Cyclic AMP phosphodiesterase (PDE) partially purified from roots of Vigna mungo exhibited optimum activity at pH 5.5 to 6.0 and maximum enzyme activity at 50 degrees C. Levels of PDE activity in roots remained relatively constant from the first to the eleventh day after germination; on the twelfth day there was a 400% increase in PDE activity. The enzyme was stable for at least 48 hours at 28 degrees C, retaining 92% of its original activity. Plant growth hormones including gibberellic acid, indoleacetic acid and kinetin at 1.0 and 10.0 microM concentrations did not have any significant effect on enzyme activity. Nucleotides tested including cyclic 2'3' AMP, cyclic 2'3' GMP completely abolished enzyme activity at 1.0mM while cyclic 3'5' GMP, cyclic 3'5' GMP, 2'deoxy 5' ATP, 2'deoxy 5'GTP and 5'ADP were also inhibitory to the enzyme. The enzyme was stimulated by Mg2+, Fe2+ and NH4+ while Cu2+ and Fe3+ were inhibitory. Theophylline, caffeine, phosphate, pyrophosphate and EDTA were inhibitory to the enzyme.
1. The activity of cAMP phosphodiesterase (PDE) was studied in a 10,000 g particulate fraction prepared from rat brain. 2. Phospholipase C such as sphingomyelin choline phosphodiesterase (SMase), phosphatidylinositol phosphodiesterase (PIase) and phosphatidylcholine phosphohydrolase (PCase) were used to deplete phospholipid(s) from the particulate fraction and their effects on PDE activity were investigated. 3. Treatment with SMase or PIase did not affect PDE activity whereas treatment with PCase resulted in inhibition. 4. It was also found that the PCase used not only hydrolyzed phosphatidylcholine but also other phospholipids such as phosphatidylethanolamine, phosphatidylserine and sphingomyelin.
Cyclic AMP phosphodiesterase (PDE) activity was assayed in the plasma membrane, mitochondrial and microsomal fractions of rat brain. The specific activity of the enzyme was highest in the plasma membrane fraction followed by mitochondrial and then the microsomal fraction. Phosphodiesterase activity of all three fractions was reduced after pretreatment with lecithinase C (PCase) from Clostridium perfringens but less markedly affected by the pretreatment with sphingomyelinase (SMase) from human placenta. The PDE activity of the plasma membrane fraction was more sensitive to PCase treatment compared with the other two particulate fractions, which showed only a slight loss of activity. Temperature seemed to affect PDE activity of the plasma membrane. The enzyme was quite stable at 30 degrees C but its activity dropped by approximately 46% at 37 degrees C after 90 min of incubation. Pretreatment of the plasma membrane at 30 degrees C with PCase at a concentration of more than 5 U caused a marked loss of PDE activity and the decrease in activity reached a plateau at concentrations above 10 U.
Absence of physiological concentrations of extracellular Ca2+ in the Krebs-Henseleit incubation buffer did not affect the ability of 10 nM glucagon (< 5%) to increase hepatocyte intracellular cyclic AMP concentrations, but severely ablated (by approximately 70%) the ability of 10 nM insulin to decrease these elevated concentrations. Cyclic AMP metabolism is determined by production by adenylate cyclase and degradation by cyclic AMP phosphodiesterase (PDE). In the absence of added extracellular Ca2+ (2.5 mM), insulin's ability to activate PDE activity was selectively compromised, showing a failure of insulin to activate two of the three insulin-stimulated activities, namely the 'dense-vesicle' and peripheral plasma-membrane (PPM) PDEs. In the absence of added Ca2+, insulin's ability to inhibit adenylate cyclase activity in intact hepatocytes was decreased dramatically. Vasopressin and adrenaline (+ propranolol) failed to elicit the activation of either the 'dense-vesicle' or the PPM-PDEs. The presence of physiological concentrations of extracellular Ca2+ in the incubation medium is shown to be important for the appropriate generation of insulin's actions on cyclic AMP metabolism.