this activity may contribute to the regulation of cAMP levels in neutrophils during activation.
D015105:3',5'-Cyclic-AMP Phosphodiesterases / Q000097:blood*; D015106:3',5'-Cyclic-GMP Phosphodiesterases / Q000097:blood; D000328:Adult; D002147:Calmodulin / Q000097:blood / Q000494:pharmacology; D002852:Chromatography, Ion Exchange; D006160:Guanosine Triphosphate / Q000494:pharmacology; D006165:Guanylyl Imidodiphosphate / Q000494:pharmacology; D006801:Humans; D007700:Kinetics; D008213:Lymphocyte Activation; D009240:N-Formylmethionine Leucyl-Phenylalanine / Q000494:pharmacology*; D009504:Neutrophils / Q000032:analysis / Q000201:enzymology*
D002147:Calmodulin; D006165:Guanylyl Imidodiphosphate; D009240:N-Formylmethionine Leucyl-Phenylalanine; D006160:Guanosine Triphosphate; D015105:3',5'-Cyclic-AMP Phosphodiesterases; D015106:3',5'-Cyclic-GMP Phosphodiesterases