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)

In the present purification of low molecular weight fractions (Mr: 2000-4000) containing basic peptides, twenty nmol of novel calmodulin binding peptide, possessing a potent affinity for calmodulin, was isolated from 18 kg of porcine brain. By analysis with gas phase sequencer, the sequence was determined to be APAEDLARYYSALRHYINLITRQRY. Carboxy terminus of the peptide was determined to be Tyr-NH2. The peptide was a carboxy terminal pentacosanepeptide of neuropeptide Y and was termed NPY-25. NPY-25 competitively inhibited the activation of cAMP-phosphodiesterase through CaM binding in a Ca++ dependent fashion, but did not inhibit the basal activity of cAMP phosphodiesterase. NPY-25 elicited a more potent activity than did neuropeptide Y. IC50 values of NPY-25 and Neuropeptide Y were 0.06 microM and 0.54 microM respectively.
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PMID:Isolation of NPY-25 (neuropeptide Y[12-36]), a potent inhibitor of calmodulin, from porcine brain. 216 29

The role of cyclic nucleotides as intracellular second messengers mediating the excitatory chronotropic and inotropic actions of octopamine (OCT) and dopamine (DA) on the neurogenic Limulus heart was investigated. Tissue levels of cAMP, but not cGMP, were significantly increased in isolated cardiac ganglia and cardiac muscle following 10 min exposure to 10(-5) M OCT or 10(-5) M DA. In both tissues, OCT elicited larger increases in cAMP than did DA. Amine-induced cAMP accumulation in the cardiac ganglion and in the cardiac muscle was prevented by the alpha-adrenergic blocker phentolamine. The adenylate cyclase activator forskolin and the phosphodiesterase inhibitor IBMX produced amine-like chronotropic and inotropic effects when applied to the isolated heart preparation. However, the kinetics of the responses differed for the two agents. Additional pharmacological agents (RO-20-1724, papaverine, SQ 20,009, and 8-parachloro-phenylthio cAMP) also had amine-like effects but to a lesser extent. The chronotropic, but not inotropic, effects of OCT and DA were potentiated in the presence of IBMX. These data suggest that a cAMP-dependent mechanism underlies the excitatory effects of the neuromodulators OCT and DA on the Limulus heart.
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PMID:Mechanism for amine modulation of the neurogenic Limulus heart: evidence for involvement of cAMP. 244 16

cGMP-stimulated phosphodiesterase (PDE) has been directly photolabeled with [32P]cGMP using UV light. Sequence analysis of peptide fragments obtained from partial proteolysis or cyanogen bromide cleavage indicate that two different domains are labeled. One site, on a Mr = 36,000 chymotryptic fragment located near the COOH terminus, has characteristics consistent with it being close to or part of the catalytic site of the enzyme. This peptide contains a region of sequence that is highly conserved in all mammalian cyclic nucleotide PDEs and has been postulated to contain the catalytic domain of the enzyme. The other site, on a Mr = 28,000 cyanogen bromide cleavage fragment located near the middle of the molecule, probably makes up part of the allosteric site of the molecule. Labeling of the enzyme is concentration dependent and Scatchard analysis of labeling yields a biphasic plot with apparent half labeling concentrations of about 1 and 30 microM consistent with two types of sites being labeled. Limited proteolysis of the PDE by chymotrypsin yields five prominent fragments that separate by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) at Mr = 60,000, 57,000, 36,000, 21,000, and 17,000. Both the Mr = 60,000 and 57,000 apparently have blocked NH2 termini suggesting that the Mr = 57,000 fragment is a subfragment of the Mr = 60,000 fragment. Primary sequence analysis indicates that both the Mr = 21,000 and 17,000 fragments are subfragments of the Mr = 36,000 fragment. Autoradiographs of photolabeled then partially proteolyzed enzyme show labeled bands at Mr = 60,000, 57,000, and 36,000. Addition of 5 microM cAMP prior to photolabeling eliminates photolabeling of the Mr = 36,000 fragment but not the Mr = 60,000 or 57,000 fragments. The labeled site not blocked by cAMP is also contained in a Mr = 28,000 cyanogen bromide fragment of the enzyme that does not overlap with the Mr = 36,000 proteolytic fragment. Limited chymotryptic proteolysis also increases basal activity and eliminates cGMP stimulation of cAMP hydrolysis. The chymotryptic fragments can be separated by either ion exchange high performance liquid chromatography (HPLC) or solid-phase monoclonal antibody treatment. A solid-phase monoclonal antibody against the cGMP-stimulated PDE removes the Mr = 60,000 and 57,000 labeled fragments and any intact, unproteolyzed protein but does not remove the Mr = 36,000 fragment or the majority of activity. Ion exchange HPLC separates the fragments into three peaks (I, II, and III). Peaks I and II contain activity of approximately 40 and 100 units/mg, respectively. Peak II is the undigested or slightly nicked native enzyme.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Direct photolabeling of the cGMP-stimulated cyclic nucleotide phosphodiesterase. 254 73

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

Extracts of Chlorella pyrenoidosa, Euglena gracilis var. bacillaris, spinach, barley, Dictyostelium discoideum and Escherichia coli form an unknown compound enzymically from adenosine 5'-phosphosulphate in the presence of ammonia. This unknown compound shares the following properties with adenosine 5'-phosphoramidate: molar proportions of constituent parts (1 adenine:1 ribose:1 phosphate:1 ammonia released at low pH), co-electrophoresis in all buffers tested including borate, formation of AMP at low pH through release of ammonia, mass and i.r. spectra and conversion into 5'-AMP by phosphodiesterase. This unknown compound therefore appears to be identical with adenosine 5'-phosphoramidate. The enzyme that catalyses the formation of adenosine 5'-phosphoramidate from ammonia and adenosine 5'-phosphosulphate was purified 1800-fold (to homogeneity) from Chlorella by using (NH(4))(2)SO(4) precipitation and DEAE-cellulose, Sephadex and Reactive Blue 2-agarose chromatography. The purified enzyme shows one band of protein, coincident with activity, at a position corresponding to 60000-65000 molecular weight, on polyacrylamide-gel electrophoresis, and yields three subunits on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis of 26000, 21000 and 17000 molecular weight, consistent with a molecular weight of 64000 for the native enzyme. Isoelectrofocusing yields one band of pI4.2. The pH optimum of the enzyme-catalysed reaction is 8.8. ATP, ADP or adenosine 3'-phosphate 5'-phosphosulphate will not replace adenosine 5'-phosphosulphate, and the apparent K(m) for the last-mentioned compound is 0.82mm. The apparent K(m) for ammonia (assuming NH(3) to be the active species) is about 10mm. A large variety of primary, secondary and tertiary amines or amides will not replace ammonia. One mol.prop. of adenosine 5'-phosphosulphate reacts with 1 mol.prop. of ammonia to yield 1 mol.prop. each of adenosine 5'-phosphoramidate and sulphate; no AMP is found. The highly purified enzyme does not catalyse any of the known reactions of adenosine 5'-phosphosulphate, including those catalysed by ATP sulphurylase, adenosine 5'-phosphosulphate kinase, adenosine 5'-phosphosulphate sulphotransferase or ADP sulphurylase. Adenosine 5'-phosphoramidate is found in old samples of the ammonium salt of adenosine 5'-phosphosulphate and can be formed non-enzymically if adenosine 5'-phosphosulphate and ammonia are boiled. In the non-enzymic reaction both adenosine 5'-phosphoramidate and AMP are formed. Thus the enzyme forms adenosine 5'-phosphoramidate by selectively speeding up an already favoured reaction.
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PMID:Purification and properties of adenylyl sulphate:ammonia adenylyltransferase from Chlorella catalysing the formation of adenosine 5' -phosphoramidate from adenosine 5' -phosphosulphate and ammonia. 627 7

Limited proteolysis of calmodulin with trypsin in the presence of ethylene glycol bis(beta-aminoethyl ether)-N, N,N',N'-tetracetic acid (EGTA) or Ca2+ was performed according to a modification of the method of Drabikowski et al. (Drabikowski, W., Kuznicki, J., and Grabarek, Z. (1977) Biochim. Biophys. Acta 485, 124-133). The resulting peptides were purified by reverse-phase high performance liquid chromatography. Tryptic digests in EGTA yielded peptides 1-106, 1-90, and 107-148 with yields of 9, 47, and 61%, respectively. The digests performed with Ca2+ yielded peptides 1-77 and 78-148 in 35 and 45% yield. Analysis by high performance liquid chromatography indicated that the purified fragments contained less than 0.1% contamination by calmodulin, thus allowing a definitive study of the ability of these fragments to activate, or interact with, calmodulin-regulated enzymes and anti-calmodulin drugs. Each of the fragments, except 107-148, bound to a phenothiazine affinity column in a Ca2+-dependent manner. Thus, calmodulin contains two interaction sites for phenothiazines: one on the NH2-terminal half (fragment 1-77) and one on the COOH-terminal half (fragment 78-148). None of the fragments activates the protein phosphatase, calcineurin, or prevents its stimulation by calmodulin, nor does any of the fragments stimulate Ca2+-dependent cAMP phosphodiesterase. A single cleavage in the middle of the calmodulin molecule results in the rapid dissociation of the two resultant fragments and a loss of ability to activate cAMP phosphodiesterase. One fragment, 78-148, interacts with phosphodiesterase and prevents its activation by calmodulin (Ki: 1.5 +/- 0.4 X 10(-6) M). The same fragment, 78-148, can fully activate phosphorylase kinase but with a lower affinity than calmodulin (Kuznicki, J., Grabarek, Z., Brzeska, H., Drabikowski, W., and Cohen, P. (1981) FEBS Lett. 130, 141-145). Thus, peptide 78-148 behaves as a calmodulin agonist or antagonist or as neither, depending on the enzyme under study.
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PMID:Agonist and antagonist properties of calmodulin fragments. 632 72

Sperm cytosolic pH, determined by the spectral properties of intracellular carboxyfluorescein, is decreased rapidly by the diffusion and subsequent dissociation of the uncharged weak acids pyruvic, lactic, or hydroxybutyric and is increased by diffusion and subsequent intracellular protonation of the weak base NH3. Metabolic and kinetic activity increases dramatically when intracellular pH is elevated above 6.8-6.9 by addition of 50 mM NH4Cl to sperm suspended in a 120 mM NaCl medium. Respiratory stimulation is not observed upon comparable additions of 50 mM Li+ or K+ or when the pH of the medium is increased from 6.5 to 8.2. However, increases of the external pH to 7.8-8.2 in medium employing 120 mM KCl result in increased metabolic and kinetic activity, comparable to the maximal stimulation induced by the phosphodiesterase inhibitor caffeine. An increase in cytosolic pH from 6.3-6.6 to 6.8 occurs concomitant with the respiratory stimulation induced by KCl in alkaline media. No change in cytosolic pH follows addition of caffeine. Cyclic AMP-dependent protein kinase activity ratios, determined in cellular extracts, are increased by caffeine treatment but are not elevated by 120 mM KCl, by alkaline pH, or by their combination. These observations indicate that cytosolic pH plays a role in the regulation of motility and metabolism of mammalian sperm that is not mediated by cyclic AMP but that may be under control of a plasma membrane voltage-dependent proton channel. However, H+ fluxes across vesicles prepared from sperm membranes are unaffected by variation in the magnitude of the transvesicular K+ concentration gradient.
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PMID:Potassium-dependent increases in cytosolic pH stimulate metabolism and motility of mammalian sperm. 657 91

The effect of VIP on phagocytosis in peritoneal macrophages was examined by means of flow cytometry (FCM). This assay revealed that VIP suppressed phagocytosis in a dose-dependent manner. VIP(1-12) did not suppress phagocytosis. VIP(10-28) was more suppressive than VIP(1-28). A known VIP-antagonist (N-Ac-Tyr1,D-Phe2)-growth hormone-releasing factor (GRF) (1-29)-NH2 suppressed phagocytosis less than VIP. Control phagocytosis was partially suppressed in Ca(2+)-free solution. Phagocytosis was suppressed by VIP further in Ca(2+)-free solution than in the normal solution. Phagocytosis was suppressed in a known phosphodiesterase inhibitor IBMX-containing solution. The degree of suppression by VIP was the same in the presence or the absence of IBMX. These results suggest that VIP suppresses extracellular Ca(2+)-dependent and -independent phagocytosis, that the C-terminal fragment of VIP is essential for VIP action, that the suppression is mediated by cAMP and that the inhibition of macrophage phagocytosis by VIP is one of the mechanisms which modulates immune responses by the nervous system.
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PMID:Inhibitory effect of vasoactive intestinal peptide (VIP) on phagocytosis in mouse peritoneal macrophages. 753 35

We have examined the effects of the natriuretic peptides on DNA synthesis in primary cultures of neonatal rat cardiac fibroblasts. Binding analysis using 125I-labeled atrial natriuretic peptide identified a single class of high-affinity binding sites (Kd = 0.03 +/- 0.01 nmol/L) in these cells. Of these sites, 80% appear to be of the natriuretic peptide C receptor subtype, with the remainder being A and B receptor subtypes. Northern blot analysis confirmed the presence of all three natriuretic peptide receptors in these cells. Atrial natriuretic peptide (10(-7) mol/L) effected a modest but consistent reduction in both agonist- and stretch-stimulated [3H]thymidine incorporation (17% to 41%). Moreover, brain natriuretic peptide (10(-7) mol/L), C-type natriuretic peptide (10(-7) mol/L), and des-[Gln18,Ser19,Gly20,Leu21,Gly22]-ANF 4-23-NH2 (10(-7) to 10(-6) mol/L) all proved capable of antagonizing growth factor-dependent [3H]thymidine incorporation (the inhibition ranged from 14% to 28%) and cell proliferation, suggesting that all three natriuretic peptide receptor subtypes are involved in the regulation of mitogenesis in these cultures. The inhibition by atrial natriuretic peptide was amplified by cotreatment with phosphodiesterase inhibitors. Similar reduction in [3H]thymidine incorporation was seen after treatment with 8-bromo-cGMP (10(-4) to 10(-3) mol/L) or nitroprusside (10(-4) to 10(-3) mol/L). These results suggest an important paracrine role for the natriuretic peptides in regulating fibroblast growth during cardiac hypertrophy.
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PMID:Natriuretic peptides inhibit DNA synthesis in cardiac fibroblasts. 784 72

Activation of cGMP phosphodiesterase (PDE) by the rod G-protein transducin is a key event in visual signal transduction in vertebrate photoreceptor cells. Interaction between the GTP-bound form of the alpha-subunit of transducin (alpha t*) and the PDE inhibitory gamma-subunit (P gamma) is a major component of PDE activation. The central polycationic region of P gamma, P gamma-24-45, has been implicated as one of the sites involved in alpha t*.P gamma interaction. Here we determine the site on alpha t* that interacts with P gamma-24-45 using a photo-cross-linking approach. The synthetic peptides Cys(ACM)Tyr-P gamma-24-45-Cys (where ACM indicates acetamidomethyl group) and Cys-P gamma-24-45 were labeled with 4-(N-maleimido)benzophenone at the COOH and NH2 termini, respectively, and then cross-linked to alpha t. When the photoprobe was attached to the COOH terminus of the peptide, a specific high yield cross-linked product (80%) was formed between the peptide and alpha t GTP gamma S (guanosine 5'-O-(thiotriphosphate)). A lower yield of cross-linking (35%) was seen between the peptide and alpha t GDP. The site of cross-linking between Cys(ACM)Tyr-P gamma-24-45-Cys and alpha t GTP gamma S was localized to within alpha t-306-310 using a variety of chemical and proteolytic cleavages of the cross-linked product, analysis of the fragments with SDS-polyacrylamide gel electrophoresis, and matrix-assisted laser desorption ionization mass spectrometry.
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PMID:A site on transducin alpha-subunit of interaction with the polycationic region of cGMP phosphodiesterase inhibitory subunit. 822 88


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