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)

X-ray studies of the proofreading 3',5'-exonuclease site of the large (Klenow) fragment of DNA polymerase I have detected a binuclear metal complex consisting of a pentacoordinate metal (site A) which shares a ligand, Asp-355, with an octahedral metal (site B) [Freemont, P. S., Friedman, J. M., Beese, L. S., Sanderson, M. R., & Steitz, T. A. (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 8924-8928; Beese, L. S., & Steitz, T. A. (1991) EMBO J. 10, 25-33]. Kinetic studies of the activation of the 3',5'-exonuclease reaction by Co2+, Mn2+, or Mg2+, at low concentrations of DNA, reveal sigmoidal activation curves for the three metal ions with Hill coefficients of 2.3-2.4 and K0.5 values of 16.6 microM, 4.2 microM, and 343 microM, respectively. The binding of Co2+ to the enzyme results in the appearance of an intense visible absorption spectrum of the metal ion with maxima at 633, 570, and 524 nm and extinction coefficients of 190, 194, and 150 M-1 cm-1, respectively, suggesting the formation of a pentacoordinate Co2+ complex. Optical titration with Co2+ yields a sigmoidal titration curve which is best fit by assuming the cooperative binding of three Co2+ ions with a K0.5 of 39.9 microM, comparable to the value of 16.6 microM obtained kinetically. Displacement of Co2+ by 1 equiv of Zn2+, which binds tightly to the A site of the 3',5'-exonuclease, shifts the optical spectrum to 524 nm and lowers the extinction coefficient to 30 -1 cm-1, indicative of octahedral coordination.2+ the formation of the binuclear complex.
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PMID:Role of divalent cations in the 3',5'-exonuclease reaction of DNA polymerase I. 165 60

The yeast Saccharomyces cerevisiae catalytic DNA polymerase I 180-kDa subunit and the tightly associated 86-kDa polypeptide have been purified using immunoaffinity chromatography, permitting further characterization of the DNA polymerase activity of the DNA primase-DNA polymerase protein complex. The subunits were purified to apparent homogeneity from separate overproducing yeast strains using monoclonal antibodies specifically recognizing each subunit. When the individual subunits were recombined in vitro a p86p180 physical complex formed spontaneously, as judged by immunoprecipitation of 180-kDa polypeptide and DNA polymerase activity with the anti-86-kDa monoclonal antibody. The 86-kDa subunit stabilized the DNA polymerase activity of the 180-kDa catalytic subunit at 30 degrees C, the physiological temperature. The apparent DNA polymerase processivity of 50-60 nucleotides on poly(dA).oligo(dT)12 or poly(dT).oligo(A)8-12 template-primer was not affected by the presence of the 86-kDa subunit but was reduced by increased Mg2+ concentration. The Km of the catalytic 180-kDa subunit for dATP or DNA primer terminus was unaffected by the presence of the 86-kDa subunit. The isolated 180-kDa polypeptide was sufficient to catalyze all the DNA synthesis that had been observed previously in the DNA primase-DNA polymerase protein complex. The 180-kDa subunit possessed a 3'----5'-exonuclease activity that catalyzed degradation of polynucleotides, but degradation of oligonucleotide substrates of chain lengths up to 50 was not detected. This exonuclease activity was unaffected by the presence of the 86-kDa subunit. Despite the striking physical similarity of the DNA primase-DNA polymerase protein complex in all eukaryotes examined, the data presented here indicate differences in the enzymatic properties detected in preparations of the DNA polymerase subunits isolated from S. cerevisiae as compared with the properties of preparations from Drosophila cells. In particular, the 3'----5'-exonuclease activity associated with the yeast catalytic DNA polymerase subunit was not masked by the 86-kDa subunit.
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PMID:Purification and characterization of the 180- and 86-kilodalton subunits of the Saccharomyces cerevisiae DNA primase-DNA polymerase protein complex. The 180-kilodalton subunit has both DNA polymerase and 3'----5'-exonuclease activities. 170 71

When the homogenate prepared from immature rat testes was incubated with [32P]NAD, several proteins (90, 39 and 20 kDa) were ADP-ribosylated in the absence of bacterial toxins. This observation suggested the existence of an endogenous ADP-ribosyltransferase and substrates. The data that the digested product by phosphodiesterase of ADP-ribosylated 20 kDa protein was 5'-AMP suggested that 20 kDa protein was mono(ADP-ribosyl)ated. In addition, the mono(ADP-ribosyl)ation of 20 kDa protein was enhanced by guanine nucleotides such as GTP, GDP and GTP[gamma S], and decreased by the concentrations of 10 mM Mg2+. In contrast, the incorporation of ADP-ribose moiety from NAD to both 90 and 39 kDa proteins was not changed by guanine nucleotides. On the other hand, mono(ADP-ribosyl)ation of 20 kDa protein was not observed in the homogenate prepared from other tissues of the same rats. Furthermore, we found that mono(ADP-ribosyl)ation of 20 kDa protein was decreased with the maturation of the rats and that an endogenous mono(ADP-ribosyl)transferase and 20 kDa protein were located in the nuclei.
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PMID:Reduction of mono(ADP-ribosyl)ation of 20 kDa protein with maturation in rat testis: involvement of guanine nucleotides. 189 5

In this study we seek to elucidate the interaction of capsaicin with the calmodulin mediated signal pathways in macrophages, by comparing its action on macrophage functions with a known calmodulin antagonist, fluphenazine. Kinetics of capsaicin uptake by macrophages (10(3) cells) revealed that a maximum of 200 microM capsaicin was taken up within 10 min. Ca2+ ionophore triggered generation of superoxide anion and hydrogen peroxide by macrophages was inhibited in a dose-dependent manner by fluphenazine (IC50, 20 microM and 12 microM, respectively) and also by capsaicin (IC50, 30 microM and 9 microM, respectively), suggesting an involvement of calmodulin in the regulation of NADPH oxidase. In vitro both fluphenazine and capsaicin inhibited Ca2(+)-Mg2+ ATPase and cAMP-phosphodiesterase from macrophages and this inhibition was reversed by exogenous addition of calmodulin. Fluorescence studies revealed a direct Ca2+ dependent interaction of capsaicin with calmodulin. From these results we suggest that capsaicin acts via calmodulin to inhibit stimulus-induced macrophage oxidative burst and also that calmodulin regulates the oxidative burst in macrophages.
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PMID:Capsaicin inhibits calmodulin-mediated oxidative burst in rat macrophages. 196 91

Conditions favouring protein phosphorylation and dephosphorylation are examined for their effects on activity and charge heterogeneity of the rat gastric mucosal histidine decarboxylase. Incubation of gastric supernatant with various combinations of ATP, Mg2+, cyclic AMP and protein kinase under the blockade of endogenous phosphodiesterase and phosphatase fails to alter significantly enzyme activity as assayed with or without pyridoxal 5'-phosphate. Similar results are found with the purified enzyme. No change occurs in the distribution of activity between the charged forms. In contrast, treatment with alkaline phosphatase both inactivates the enzyme with preservation of heterogeneity, full reactivation being achieved by pyridoxal 5'-phosphate, and reduces the number of forms and converts forms II and III to form I with preservation of the catalytic potentialities. The data suggest that the enzyme heterogeneity may be related in part to the phosphorylation state; the possibility that the gastric enzyme is susceptible to several post-translational modifications is discussed.
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PMID:Relationship between the multiple forms of rat gastric histidine decarboxylase: effects of conditions favouring phosphorylation and dephosphorylation. 215 9

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 possible involvement of chemiosmotic lysis of secretory granules in the exocytosis of insulin from pancreatic beta cells was investigated by comparing insulin release from isolated secretory granules, from intact islets of Langerhans, and from electrically permeabilised islets. Lysis of isolated granules was stimulated by ATP in the presence of Mg2+. ATP-induced granule lysis was pH and temperature dependent and was inhibited by collapsing the pH gradient across the granule membrane by removal of permeant anions, or by increasing the extragranular osmolarity. However, insulin secretion from intact islets in response to glucose, a phosphodiesterase inhibitor or a Ca2+ ionophore was only partially inhibited by anion replacement, while Ca2+ -induced insulin release from electrically permeabilised islets was not affected by altering the extragranular or intragranular pH. These results suggest that studies of the stability of isolated granules in vitro do not necessarily relate to insulin release from whole cells, and do not support a major role for chemiosmotic lysis of secretory granules in the exocytotic release of insulin.
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PMID:Chemiosmotic lysis and insulin secretion: studies of isolated granules, intact and permeabilised rat islets of Langerhans. 244 Apr 82

We used pertussis toxin to study the mechanism(s) by which divalent cations lower cellular cAMP content in bovine parathyroid cells. In cultured parathyroid cells, high extracellular Ca2+ (5 mM) or Mg2+ (5-10 mM) lowers dopamine-stimulated cAMP content by 70-90%. Pertussis toxin (0.5 microgram/ml) totally blocks the inhibitory effects of Ca2+ and Mg2+ on cAMP content. Ba2+ and Sr2+ (5 mM) also lower cAMP content by 80-90%, and this effect is, likewise, blocked by pertussis toxin. Pretreatment with pertussis toxin had no effect on the release of cAMP into the extracellular fluid. The toxin also did not modify phosphodiesterase activity in sonicates of parathyroid cells (42.68 +/- 3.26 vs. 47.00 +/- 2.82 pmol cAMP hydrolyzed/10(6) cells.20 min in control and toxin-treated cells, respectively). Moreover, addition of the phosphodiesterase inhibitor isobutyl-methylxanthine did not modify the inhibition of dopamine-stimulated cAMP accumulation by 5 mM Ca2+ in control cells (85% vs. 86% inhibition, respectively, with and without isobutylmethylxanthine). Pertussis toxin-catalyzed ADP ribosylation in homogenates of control cells demonstrated the presence of two substrates with mol wt of 40K and 41K. Preexposure of cells to pertussis toxin overnight resulted in the complete loss of both substrates on subsequent ADP ribosylation with [32P]NAD. Pertussis toxin pretreatment did not enhance adenylate cyclase activity indirectly via reducing the extracellular Ca2+-induced rise in cytosolic Ca2+, since the cytosolic Ca2+ level at 5 mM Ca2+ was about 60% higher in pertussis toxin-treated than in control cells (531 +/- 85 vs. 326 +/- 35 nM; P less than 0.05). In addition, ionomycin had no significant effect on cellular cAMP levels in control cells despite increasing the cytosolic Ca2+ concentration to levels as high as 1700 nM at 10(-5) M. Thus, changes in cytosolic Ca2+ phosphodiesterase activity, or efflux of cAMP from the cell cannot explain the inhibition of cAMP accumulation by divalent cations or the reversal of this effect by pertussis toxin. Instead, the present data suggest that extracellular divalent cations modulate the formation of cellular cAMP in parathyroid cells by a process involving a pertussis toxin-sensitive guanine nucleotide regulatory protein, presumably inhibition of adenylate cyclase by Gi via a receptor-like mechanism.
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PMID:Divalent cations suppress 3',5'-adenosine monophosphate accumulation by stimulating a pertussis toxin-sensitive guanine nucleotide-binding protein in cultured bovine parathyroid cells. 246 88

Aluminum ion perturbs the activity of a number of physiologically important enzymes, including members of a family of guanine nucleotide-binding proteins (G-proteins). G-proteins couple cellular receptor proteins to a variety of effector enzymes (including adenylate cyclase, phospholipase C, and the rod photoreceptor phosphodiesterase). We show herein that subnanomolar concentrations of free aluminum ion, produced in a carefully defined and kinetically stable manner through the buffering of total aluminum at 0.1-1.0 mM with calculated ratios of chelating agents, inhibit both the receptor-mediated activation and the self-inactivating GTPase activity of the rod photoreceptor G-protein, Gv. In the presence of 4 X 10(-10) M free aluminum ion, GTPase activity is inhibited from about 25-60% as the magnesium ion concentration is reduced from 10(-3) to about 5 X 10(-5) M. The principal effect of aluminum ion upon Gv is to inhibit receptor catalyzed nucleotide exchange. Binding of the GTP analog 5'-guanylyl imidodiphosphate can be reduced by as much as 90% by aluminum ion following subsaturating rhodopsin stimulation. Aluminum ion can produce either competitive or mixed noncompetitive inhibition of rhodopsin-catalyzed Gv activation and GTPase activity, as a function of whether Gv undergoes single (competitive), or multiple (mixed noncompetitive) nucleotide exchanges. The rod photoreceptor phosphodiesterase is only slightly inhibited by similar aluminum ion activities. Light- and Gv-coupled phosphodiesterase activation exhibits both a lower maximum rate of cyclic guanosine monophosphate hydrolysis and a slower inactivation in the presence of aluminum ion activities from about 10(-12) - 10(-10) M. These data suggest that intracellular free aluminum ion concentrations in the subnanomolar range could markedly affect the ability of cells to transduce extracellular signals. Interestingly, the combination of Al3+ and F- to produce the fluoro-aluminate species (AlFx) also inhibits the GTPase of G-proteins, although the mechanism of inhibition (e.g. binding to the G-protein.Mg2+.GDP complex) is totally distinct from that observed for free Al3+ and the overall effect on signal transduction (e.g. enhanced signal amplification) is in complete opposition to that observed for free Al3+.
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PMID:Inhibition of transducin activation and guanosine triphosphatase activity by aluminum ion. 253 40


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