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)

Peptide hormones can stimulate cyclic GMP synthesis through either of two general mechanisms: some peptides activate the cytoplasmic form of guanylate cyclase via a coupling factor called EDRF (endothelium-derived relaxation factor), while others activate the membrane form by interacting directly with an extracellular binding domain of the cyclase molecule itself. We have investigated the mechanism(s) by which crustacean hyperglycemic hormone (CHH), a neuropeptide that regulates energy metabolism in crustaceans, elevates cyclic GMP levels in lobster muscle. Phosphodiesterase inhibitors potentiate the response in intact tissue. This indicates that the primary effect of the peptide is to activate a cyclase rather than inhibit a phosphodiesterase. Methylene blue, a specific inhibitor of the EDRF pathway, does not block the actions of CHH. In addition, nitroprusside, an agent that directly activates the EDRF pathway in vertebrate animals, does not activate guanylate cyclase either in intact or homogenized lobster muscle. This indicates that the EDRF pathway, although prominent in vertebrate muscle, is not found in crustaceans and further suggests that the membrane cyclase is the most likely target of CHH. Membrane and soluble cyclases can be isolated from homogenates of lobster muscle (in a 3.5:1 ratio), and both are stimulated by Mn2+ and inhibited by Ca2+. CHH has no effect on the soluble enzyme. Coupling of CHH receptors to the particulate cyclase, however, remains intact in isolated membranes, thus providing a new model system for the study of receptor/cyclase interactions.
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PMID:Activation of membrane guanylate cyclase by an invertebrate peptide hormone. 170 Jul 84

Previous studies have suggested that the platelet glycoprotein complex GPIIb-IIIa, which is the putative fibrinogen receptor, regulates Ca2+ influx into platelets, possibly operating as a Ca2+ channel. We have used RGD-peptides (peptides containing the sequence Arg-Gly-Asp; disintegrins), isolated from snake venoms, that have a high affinity and specificity for the fibrinogen-binding site of GPIIb-IIIa to address the question of whether blocking this site inhibits Ca2+ movement from the extracellular medium to the cytosol. Using fura-2-loaded human platelets, we found that neither disintegrins nor a monoclonal antibody (M148) to the GPIIb-IIIa complex altered the level of cytosolic Ca2+ obtained when the cells were stimulated with various agonists in the presence of either nominal or 1 mM extracellular Ca2+. In the presence of Mn2+, an ion that quenches fura-2 fluorescence, fura-2-loaded platelets were stimulated with thrombin or ADP. Neither disintegrins nor the monoclonal antibody altered the kinetics or the amount of quenching of fura-2 fluorescence by Mn2+. These data indicate that the binding of ligands to the fibrinogen receptor is not associated with an inhibition of Ca2+ movement through a receptor-operated channel. Furthermore, the disintegrins have no effect on platelet cyclic AMP metabolism in either the presence or the absence of phosphodiesterase inhibitors.
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PMID:Ligands to the platelet fibrinogen receptor glycoprotein IIb-IIIa do not affect agonist-induced second messengers Ca2+ or cyclic AMP. 216

A cyclic GMP-stimulated cyclic nucleotide phosphodiesterase was purified to near homogeneity from the 150,000 g supernatant fraction of human platelets by a combination of DEAE-cellulose chromatography and cyclic GMP affinity chromatography. Overall purification was about 7400-fold with a 10% to 15% recovery of activity. On NaDodSO4-containing polyacrylamide gels, the purified enzyme migrates as a single band Mr = 105,000. Phosphodiesterase activity co-migrates with the protein band on native polyacrylamide gels. Both Mg2+ and Mn2+ support the activity of this phosphodiesterase. The enzyme hydrolyzes both cyclic AMP and cyclic GMP with similar maximal rates. The hydrolysis of both nucleotides exhibits positive homotropic cooperativity with S0.5 values of 50 +/- 12 microM for cyclic AMP and 35 +/- 15 microM for cyclic GMP and Hill coefficients of 1.2 to 1.5 for both nucleotides. Low levels of cyclic GMP stimulate the rate of cyclic AMP hydrolysis from 3- to 10-fold. The activity of this phosphodiesterase is not stimulated by the calcium binding protein, calmodulin. The cyclic GMP stimulation of cyclic AMP hydrolysis by this phosphodiesterase may provide a possible regulatory link between the metabolism of these two nucleotides in platelets.
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PMID:Purification and characterization of a cyclic GMP-stimulated cyclic nucleotide phosphodiesterase from the cytosol of human platelets. 216 75

DNA polymerase I (Pol I) is an enzyme of DNA replication and repair containing three active sites, each requiring divalent metal ions such as Mg2+ or Mn2+ for activity. As determined by EPR and by 1/T1 measurements of water protons, whole Pol I binds Mn2+ at one tight site (KD = 2.5 microM) and approximately 20 weak sites (KD = 600 microM). All bound metal ions retain one or more water ligands as reflected in enhanced paramagnetic effects of Mn2+ on 1/T1 of water protons. The cloned large fragment of Pol I, which lacks the 5',3'-exonuclease domain, retains the tight metal binding site with little or no change in its affinity for Mn2+, but has lost approximately 12 weak sites (n = 8, KD = 1000 microM). The presence of stoichiometric TMP creates a second tight Mn2+ binding site or tightens a weak site 100-fold. dGTP together with TMP creates a third tight Mn2+ binding site or tightens a weak site 166-fold. The D424A (the Asp424 to Ala) 3',5'-exonuclease deficient mutant of the large fragment retains a weakened tight site (KD = 68 microM) and has lost one weak site (n = 7, KD = 3500 microM) in comparison with the wild-type large fragment, and no effect of TMP on metal binding is detected. The D355A, E357A (the Asp355 to Ala, Glu357 to Ala double mutant of the large fragment of Pol I) 3',5'-exonuclease-deficient double mutant has lost the tight metal binding site and four weak metal binding sites. The binding of dGTP to the polymerase active site of the D355A,E357A double mutant creates one tight Mn2+ binding site with a dissociation constant (KD = 3.6 microM), comparable with that found on the wild-type enzyme, which retains one fast exchanging water ligand. Mg2+ competes at this site with a KD of 100 microM. It is concluded that the single tightly bound Mn2+ on Pol I and a weakly bound Mn2+ which is tightened 100-fold by TMP are at the 3',5'-exonuclease active site and are essential for 3',5'-exonuclease activity, but not for polymerase activity. Additional weak Mn2+ binding sites are detected on the 3',5'-exonuclease domain, which may be activating, and on the polymerase domain, which may be inhibitory. The essential divalent metal activator of the polymerase reaction requires the presence of the dNTP substrate for tight metal binding indicating that the bound substrate coordinates the metal.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Metal binding to DNA polymerase I, its large fragment, and two 3',5'-exonuclease mutants of the large fragment. 220 84

The aim of this study was to establish the mechanism by which adrenalectomy promotes the antilipolytic effect of the adenosine analog (-)-N6-(R-phenyl-isopropyl)adenosine (R-PIA) in rat fat cells. This action of adrenalectomy was not specific for R-PIA, since it was also observed with nicotinic acid and was prevented by phosphodiesterase inhibitors. In contrast, the inhibitory effect of R-PIA and nicotinic acid toward isoproterenol-stimulated cAMP accumulation was unaltered by adrenalectomy regardless of whether phosphodiesterase inhibitors were present. Whatever the conditions used, however, the cAMP levels in adrenalectomized rat adipocytes were one quarter to one third of those in sham-operated rats and remained below the limit over which variations in cAMP had no more influence in lipolysis. Both total and particulate low Km cAMP phosphodiesterase activities per adipocyte were decreased in adrenalectomized rats, but the stimulatory responses of the particulate enzyme to R-PIA remained unchanged. Pertussis toxin-catalyzed ADP ribosylation studies revealed a marked decrease in the total amount of the alpha-subunits of Go and the adenylate cyclase inhibitory regulatory protein Gi after adrenalectomy. However, the inhibitory dose-response curves of adenylate cyclase to R-PIA, nicotinic acid, GTP, guanylylimidodiphosphate, and guanosine 5'-O-(3-thiotriphosphate) were unaltered by adrenalectomy, indicating that the inhibitory function of Gi is unimpaired by adrenalectomy. Lastly, adrenalectomy resulted in a 60% reduction of the Mn2+-stimulated adenylate cyclase activity/adipocyte, which indicates that adrenalectomy causes a defect in adenylate cyclase catalytic activity. Thus, enhanced antilipolytic effects of R-PIA induced by adrenalectomy do not involve increased function of the adenosine receptor Gi-coupled adenylate cyclase inhibitory pathway, but are related to abnormally low intracellular cAMP levels due to defective adenylate cyclase catalytic activity.
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PMID:Role of adenosine 3',5'-monophosphate and the Ri-receptor Gi-coupled adenylate cyclase inhibitory pathway in the mechanism whereby adrenalectomy increases the adenosine antilipolytic effect in rat fat cells. 246 35

[3H]LY186126, an analogue of the cardiotonic agent indolidan, was shown to bind reversibly and with high affinity (Kd = 4 nM) to a single class of binding sites within canine myocardial vesicles. Binding site density measured in various cardiac membrane fractions correlated well with Ca2+-ATPase activity (r = 0.94; p less than 0.01), but not with Na+,K+-ATPase or azide sensitive ATPase, indicating a localization of these sites within sarcoplasmic reticulum membranes. Divalent cations were required for binding and displayed the following order of activation: Zn2+ greater than Mn2+ greater than Mg2+ greater than Ca2+. Differential activation of [3H]LY186126 binding by various divalent cations was due to alterations in binding site density, rather than affinity. cGMP and selective inhibitors of type IV membrane-bound phosphodiesterase (SR-PDE), for example, indolidan, milrinone, imazodan, and enoximone, selectively displaced bound [3H]LY186126 caffeine, theophylline, and rolipram were relatively impotent as inhibitors of radiolabel binding. Kd values from displacement curves were highly correlated with IC50 values for inhibition of SR-PDE (r = 0.92; p less than 0.001). In addition, Kd values correlated well with published ED50 values for increases in cardiac contractility in pentobarbital-anesthetized dogs (r = 0.94; p less than 0.001). The results support the hypothesis that [3H]LY186126 labels the pharmacological receptor for the class of positive inotropic agents characterized as isozyme-selective phosphodiesterase inhibitors. Furthermore, the data suggest that the identity of the site labeled by [3H]LY186126 is SR-PDE, the type IV isozyme of cardiac phosphodiesterase located in the sarcoplasmic reticulum.
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PMID:Characterization and pharmacological relevance of high affinity binding sites for [3H]LY186126, a cardiotonic phosphodiesterase inhibitor, in canine cardiac membranes. 254 18

We have investigated the role of Ca2+ and calmodulin in the stimulation of cGMP formation by mouse Leydig cells in response to rat atriopeptin-II (rAP-II). Removal of extracellular Ca2+ had no influence on the levels of cGMP accumulated by the cells stimulated with rAP-II. The amounts of testosterone produced by unstimulated and rAP-II-stimulated cells were, however, reduced by 50% in the absence of Ca2+ from the incubation medium. Addition of ionomycin to the Leydig cells led to a dose-related inhibition of rAP-II-stimulated cGMP formation, but the basal cGMP level was not affected. These experiments were carried out in the presence of a phosphodiesterase inhibitor. The inhibitory effect of ionomycin was absolutely dependent upon the presence of Ca2+ in the medium. The guanylate cyclase activity required the presence of a cation, and Mn2+, Mg2+, or Ca2+ could function as the required cation. There was no direct inhibition of the cyclase activity by Ca2+ up to as high a concentration as 8 mM. Furthermore, three structurally unrelated calmodulin antagonists, W7, trifluoperazine, and calmidazolium, but not W5, caused a dose-related inhibition of rAP-II-stimulated cGMP accumulation by the cells. The inhibitory effect of calmodulin antagonists was not exerted directly at the level of guanylate cyclase activity, since the particulate enzyme was not inhibited by any of these drugs. We conclude, therefore, that extracellular Ca2+ is not essential for rAP-II-mediated stimulation of cGMP formation by mouse Leydig cells, at least under the short term incubation conditions used. An excessive ionophoretic influx of Ca2+ into the cells impairs the ability of rAP-II to stimulate cGMP formation. Therefore, it appears that a finely regulated level of intracellular Ca2+ is required for optimal activation of atrial natriuretic peptide-responsive guanylate cyclase in mouse Leydig cells, and calmodulin plays an important role in this process.
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PMID:The role of Ca2+ and calmodulin in the regulation of atrial natriuretic peptide-stimulated guanosine 3',5'-cyclic monophosphate accumulation by isolated mouse Leydig cells. 254 43

Patients with pseudohypoparathyroidism type Ia have resistance to multiple hormones because of deficient activity of the stimulatory guanine nucleotide-binding protein (Gs) that couples membrane receptors to activation of adenylate cyclase. However, in a subset of patients with pseudohypoparathyroidism who have resistance to multiple hormones yet possess normal erythrocyte membrane Gs activity, the biochemical abnormality responsible for hormone resistance has remained undefined. Cultured skin fibroblasts were derived from a patient with this atypical form of pseudohypoparathyroidism. In the patient's fibroblast membranes, adenylate cyclase stimulation mediated by Gs after fluoride ion treatment produced only 52% of normal activity, yet fibroblast membrane Gs activity measured by cyc- complementation was normal. Activation of the catalytic unit of adenylate cyclase with manganese produced 49% of normal activity; manganese plus forskolin produced 54% of normal adenylate cyclase activity. beta-Adrenergic receptor coupling to Gs and phosphodiesterase activity were normal. A defect in the catalytic unit of adenylate cyclase can account for these results and may be a mechanism for clinical resistance to multiple hormones that act through adenylate cyclase.
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PMID:New form of pseudohypoparathyroidism with abnormal catalytic adenylate cyclase. 254 94

The modification of Klenow fragment of DNA polymerase I E. coli was investigated by the affinity reagents d(Tp)2C[Pt2+(NH3)2OH](pT)7 and d(pT)2pC[Pt2+(NH3)2OH](pT)7. The template binding site of the enzyme was modified by these reagents in the presence of NaF (5 mM), which inhibits selectively the 3'----5'-exonuclease activity of the enzyme and therefore prevents the reagent from degradation. NaCN destroyed covalent bonds between reagents and enzyme, restoring activity of the Klenow fragment. The affinity of different ligands (inorganic phosphate, nucleoside monophosphates, oligonucleotides) to the template binding site of Klenow fragment was estimated. Minimal ligands capable to bind with the template site were shown to be triethylphosphate (Kd 290 microM) and phosphate (Kd 26 microM). Ligand affinity increases by the factor 1.76 per an added (monomer unit from phosphate to d(pT) and then for oligonucleotides d(Tp)nT (n 1 to 19-20). At n greater than 19-20, the ligand affinity remained constant. The complete ethylation of phosphodiester groups lowers affinity of the oligothymidylates to the enzyme by approximately 10 times, and comparable decrease of Pt2+-oligonucleotide affinity to polymerase is caused by the absence of Mn2+-ions. The data obtained led to suggestion that one Me2+-dependent electrostatic contact of the template phosphodiester group with the enzyme takes place (delta G = -1.45...-1.75 kcal/mole). Formation of a hydrogen bond with the oxygen atom of P = O group of the same template phosphate is also assumed (delta G = -4.8...-4.9 kcal/mole). Other template internucleotide phosphates do not interact with the enzyme but the bases of oligonucleotides take part in hydrophobic interactions with the template binding site. Gibbs energy changes by -0.34 kcal/mole when the template is lengthened by one unit.
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PMID:[Klenow fragment of DNA-polymerase I from E. coli. III. The role of internucleotide phosphate groups of the matrix in its binding with the enzyme]. 266 77

Release of the neuropeptide egg-laying hormone (ELH) from Aplysia bag cell neurons augments the endogenous bursting pacemaker activity of neuron R15. We have studied the ionic mechanisms underlying the effect of ELH in voltage-clamped R15 neurons. Both electrical discharge of the bag cells, which releases endogenous ELH, and application of synthetic ELH on cell R15 result in an increase in two discrete ionic currents. One of these currents activates with hyperpolarization, reverses near the K+ equilibrium potential, is sensitive to the external K+ concentration, and is blocked by addition of 5 mM Rb+ or 1 mM Ba2+ to the bathing medium. This current appears to be identical to the inwardly rectifying K+ current IR. The other current activates with depolarization and is blocked by replacement of external Ca2+ with Co2+ or Mn2+. This current appears to be a voltage-gated Ca2+ current ICa. Both ICa and IR in R15 have previously been shown to be enhanced by the neurotransmitter serotonin, acting via intracellular cyclic AMP. We now report that increasing cyclic AMP in R15, by applying either serotonin or the adenylate cyclase activator forskolin together with a phosphodiesterase inhibitor, mimics and occludes the action of ELH on neuron R15. Furthermore, application of ELH increases the cyclic AMP content of single R15 neurons, as measured by radioimmunoassay. Finally, the effects of ELH are potentiated by a phosphodiesterase inhibitor. These results suggest that ELH augments bursting activity in R15 by causing cyclic AMP-mediated increases in IR and ICa.
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PMID:Augmentation of bursting pacemaker activity by egg-laying hormone in Aplysia neuron R15 is mediated by a cyclic AMP-dependent increase in Ca2+ and K+ currents. 281 71


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