Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.1.4.1 (phosphodiesterase)
18,767 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

3',5'-CAMP phosphodiesterase was partially purified from bovine cerebral cortex. A heat-stable activating factor was separated from the enzyme by chromatography on DEAE-cellulose. The enzyme in crude ammonium sulfate fractions was stimulated by 5 mM CaCl2. This stimulation was reversed by the calcium chelator EGTA. The main phosphodiesterase peak obtained by DEAE-cellulose chromatography was not stimulated by Ca2+. Upon addition of column effluent containing a heat stable factor, Ca2+ activation was restored. Protein activator was inactive when endogenous contaminating Ca2+ was complexed with EGTA. It was concluded that activation of phosphodiesterase requires the presence of both activator and Ca1+. From an analysis of activation of cGMP hydrolysis a kinetic model for the interaction of Ca2+ and protein activator with the phosphodiesterase was developed. Heterotropic cooperativity between the binding of Ca2+ and protein activator to the phosphodiesterase was observed, i.e., Ca1+ decreased the apparent dissociation constant for protein activator and protein activator decreased the apparent dissociation constant for Ca2+.
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PMID:Activation of 3',5'-cyclic adenosine monophosphate phosphodiesterase by calcium ion and a protein activator. 16 41

Microwave irradiation is shown to be a useful method for simultaneously killing chicks and fixing tissues. Renal adenylate cyclase and phosphodiesterase activities were rapidly abolished by microwaving. The increase in chick kidney cyclic adenosine 3',5'-monophosphate (cyclic AMP) content produced by intravenous bovine parathyroid hormone (PTH) injection was much greater in microwaved birds than in those killed by cervical dislocation with subsequent tissue fixation in liquid nitrogen. After PTH injection there was a prolonged elevation of renal cyclic AMP content. At the time of maximum response (2 minutes), log. dose-response curves were linear in the dose range 0.1-10 U. The responses to three different bovine PTH preparations were indistinguishable. Arginine vasopressin, arginine vasotocin, salmon calcitonin and prostaglandin E1 did not affect kidney cyclic AMP content within 2 minutes. Because of its specificity and precision, the method is of use for the in vivo bioassay of PTH. Injection of CaCl2 (20 mumoles) 1 minute before, or conjointly with, bovine PTH inhibited the subsequent increase in kidney cyclic AMP content. The synthetic bovine PTH peptide fragments BPTH (1-34) and BPTH (2-34) both increased chick kidney cyclic AMP content. The use of such fragments allows investigation of the structural requirements of PTH for interaction with the systems regulating cyclic AMP metabolism in the kidney in vivo.
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PMID:Studies in vivo on the effects of parathyroid hormone upon kidney cyclic adenosine 3',5'-monophosphate content using rapid tissue fixation by microwave irradiation. 18 35

A Ca2+-activatable cyclic nucleotide phosphodiesterase from bovine heart can be eluted from a DEAE-cellulose column either in the free form by buffers containing 0.1 mM ethylene glycol bis(beta-aminoethyl ether)N-N,N'N'-tetraacetic acid (EGTA) or as a complex of the enzyme with its protein modulator by buffers containing 0.01 mM CaCl2. A purification procedure based primarily on the significantly different affinity of the two forms of the enzyme for DEAE-cellulose was developed for the purification of the enzyme from bovine heart. The procedure involves ammonium sulfate fractionation, three chromatographic steps on DEAE-cellulose, and gel filtration on Sephadex G-200 with a 5000-fold purification over the crude extract. The purified enzyme has a specific activity of 120 mumol of cAMP/mg/min, can be activated 5-fold by Ca2+, but is only 80% pure as judged by analytical disc gel electrophoresis. The purified enzyme is unstable but can be stabilized by addition of Ca2+ and the protein modulator; this is in contrast to the less pure preparations of Ca2+-activatable phosphodiesterase which are destabilized by the protein modulator in the presence of Ca2+.
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PMID:Purification of a Ca2+-activatable cyclic nucleotide phosphodiesterase from bovine heart by specific interaction with its Ca2+-dependent modulator protein. 18 13

Phosphodiesterase activities for adenosine and guanosine 3':5'-monophosphates (cyclic AMP and cyclic GMP) were demonstrated in particulate and soluble fractions of rat anterior pituitary gland. Both fractions contained higher activity for cyclic GMP hydrolysis than that for cyclic AMP hydrolysis when these activities were assayed at subsaturating substrate concentrations. Addition of protein activator and CaCl2 to either whole homogenate, particulate or supernatant fraction stimulated both cyclic AMP and cyclic GMP phosphadiesterase activities. Almost 80% of cyclic AMP and 90% of cyclic GMP hydrolyzing activities were localized in soluble fraction. Particulate-bound cyclic nucleotide phosphodiesterase activity was completely solubilized with 1% Triton X-100. Detergent-dispersed particulate and soluble enzymes were compared with respect to Ca2+ and activator requirements and gel filtration profiles. Particulate, soluble and partially purified phosphodiesterase activities were also characterized in relation to divalent cation requirements, kinetic behavior and effects of Ca2+, activator and ethyleneglycol-bis-(2-aminoethyl)-N,N'-tetraacetic acid. Gel filtration of either sonicated whole homogenate or the 10500 X g supernatant fraction showed a single peak of activity, which hydrolyzed both cyclic AMP and cyclic GMP and was dependent upon Ca2+ and activator for maximum activity. Partially purified enzyme was inhibited by 1-methyl-3-isobutylxanthine and papaverine with the concentration of inhibitor giving 50% inhibition at 0.4 muM substrate being 20 muM and 24 muM for cyclic AMP and 7 muM and 10 muM for cyclic GMP, respectively. Theophylline, caffeine and theobromine were less effective. The rat anterior pituitary also contained a protein activator which stimulated both pituitary cyclic nucleotide phosphodiesterase(s) as well as activator-deficient brain cyclic GMP and cyclic AMP phosphodiesterases. Chromatography of the sonicated pituitary extract on DEAE-cellulose column chromatography resolved the phosphodiesterase into two fractions. Both enzyme fractions hydrolyzed cyclic AMP and cyclic GMP and had comparable apparent Km values for the two nucleotides. Hydrolysis of cyclic GMP and cyclic AMP by fraction II enzyme was stimulated 6--7-fold by both pituitary and brain activator in the presence of micromolar concentrations of Ca2+.
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PMID:Cyclic nucleotide phosphodiesterases from rat anterior pituitary. Characterization of multiple forms and regulation by protein activator and Ca+. 19 11

1. Continuous recording of cardiac force of contraction, heart rate and coronary flow from isolated perfused hearts of rats was used to study coronary reactions: (a) to cardiostimulation with noradrenaline, CaCl2, or electrically induced tachycardia; (b) to short duration stoppage of coronary inflow (hypoxia). 2. The heart rate was controlled by electrical pacing. Coronary vasodilatation resulted from cardiostimulation or hypoxia. This coronary response was greater at higher heart rates. 3. In parallel experiments administration of noradrenaline to hearts paced at different frequencies resulted in a rate-dependent elevation of adenosine-3',5'-cyclic monophosphate (cyclic AMP). 4. Duration of hypoxia leading to different degrees of reactive hyperaemia did not change the cardiac cyclic AMP levels. 5. Coronary vasodilatation due to increased cardiac metabolism produced by noradrenaline, Ca2+ or tachycardia was enhanced by the phosphodiesterase inhibitors diazoxide and papaverine while it was inhibited during the administration of prostaglandin E2.6. Reactive hyperaemia was unaffected by diazoxide, papaverine or prostaglandin E2. 7. Catecholamine depletion by reserpine did not influence metabolic coronary dilatation nor the reactive hyperaemic responses. 8. We postulate that there are at least two types of coronary reactions: one in response to hypoxia, 'reactive hyperaemia', and another resulting from cardiac hyperactivity, 'metabolic coronary dilatation'. The latter, blocked by prostaglandin E2 and enhanced by diazoxide or papaverine, would be triggered by cyclic AMP while reactive hyperaemia would result from other mechanisms.
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PMID:Coronary reactions to cardiac hyperactivity and to hypoxia in isolated perfused heart of rat. 20 21

The temperature optimum for the positive inotropic response of guinea-pig isolated atria to single submaximal doses of isoprenaline was 25 degrees C. This was well separated from that for rate responses (37.5 degrees C). This separation was not due to changes in catechol-0-methyl-transferase or phosphodiesterase activity since it occurred with orciprenaline alone and in the presence of theophylline. The rate optima for aminophylline, histamine, glucagon, ouabain, calcium chloride and dibutyryl cAMP were essentially the same as for isoprenaline. The temperature-dependences therefore lie at a common ultimate pathway leading to the rate response. The site of temperature-dependence of the inotropic response to isoprenaline is not at the common contractile mechanisms since its optimum differed from those of ouabain and CaCl2. Activity of cAMP and its production were also eliminated as possible sites from differing optima of aminophylline, histamine and dibutyryl cAMP. The temperature-dependence may lie at the beta-adrenoceptor itself, possibly adenyl cyclase. This may be shared by glucagon although tachyphaylaxis made its optimum difficult to determine.
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PMID:Possible sites of temperature-dependent changes in sensitivity of the positive inotropic and chronotropic responses to sympathomimetic amines by comparisons of the temperature optima for a range of agonists. 64 19

A yeast-mycelium (Y-M) transition in Candida albicans was induced by exogenous yeast extract, adenosine, adenosine 5'-monophosphate (AMP), adenosine 5'-diphosphate (ADP), adenosine 3':5' cyclic monophosphate (cAMP) and its analogue N6, O2'-dibutyryl adenosine 3':5'-cyclic monophosphate (dbcAMP) in defined liquid medium at 25 degrees C. Adenosine 5'-triphosphate (ATP) was found to delay germ tube formation in yeast cells, whereas the cAMP phosphodiesterase inhibitors, theophylline and caffeine, induced a Y-M transition. Intracellular and extracellular cyclic AMP levels increased during the yeast-mycelium transition and maximum levels of intracellular cyclic AMP coincided with maximum germ tube formation. Of the many inducers and inhibitors of germ tube and mycelium formation in C. albicans tested, including incubation at 37 degrees C or in the presence of 1.5 mM CaCl2, the calmodulin inhibitor calmidazolium (R24571) added together with CaCl2 induced the highest intra- and extracellular cyclic AMP levels. These results confirm the involvement of cyclic AMP in the yeast-mycelium transition of C. albicans.
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PMID:Effect of nucleosides and nucleotides and the relationship between cellular adenosine 3':5'-cyclic monophosphate (cyclic AMP) and germ tube formation in Candida albicans. 133 93

Experiments have been performed to characterize guinea-pig peritoneal eosinophil cyclic nucleotide phosphodiesterase (PDE) activity and establish whether it is involved in regulating superoxide (.O2-) generation. Eosinophils were found to contain a predominantly membrane-bound cAMP PDE(s) (92.5 +/- 2.4% of total activity) which was resistant to solubilization with Triton X-100 (1%). This particulate PDE exhibited complex kinetics (Km = 1.3 and 31.4 microM) and was unaffected by cGMP (IC50 greater than 100 microM) or CaCl2 (2 mM) + calmodulin (10 units/mL). Little cGMP PDE activity was detected in either the soluble or particulate fractions. Inhibitors of the Ro-20-1724-inhibited (Type IV) cAMP PDE, namely Ro-20-1724 (IC50 = 0.92 +/- 0.43 microM), rolipram (IC50 = 0.20 +/- 0.04 microM) and denbufylline (IC50 = 0.20 +/- 0.01 microM), potently inhibited the particulate cAMP PDE, as did the non-selective inhibitors trequinsin (IC50 = 0.11 +/- 0.02 microM) and AH-21-132 (IC50 = 2.57 +/- 0.02 microM). Eosinophil cAMP PDE was resistant to SK&F 94120 (IC50 greater than 1000 microM), the cGMP-inhibited (Type III) cAMP PDE inhibitor, and the cGMP PDE (Type I) inhibitor, zaprinast, was only weakly active (IC50 = 35.33 +/- 10.74 microM). .O2- release from resting cells was potently inhibited by rolipram (IC50 = 0.05 +/- 0.03 microM) and denbufylline (IC50 = 0.06 +/- 0.04 microM) but surprisingly, in view of its potent cAMP PDE inhibitory activity, was only weakly decreased by trequinsin (IC50 = 8.0 +/- 2.7 microM). AH-21-132 (IC50 greater than 10 microM), SK&F 94120 (IC50 greater than 10 microM) and zaprinast (IC50 greater than 10 microM) were without effect. Rolipram and denbufylline alone exerted little effect on cAMP in intact cells but, in the presence of 10 microM isoprenaline, potently increased intracellular accumulation (EC50 = 0.45 +/- 0.16 and 0.28 +/- 0.08 microM, respectively). Trequinsin and AH-21-132 only weakly enhanced isoprenaline-stimulated cAMP accumulation. Although it induced a marked rise in cAMP only in the presence of isoprenaline, rolipram (50 microM) alone was able to increase the activity ratio of cAMP-dependent protein kinase from 0.24 to 0.84. The results suggest that Ro-20-1724-inhibited cAMP PDE plays a role in regulating eosinophil .O2- generation. The poor correlation between the PDE inhibitory actions of certain compounds and their effectiveness in elevating cAMP and inhibiting .O2- suggests the existence of a barrier impeding access to the enzyme.
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PMID:Characterization of guinea-pig eosinophil phosphodiesterase activity. Assessment of its involvement in regulating superoxide generation. 165 Oct 83

The involvement of rolipram-sensitive phosphodiesterase (PDE IV) in regulation of cardiac contraction was investigated by studying the effect of selective inhibitors (rolipram, denbufylline, Ro 20-1724) on guinea pig left atria contraction. In contrast to milrinone and SK&F 94120 (inhibitors of the cyclic GMP-inhibited PDE, PDE III), (+/-)-rolipram and denbufylline (0.1-30 microM) did not produce any positive inotropic effect in normal (2.5 mM) or elevated (3-3.2 mM) external CaCl2 concentration. In these conditions, Ro 20-1724 produced only a slight but significant increase of contraction over control levels. In the presence of forskolin (an adenylate cyclase activator) or SK&F 94120 (a PDE III inhibitor), which produced an increase of the response to electrical stimulation of approximately 10%, (+/-)-rolipram, denbufylline, and Ro 20-1724 all exerted concentration-dependent positive inotropic effects (mean EC50 values were 20, 25, and 125 nM, respectively, in the presence of forskolin). Rolipram exhibited stereospecificity: the (-)-enantiomer was 10 times more potent than the (+)-enantiomer. Neither preincubation of the atria with atenolol nor pretreatment of the guinea pigs with reserpine significantly modified the effect of PDE IV inhibitors obtained in the presence of forskolin. These data show that in the presence of cyclic AMP-dependent positive inotropic agents, PDE IV inhibitors exert a positive inotropic effect which probably does not involve enhanced catecholamine release from sympathetic nerve endings. This suggests that PDE IV may play a role in regulation of cardiac contraction in physiologic conditions in which the sympathetic outflow produces a stimulation of adenylate cyclase in cardiac cells.
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PMID:Involvement of rolipram-sensitive cyclic AMP phosphodiesterase in the regulation of cardiac contraction. 170 3

Partially permeabilized rat adipocytes with a high responsiveness to insulin were prepared by electroporation and used to study the effect of 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H-7) on insulin actions in adipocytes. H-7 is a well-documented inhibitor of several protein kinases, including protein kinase C; however, it does not rapidly enter adipocytes protected with the intact plasma membrane. The cells were suspended in Buffer X [4.74 mM NaCl, 118.0 mM KCl, 0.38 mM CaCl2, 1.00 mM EGTA, 1.19 mM Mg2SO4, 1.19 mM KH2PO4, 25.0 mM Hepes/K, 20 mg/ml bovine serum albumin, and 3 mM pyruvate/Na, pH 7.4] and electroporated six times with a Gene-Pulser (from Bio-Rad) set at 25 microF and 2 kV/cm. In cells electroporated as above, insulin stimulated (a) membrane-bound, cAMP phosphodiesterase approximately 2.6-fold when the hormone concentration was 10 nM and (b) glucose transport activity approximately 4.5-fold when the hormone concentration was raised to 100 nM. H-7 strongly inhibited the actions of insulin on both glucose transport (apparent Ki = 0.3 mM) and cAMP phosphodiesterase (apparent Ki = 1.2 mM) in electroporated adipocytes. H-7 also inhibited lipolysis in adipocytes; the apparent Ki value for the reaction in intact cells was 0.45 mM, and that in electroporated cells was 0.075 mM. It is suggested that a certain protein kinase or kinases that are significantly sensitive to H-7 may be involved in the insulin-dependent stimulation of glucose transport and that of phosphodiesterase. However, protein kinase C (or Ca2+/phospholipid-dependent protein kinase) may not be involved, at least, in the hormonal action on phosphodiesterase since the apparent Ki value of H-7 for the reaction is too high.
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PMID:Evidence that protein kinase C may not be involved in the insulin action on cAMP phosphodiesterase: studies with electroporated rat adipocytes that were highly responsive to insulin. 184 37


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