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)

A bovine lung cGMP-binding phosphodiesterase (cG-BPDE) was purified to homogeneity and exhibited specific cGMP hydrolytic (Km = 5.6 microM) and cGMP binding (half-maximum approximately 0.2 microM) activities which comigrated throughout the purification. A chimeric structure was suggested for cG-BPDE since DEAE chromatography of a partial alpha-chymotryptic digest of cG-BPDE separated cGMP-binding fragments from a cGMP hydrolytic fragment. Native cG-BPDE (178 kDa) appeared to be a homodimer comprised of two 93-kDa subunits. The order of potency of inhibitors of cG-BPDE hydrolysis of cGMP was as follows: zaprinast greater than dipyridamole greater than 3-isobutyl-1-methyl-8-methoxymethylxanthine greater than 3-isobutyl-1-methylxanthine greater than cilostamide greater than theophylline greater than rolipram. Minimum [3H]cGMP binding stoichiometry was 0.93 mol of cGMP bound/mol of monomer, but [3H]cGMP dissociation from cG-BPDE in the presence of excess unlabeled cGMP was curvilinear, suggesting multiple cGMP-binding sites. Two chymotryptic cGMP-binding fragments of 35 and 45 kDa were specifically photoaffinity labeled with [32P] cGMP, exhibited [3H]cGMP association and dissociation behavior indistinguishable from native cG-BPDE, and each had the amino-terminal sequence: Thr-Ser-Pro-Arg-Phe-Asp-Asn-Asp-Glu-Gly-. Cochromatography of the two cGMP-binding fragments suggested that both a dimerization domain and a cGMP-binding domain were located in a 35-kDa segment of cG-BPDE. Increased [3H]cGMP binding to or [32P]cGMP photoaffinity labeling of cG-BPDE binding sites in the presence of hydrolytic site-specific cyclic nucleotide analogs suggested communication between hydrolytic and binding sites. The principle of reciprocity thus predicts that cGMP binding to the binding sites may affect the hydrolytic site. In the presence of cGMP, the binding fragments or native cG-BPDE exhibited an electronegative shift on high performance liquid chromatography-DEAE, consistent with a cGMP-induced change in cG-BPDE conformation.
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PMID:Characterization of a purified bovine lung cGMP-binding cGMP phosphodiesterase. 169 84

Studies of cGMP binding to both the native cyclic GMP-stimulated phosphodiesterase and to two unique isolated chymotryptic fragments lacking the catalytic domain suggest that the enzyme contains two noncatalytic cGMP-binding sites/homodimer. In the presence of high concentrations of ammonium sulfate, 2 mol of cGMP are bound/mol of cGMP-stimulated phosphodiesterase homodimer. Under these conditions, linear Scatchard plots of binding are obtained that give an apparent Kd of approximately 2 microM. The inclusion of 3-isobutyl-1-methylxanthine produces a curvilinear plot. In the absence of ammonium sulfate, the dissociation of cGMP from the holoenzyme is rapid, having a t1/2 of less than 10 s, and addition of ammonium sulfate to the incubation greatly decreases this rate of dissociation. The native enzyme is resistant to degradation by chymotrypsin in the absence of cGMP; however, in its presence, chymotrypsin treatment produces several discrete fragments. Similarly, in the presence but not in the absence of cGMP, dicyclohexylcarbodiimide causes an irreversible activation of the enzyme without cross-linking the nucleotide to the phosphodiesterase. Both observations provide evidence that a different conformation in the enzyme results from cGMP binding. Only the conformation formed upon cGMP binding is easily attacked by chymotrypsin or permanently activated by treatment with dicyclohexylcarbodiimide. One major chymotryptic cleavage site exposed by cGMP binding is at tyrosine 553, implying that this region takes part in the conformational change. Limited proteolysis experiments indicate that these noncatalytic binding sites are located within a region of internal sequence homology previously proposed to include the cGMP-binding site(s) and that they retain a high affinity and specificity for cGMP independent of the catalytic domain of the enzyme. The products formed by partial proteolysis can be separated into individual catalytically active and cGMP-binding fractions by anion exchange chromatography. Gel filtration and electrophoresis analysis of the isolated fractions suggest that the cGMP-binding peak has a dimeric structure. Moreover, it can be further resolved by polyethyleneimine high performance liquid chromatography into two peaks (Peaks IIIA and IIIB). Peak IIIA binds 2 mol of cGMP/mol of dimer with an apparent Kd of 0.2 microM. Peak IIIB, however, has greatly reduced cGMP binding. Further digestion of these fragments with cyanogen bromide show that the differences between Peaks IIIA and IIIB are due to one or more additional proteolytic nicks in IIIB that remove a few residues near its C terminus, most probably residues 523-550 or 534-550. This in turn suggests that this region is essential for cGMP-binding activity.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Structure and function studies of the cGMP-stimulated phosphodiesterase. 172 Oct 55

The complete amino acid sequence of the cyclic GMP stimulated cyclic nucleotide phosphodiesterase (cGS-PDE) of bovine heart has been determined by analysis of five digests of the protein; placement of the C-terminal 330 residues has been confirmed by interpretation of the corresponding partial cDNA clone. The holoenzyme is a homodimer of two identical N alpha-acetylated polypeptide chains of 921 residues, each with a calculated molecular weight of 103,244. The C-terminal region, residues 613-871, of the cGS-PDE comprises a catalytic domain that is conserved in all phosphodiesterase sequences except those of PDE 1 from Saccharomyces cerevisiae and a secreted PDE from Dictyostelium. A second conserved region, residues 209-567, is homologous to corresponding regions of the alpha and alpha' subunits of the photoreceptor phosphodiesterases. This conserved domain specifically binds cGMP and is involved in the allosteric regulation of the cGS-PDE. This regulatory domain contains two tandem, internal repeats, suggesting that it evolved from an ancestral gene duplication. Common cyclic nucleotide binding properties and a distant structural relationship provide evidence that the catalytic and regulatory domains within the cGS- and photoreceptor PDEs are also related by an ancient internal gene duplication.
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PMID:Amino acid sequence of the cyclic GMP stimulated cyclic nucleotide phosphodiesterase from bovine heart. 217 66

Purified calmodulin-stimulated cyclic nucleotide phosphodiesterase from brain, a homodimer of 59-kDa subunits, was activated by limited proteolysis with trypsin, alpha-chymotrypsin, Pronase, or papain and could not be further stimulated by addition of Ca2+ and calmodulin. Proteolysis increased Vmax and had little effect on the Km for cGMP. Treatment with alpha-chymotrypsin in the presence of ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) produced, sequentially, 57- and 45-kDa peptides from the bovine and 55-, 53-, and 38-kDa peptides from the ovine enzyme. This protease-treated phosphodiesterase exhibited a Stokes radius of 3.9 nm and an S20,w value of 4.55; comparison with the hydrodynamic properties observed for native enzyme (4.3 nm, 5.95 S) strongly suggests a dimeric protein of Mr approximately 80,000-90,000. The proteolyzed species does not interact significantly with calmodulin immobilized on agarose, nor does it show complex formation with 2-dimethylaminonaphthalene-1-sulfonyl-calmodulin even at micromolar concentrations of protein. Proteolysis, in the presence of calmodulin plus Ca2+, fully activated phosphodiesterase, producing the same intermediate peptides; however, final peptides from the bovine and ovine enzymes were 47 and 42 kDa, respectively, indicating a new, specific conformation of the enzyme. When EGTA was added to such incubations, these peptides were cleaved to those of the size seen when proteolysis was carried out entirely in the presence of EGTA. The initial rate of activation was increased by the presence of Ca2+ and calmodulin, suggesting that, in complex, phosphodiesterase exhibits a site with increased susceptibility to proteolysis. Since calmodulin can still interact with a fully activated form of the enzyme, it appears that retention of calmodulin binding can occur concomitantly with damage to that portion of the phosphodiesterase molecule responsible for suppression of its basal catalytic activity.
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PMID:Proteolytic activation of calmodulin-dependent cyclic nucleotide phosphodiesterase. 299 Dec 33

1. The effect on cyclic nucleotide contents of selective inhibitors of cyclic nucleotide phosphodiesterase (PDE) isoforms III and IV (respectively SK&F 94120 and rolipram) and their interactions with endothelium and NO have been studied in rat aorta in the presence of indomethacin (10 microM). The participation of NO was assessed by using either NG-nitro-L-arginine methyl ester (L-NAME) (NO synthase inhibitor: 30 microM) or 3-morpholinosydnonimine (SIN-1, NO donor: 10 microM with SOD 100 units ml-1). 2. The presence of endothelium significantly increased both adenosine 3':5'-cyclic monophosphate (cyclic AMP, 1.7 fold) and guanosine 3':5'-cyclic monophosphate (cyclic GMP, 2.2 fold) contents. Cyclic GMP was largely affected by L-NAME or SIN-1 treatment, this was not the case for cyclic AMP suggesting that the presence of endothelium modified cyclic AMP content in aorta independently of the NO production. 3. In the presence or absence of endothelium, neither SK&F 94120 nor rolipram, alone or combined, significantly modified cyclic GMP content. 4. The PDE III inhibitor significantly affected cyclic AMP content only in non treated aorta without endothelium. In contrast, the PDE IV inhibitor increased cyclic AMP in all conditions. These increases were generally about 2 fold but markedly higher in aorta treated with SIN-1 and superoxide dismutase (SOD, 6 fold). Association of a low concentration of the PDE III inhibitor (5 microM) with the PDE IV inhibitor (30 microM) potentiated the effect of the PDE IV inhibitor on cyclic AMP content, except for aorta without endothelium treated with SIN-1 plus SOD. 5. These data indicate that the presence of the endothelium could increase cyclic AMP content independently of NO and prostacyclin (PGI2) production. Furthermore, an increase in cyclic GMP content (modulated by NO production) could enhance the cyclic AMP accumulation induced by the PDE IV inhibitor. This result supports the hypothesis that PDE III inhibition by endogenous cyclic GMP may potentiate the effect of PDE IV inhibition on cyclic AMP content. Taken together with our previous studies on relaxation, these results suggest that the NO/cyclic GMP pathway could induce PDE IV-dependent regulation of cyclic AMP via PDE III inhibition.
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PMID:Role of phosphodiesterases III and IV in the modulation of vascular cyclic AMP content by the NO/cyclic GMP pathway. 783 94

1. The relaxant responses of S-nitroso-L-cysteine (CysNO), S-nitroso-N-acetyl-D,L-penicillamine (SNAP), S-nitroso-N-acetyl-L-cysteine (SNAC) and S-nitrosoglutathione (GSNO) in the rat gastric fundus (forestomach) were studied and compared to the relaxant responses obtained in response to nitric oxide (NO) and electrical field stimulation (EFS, 10 s strains) of non-adrenergic non-cholinergic (NANC) nerves. 2. CysNO (10(-7)-3 x 10(-4) M) caused transient relaxation of the precontracted rat gastric fundus, comparable to the response to NO (10(-6)-10(-4) M) and EFS. SNAP, SNAC and GSNO elicited more sustained relaxations. 3. The cyclic GMP-specific phosphodiesterase inhibitor, zaprinast (3 x 10(-5) M) increased the relaxant effect of CysNO, SNAP and GSNO while the NO-synthase inhibitor, NG-nitro-L-arginine (L-NOARG, 3 x 10(-4) M) had no influence. 4. In the presence of LY 83583 (10(-5) M), which releases superoxide anions, the relaxant response to NO and CysNO was decreased, whereas that to all other stimuli was unaltered. The inhibitory effect of LY 83583 on CsNO-induced relaxations was prevented by superoxide dismutase (SOD, 1000 u ml-1). 5. Tissues incubated for 1 h with 5.5 x 10(-4) M nitroglycerin (GTN) became tolerant to GTN. In this condition, the relaxant response to 10(-5) M NO was maintained, while the relaxations by EFS (8 Hz) and 3 x 10(-5) M SNAP were significantly decreased. The reduction of the response to the other S-nitrosothiols was not significant. 6. The combination of nitrate tolerance and 10-5 M LY 83583 caused a significantly larger inhibition of the relaxant response to EFS (8 Hz) than nitrate tolerance alone. The combination of LY 83583 and GTN tolerance reduced the relaxant effect of 10-5 M NO to a similar extent to LY 83583 alone, while the relaxant response to 10-4 M GTN was reduced to the same extent as after 1 h exposure to 5.5 x 10-4 M GTN alone.7. It is concluded that S-nitrosothiols potently relax the rat gastric fundus, possibly by a cyclic GMP-dependent mechanism and S-nitrosothiols such as SNAC and GSNO may be involved in NANC neurotransmission.
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PMID:Influence of S-nitrosothiols and nitrate tolerance in the rat gastric fundus. 803 15

1. Nitric oxide (NO)-mediated, endothelium-dependent vasodilator function in rat aortic smooth muscle was investigated in an in vitro model of endogenous vascular superoxide anion stress, generated by pretreatment with the Cu/Zn superoxide dismutase (SOD, EC 1.15.1.1) inhibitor, diethyldithiocarbamate (DETCA). 2. Contraction to noradrenaline (NA, 1 nM - 1 microM) in endothelium-intact vessels was augmented after a 30 min pretreatment with DETCA (10 mM) followed by 30 min washout. This effect was abolished by N(G)-nitro-L-arginine methyl ester (L-NAME, 0.3 mM) and removal of the endothelium and partially reversed by exogenous Cu/Zn SOD (200 u ml(-1)). 3. Endothelium- and basal NO-dependent vasorelaxation to the phosphodiesterase (PDE) type V inhibitor ONO- 1 505 (4-[2-(2-hydroxyethoxy)ethylamino]-2-(1H-imidazol-1-yl)-6-methoxyquin azoline methanesulphonate) (0.1-10 microM) was inhibited after DETCA (10 mM) pretreatment. In addition, the ability of L-NAME (0.3 mM) to enhance established contractile tone was effectively absent. 4. In contrast, DETCA pretreatment did not significantly affect vasorelaxation to acetylcholine (ACh, 1 nM - 3 microM) or S-nitroso-N-acetyl penicillamine (SNAP, 0.03-30 microM). However, L-NAME (0.3 mM) unmasked an inhibitory effect of DETCA pretreatment on vasorelaxation to SNAP in endothelium-intact vessels while markedly potentiating vasorelaxation to SNAP in control tissue. 5. L-NAME (0.3 mM)- and exogenous catalase (200 u ml(-1))-sensitive vasorelaxation to exogenous Cu/ Zn SOD (200 u ml(-1)) was greater after DETCA (10 mM) pretreatment in endothelium-intact aortic rings. This difference was abolished by catalase (200 u ml(-1)). 6. In conclusion, tissue Cu/Zn SOD inhibition elicited a selective lesion in basal endothelial function in rat isolated aortic smooth muscle, consistent with the inactivation of basal NO by superoxide anion. The resulting leftward shift in nitrovasodilator reactivity, due to the loss of the tonic depression by basal NO, is likely to mask the inhibitory effect of superoxide anion on agonist-stimulated endothelial function and nitrovasodilator-derived NO, thereby accounting for the differential pattern of endothelial dysfunction after DETCA pretreatment.
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PMID:Interaction between superoxide anion and nitric oxide in the regulation of vascular endothelial function. 963 Mar 65

ACh-induced vasodilation was investigated in pulmonary arteries from 8 and 2 day pre-term foetal, neonatal (0-12 h and 4 day old) and adult rabbits. The effects of superoxide anion generation [with hypoxanthine (HX, 0.1 mM)/xanthine oxidase (XO, 15 mu ml(-1))], endogenous superoxide dismutase (SOD) inhibition [with the Cu-Zn SOD inhibitor triethylenetetramine (TETA, 1 mM)], endogenous superoxide anion scavenging [by superoxide dismutase (SOD, 50 u ml(-1))] and inhibition of endothelial nitric oxide synthase (eNOS) [with, Nomega-nitro-L-arginine methylester (L-NAME, 0.1 mM)], on basal and ACh-induced NO activity were studied by examining phenylephrine-induced contraction and ACh-induced vasodilation respectively. L-NAME and endothelium removal abolished all ACh-induced vasodilation and 1 microM sodium nitroprusside fully dilated all vessels. ACh-induced vasodilation was absent in the 8 day pre-term foetus and 0-12 h neonate but present at all other ages. L-NAME itself contracted 2 day pre-term foetal vessels. At 0 12 h, SOD, but not the phosphodiesterase 5 inhibitor zaprinast (1 microM), uncovered ACh-induced vasodilation. At this age SOD reduced phenylephrine-induced contraction which was not influenced by TETA, L-NAME or HX/XO, and L-NAME itself did not cause contraction. This suggests both ACh-induced and basal NO activity are compromise in these vessels by endogenous superoxide anion production and deficiencies in endogenous SOD activity. In 4 day vessels, but not adult vessels, L-NAME, TETA and HX/XO augmented contractions to phenylephrine, and L-NAME itself induced vasoconstriction, suggesting that basal NO and SOD activities were present by 4 days but were not evident in the adult. ACh-induced NO activity, and the influence of endogenous SOD on this, were present in the adult (and 4 day) vessels as superoxide generation with HX/XO significantly reduced ACh-induced vasodilation and this effect was inhibited by SOD and augmented by TETA. Increased oxygen tensions > 500 mmHg attenuated ACh-induced vasodilation in the foetal but not neonatal rabbits. Raising the oxygen tension from approximately 20 to approximately 120 mmHg revealed ACh-induced vasodilation in the 8 day pre-term vessels. In summary, superoxide anion accumulation combined with deficiencies in SOD activity may transiently compromise basal and ACh-induced NO activity at birth. Experimental oxygen tensions markedly influence ACh-induced vasodilation in foetal rabbit pulmonary arteries.
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PMID:Developmental changes in endothelium-dependent vasodilation and the influence of superoxide anions in perinatal rabbit pulmonary arteries. 988 88

PC-1 is a type II membrane-bound glycoprotein consisting of a short N-terminal cytoplasmic domain and a large C-terminal extracellular domain, which contains phosphodiesterase/pyrophosphatase activity. When Jurkat T cells were cultured with dibutyryl cAMP, the membrane-bound PC-1 and its soluble form were induced. They were purified as a homodimer of a 130 kDa peptide and a 120 kDa monomer, respectively, and the same two forms could also be obtained from COS-7 cells that had been transfected with PC-1 cDNA. The membrane-bound and soluble forms of PC-1 were indistinguishable from each other in terms of their enzyme kinetics and N-glycosylated moieties. Thus, the enzymatically active and fully glycosylated form of soluble PC-1 was utilized to search for its interacting molecules. The phosphodiesterase/pyrophosphatase activity of PC-1 was competitively inhibited by glycosaminoglycans, such as heparin and heparan sulfate, which are the major components of the extracellular matrix. PC-1 was capable of binding to heparin-Sepharose and the binding was inhibited in the presence of the enzyme substrate, ATP or its nonhydrolyzable analog. The enzyme activity of PC-1 itself, however, was not required for the binding to heparin-Sepharose. These results suggest that PC-1 might function as an adhesion molecule independent of its enzyme activity to associate with glycosaminoglycans in the extracellular matrix.
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PMID:Inhibition of phosphodiesterase/pyrophosphatase activity of PC-1 by its association with glycosaminoglycans. 1050 8

The present study examined some possible mechanisms underlying the previously demonstrated release of adenosine by nitric oxide (NO) donors. Perfusion with the NO-donor S-nitroso-N-acetyl penicillamine (SNAP; 300 microM) led to a significant increase in the release of [3H]purines from both unstimulated and electrically stimulated hippocampal slices prelabeled with [3H]adenine. The NO-donor also evoked the release of endogenous ATP and ADP from unstimulated slices and, when combined with electrical stimulation, the release of ATP, AMP and adenosine. The SNAP-induced [3H]purine release was calcium-dependent, but not affected by the glutamate receptor antagonists MK-801 ((+)-5-methyl-10,11-dihydro-5H-dibenzo[a, d]-cyclohepten-5,10-imine;100 nM) and CNQX (6-cyano-7-nitroquinoxaline-2,3-dione; 10 microM). Zaprinast (5 microM), an inhibitor of the cyclic GMP-dependent phosphodiesterase and 8-Br-cyclic GMP (0.01-1 mM) failed to evoke the release of purines, whereas generation of oxygen free radicals by xanthine plus xanthine oxidase did evoke purine release. Coperfusion of SNAP with the free radical scavengers superoxide dismutase (SOD; 60 microg/ml) and catalase (50 microg/ml) reduced or eliminated the ability of the NO-donor to enhance [3H]purine release, but the poly (ADP-ribosyl) synthetase (PARS) inhibitor benzamide (500 microM) did not affect it. These data indicate that NO interacts with superoxide, likely forming peroxynitrite, which subsequently acts to release adenosine and adenine nucleotides from hippocampal tissue.
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PMID:Nitric oxide interacts with oxygen free radicals to evoke the release of adenosine and adenine nucleotides from rat hippocampal slices. 1086 5


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