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)

New spin-labeled analogs of nucleoside triphosphates, 8-amino(2,2,6,6-tetramethylpiperidine-N-oxyl)adenosine 5'-triphosphate ((8-AmTEMPO)ATP) and 5-amino(2,2,6,6-tetramethylpiperidine-N-oxyl)uridine 5'-triphosphate ((5-AmTEMPO)UTP), with the probe 4-amino(2,2,6,6-tetramethylpiperidine-N-oxyl) (4-AmTEMPO) attached to C-8 of ATP and C-5 of UTP via a secondary amine bond, were synthesized in 50 and 40% yield, respectively. These analogs showed a single spot by thin layer chromatographic analysis. The absorption spectra of (8-Am-TEMPO)ATP and (5-AmTEMPO)UTP exhibit maxima at 310 and 265 nm, respectively; their X-band EPR spectra have a typical three-line pattern with lines at 3,221, 3,239, and 3,257 Gauss. The intensity ratios for mid to high field lines of the EPR derivative lines were found to be 1.03 +/- 0.02, 1.08 +/- 0.04, and 1.15 +/- 0.07 for 4-AmTEMPO, (8-AmTEMPO)ATP, and (5-AmTEMPO)UTP, respectively. The immobilization of 4-AmTEMPO bound to C-8 of ATP or bound to C-5 of UTP was observed to be 5 and 11%, respectively, as compared with free 4-AmTEMPO. The initial velocity (s-1) of [3H]UMP incorporation into RNA in the presence of [3H]UTP, CTP, GTP, and (8-AmTEMPO)ATP or ATP was measured. The percent incorporation of (8-AmTEMPO)ATP into RNA product by Escherichia coli RNA polymerase using various DNA templates is 68, 66, and 61% for pAR1435 (plasmid containing A1 promoter from T7 DNA), calf thymus DNA, and poly(dA-dT) respectively, as compared with ATP incorporation. The polymerase-catalyzed reaction of (8-AmTEMPO)ATP with (3'-OCH3)UTP yielded 5'-triphosphate delta-amino(2,2,6,6-tetramethylpiperidine-N-oxyl)adenylyl (3'-5')3'-methoxy uridine in the presence of poly(dA-dT). The structure of this spin-labeled dinucleotide was identified by paper chromatographic analysis of the products of phosphodiesterase digestion. These analogs also can be used for the study by EPR spectroscopy of the dynamics of gene transcription catalyzed by RNA polymerases or of other nucleotide-utilizing enzymes.
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PMID:Spin-labeled nucleotide substrates for DNA-dependent RNA polymerase from Escherichia coli. 165 31

New fluorescent derivatives of dinucleoside monophosphates, (5'-AmNS)UpA/ApU/GpU/CpA, with a fluorophore, 1-aminonaphthalene-5-sulfonic acid (AmNS), attached to the first nucleotide of the dinucleoside monophosphates via a 5'-secondary amine linkage were synthesized in good yield. The chemical structure of (5'-AmNS)ApU was proved by the phosphodiesterase digestion followed by Whatman No. 3MM paper chromatographic and spectroscopic analysis of the digested products. The ability of these analogs to be incorporated into the 5' terminus of RNA chain forming fluorescent oligonucleotides by Escherichia coli RNA polymerase was studied in the presence of a synthetic DNA template. The enzymatic reaction of (5'-AmNS)UpA and [3H]UTP in the presence of poly(dA-dT) yielded (5'-AmNS)UpAp[3H]U in greater than 30% yield with the Km values of 5 and 2.5 microM and Vmax values of 17 and 25 nmol/min/mg of enzyme for (5'-AmNS)UpA and UpA, respectively. The structure of this fluorescent trinucleotide was identified by RNase A digestion and paper chromatographic analysis of the digested products. (5'-AmNS)UpA or (5'-AmNS)ApU exhibits two absorption maxima around 270 and 340-350 nm and a fluorescent emission maximum at 445 nm when excited at 340 nm. These spectral characteristics permit their use as energy donors for the transfer of energy to the intrinsic cobalt of the cobalt-substituted RNA polymerases. Upon hydrolysis of the phosphodiester bond of these analogs by venom phosphodiesterase, the absorption at 340 and 270 nm increased by 5 and 20%, respectively, while their fluorescence at 445 nm was enhanced by 25%. Thus, these analogs can be used for studying the dynamics of initiation and elongation reactions catalyzed by DNA-dependent RNA polymerases by absorption and fluorescence spectroscopies.
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PMID:Synthesis and characterization of fluorescent dinucleotide substrate for the DNA-dependent RNA polymerase from Escherichia coli. 244 Aug 71

Ventricular myocytes isolated from the hypertrophied hearts of thyrotoxic adult rats have an increase in mean protein content per myocyte (6.3 +/- 0.2 vs. 4.4 +/- 0.2 ng) compared with euthyroid cells. Viability and adenine nucleotide profiles are similar in both populations, but NAD content of the hyperthyroid myocytes is depressed (4.9 +/- 0.2 vs. 5.5 +/- 0.2 nmol/mg for controls) and UTP is higher (1.2 +/- 0.09 vs. 0.9 +/- 0.04 nmol/mg). Binding of (-)-[125I]iodocyanopindolol to intact hyperthyroid myocytes is increased by 42% compared with controls, with no change in the dissociation constant (Kd). This elevation in beta-receptor number is correlated to enhanced beta-agonist-induced adenosine 3',5'-cyclic monophosphate (cAMP) production. The half-maximal effective concentration (EC50) for the euthyroid isoproterenol dose-response curve is 2.14 x 10(-7) M but is decreased to 2.51 x 10(-8) M in hyperthyroid cardiac cells. Basal adenylate cyclase activity is apparently not affected by thyroid hormones, since basal cAMP levels for both groups are identical (5 pmol/mg) and both rise roughly twofold in the presence of a phosphodiesterase inhibitor. Forskolin-induced cAMP production and cAMP-specific phosphodiesterase activity are similar as well. In contrast to beta-adrenergic response, there are no significant differences in alpha 1-antagonist [3H]prazosin binding parameters between hyperthyroid and euthyroid cardiomyocytes.
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PMID:Hyperthyroid adult rat cardiomyocytes. I. Nucleotide content, beta- and alpha-adrenoreceptors, and cAMP production. 248 Jul 17

We report the purification of a CaATPase of high specific activity from Paramecium tetraurelia. The enzyme is preferentially released into solution upon deciliation of cells by a Ca2+ shock procedure. Purification by ion exchange and gel filtration chromatography yields major peptides of 68 and 53 kDa and a minor peptide of 58 kDa, as determined by electrophoresis on sodium dodecyl sulfate polyacrylamide gels. These three peptides yield similar proteolytic peptide maps. Rabbit antisera to the purified enzyme inhibit enzyme activity and specifically label 68- and 53-kDa bands on nitrocellulose blots of the deciliation supernatant from which the enzyme is isolated. Concanavalin A-Sepharose precipitates about 60% of ATPase activity; only the 53-kDa band binds concanavalin A on nitrocellulose blots. The purified enzyme has a specific activity of 620 +/- 70 mumol/min/mg with ATP as substrate in the presence of Ca2+, which is required for enzyme activity. As substrates, ATP and GTP are strongly preferred to UTP and CTP. The Km for ATP in the presence of 3 mM Ca2+ is approximately 20 microM. Enzyme activity is strongly inhibited by the calmodulin antagonists trifluoperazine, fluphenazine, W7, and calmidazolium. However, calmodulin is not associated with the purified enzyme, based on the enzyme's inability to bind anti-calmodulin antibodies or to stimulate brain phosphodiesterase. The intracellular origin of this ATPase, its possible function, and its relationship to several other ATPases of Paramecium are discussed.
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PMID:Purification and characterization of a calcium-dependent ATPase from Paramecium tetraurelia. 252 45

UDP-glucose:glycoprotein glucose-1-phosphotransferase (Glc-phosphotransferase) catalyzes the transfer of alpha-Glc-1-P from UDP-Glc to mannose residues on acceptor glycoproteins. The predominant acceptor for this transfer in both mammalian cells and Paramecium is a cytoplasmic glycoprotein of 62-63 kDa. When cytoplasmic proteins from rat liver were fractionated by preparative isoelectric focusing following incubation of a liver homogenate with the 35S-labeled phosphorothioate analogue of UDP-Glc ([beta-35S]UDP-Glc), the acceptor was found to have a pI of about 6.0. This fraction, when not labeled prior to the focusing, became very heavily labeled when mixed with [beta-35S]. UDP-Glc and intact liver microsomes, a rich source of the Glc-phosphotransferase. In addition, it was observed that the isoelectric fractions of the cytosol having pI values of 2-3.2 contained a degradative activity, alpha-Glc-1-P phosphodiesterase, that was capable of removing alpha-Glc-1-P, monitored through radioactive labeling both in the sugar and the phosphate, as an intact unit from the 62-kDa acceptor. Identification of the product of this cleavage was substantiated by its partial transformation to UDP-Glc in the presence of UTP and UDP-Glc pyrophosphorylase. The alpha-Glc-1-P phosphodiesterase had a pH optimum of 7.5 and was not effectively inhibited by any of the potential biochemical inhibitors that were tested. Specificity for the Glc-alpha-1-P-6-Man diester was suggested by the diesterase's inability to degrade UDP-Glc or glucosylphosphoryldolichol. This enzyme may be important in the regulation of secretion since the alpha-Glc-1-P present on the 62-kDa phosphoglycoprotein appears to be removed and then rapidly replaced in response to secretagogue.
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PMID:An alpha-glucose-1-phosphate phosphodiesterase is present in rat liver cytosol. 255 63

The non-differentiated HL60 cell can be stimulated to secrete when Ca2+ and guanosine 5'-[gamma-thio]-triphosphate (GTP gamma S) are introduced into streptolysin-O-permeabilized cells. Secretion is accompanied by activation of polyphosphoinositide phosphodiesterase (PPI-pde). Both responses show a concentration-dependence on Ca2+ between pCa 8 and pCa 5. The half-maximal requirements for Ca2+ for PPI-pde activation and secretion are pCa 6.4 +/- 0.1 and pCa 6.2 +/- 0.2 respectively. The rank order of potency of the GTP analogues to stimulate PPI-pde activation and secretion is similar; GTP gamma S greater than guanosine 5'-[beta gamma-imido]-triphosphate greater than guanosine 5'-[beta gamma-methylene]triphosphate greater than XTP approximately equal to ITP, but the maximal response achieved by each compound compared with GTP gamma S is much greater for secretion than for PPI-pde activation. A dissociation of the two responses is obtained with 10 mM-XTP and -ITP; secretion is always observed but not inositol trisphosphate formation at this concentration. GTP, dGTP, UTP and CTP are inactive for both secretion and PPI-pde activation. Both GDP and dGDP are competitive inhibitors of both GTP gamma S-induced secretion and PPI-pde activation. Phorbol 12-myristate 13-acetate could not fully substitute for GTP gamma S in stimulating secretion, suggesting that the effect of GTP gamma S cannot result simply from the generation of diacylglycerol. In the absence of MgATP, secretion and PPI-pde activation is still evident, albeit at a reduced level. This also supports the hypothesis that protein kinase C-dependent phosphorylation is not essential for secretion. The effect of MgATP is to enhance secretion, and to reduce both the Ca2+ and GTP gamma S requirement for secretion. In conclusion, two roles for guanine nucleotides can be identified; one for activating PPI-pde (GP) and the other for activating exocytosis (GE), acting in series.
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PMID:Guanine nucleotides stimulate polyphosphoinositide phosphodiesterase and exocytotic secretion from HL60 cells permeabilized with streptolysin O. 283 41

1. The particulate form of lactating bovine mammary lactose synthetase activity is shown to be more highly organized than previously reported. 2. A novel method of shattering frozen mammary tissue with effective cell disruption is described. 3. The apparent subcellular distribution of lactose synthetase was shown to reflect the method of homogenization. 4. After mild homogenization particles associated with a high content of intact lactose synthetase activity sedimented in the lysosome size range between 5x10(4) and 3x10(5)g-min. 5. Lactose synthetase was dissociated and solubilized by VirTis homogenization and ultrasonic treatment. The activities and behaviour of UDP-galactose hydrolase, succinate dehydrogenase, beta-glucuronidase and phosphodiesterase I were compared. 6. Inhibition of UDP-galactose hydrolase by UTP and alpha-lactalbumin was observed.
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PMID:The lactose synthetase particles of lactating bovine mammary gland. Preparation of particles with intact lactose synthetase. 430 May 6

Mg2+-ATPase activity was identified in the cytosol of human erythrocytes. A partial purification of this activity was achieved by an initial DEAE-Sephadex column chromatography, followed by gel filtration on Sephadex G-100 and then a second DEAE-Sephadex chromatography procedure. The enzyme appeared in the void volume of the Sephadex G-100 column and was retained on an Amicon XM100A ultrafiltration membrane. The molecular weight of the enzyme was estimated to be 113 000 from SD gels. The above purification protocol yielded an enzyme with an optimal pH between 7.6 and 8.2. The enzyme activity increased linearly between 30 and 44 degrees C. It was stable for several months at -20 degrees C. Magnesium was essential for activity, but the rate attainable with Mn2+ was at least as great as that due to Mg2+. No other divalent cation was able to substitute for Mg2+ or Mn2+. Neither low nor high Ca2+ concentrations significantly affected the enzymatic activity. Substrate specificity studies showed that ATP was the preferred substrate followed by CTP (46% of the rate produced by ATP). Hydrolysis of GTP, UTP, ITP and ADP was less than 10% of the rate seen with ATP. No phosphatase, pyrophosphatase, phosphodiesterase, hexokinase, phosphofructokinase or adenylate cyclase activity could be detected in this enzyme preparation. Calmodulin, which stimulates the (Ca2+ + Mg2+)-ATPase of the human erythrocyte membrane, failed to enhance the Mg2+-ATPase activity. Of considerable interest, the activity of this Mg2+-ATPase was enhanced approximately 5-fold by low concentrations of mercuric ion, p-hydroxymercuribenzoate and DTNB, but was much less sensitive to iodoacetamide.
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PMID:Partial purification and characterization of a novel Mg2+-dependent ATPase present in the cytosol from human erythrocytes. 615 Jul 30

The in vitro effects of cyclic AMP on amino acid transport and synthesis of macromolecules in the Swarm rat chondrosarcoma were investigated using the cyclic AMP analogue, N6-monobutyryl cyclic AMP (MBcAMP), and the phosphodiesterase inhibitor, 3-isobutyl-1-methylxanthine (MIX). Amino acid transport was assessed by measuring alpha-amino-[1-14C]isobutyrate (AIB) uptake. The synthesis of macromolecules was estimated by measuring radiolabeled precursor incorporation into total proteins, proteoglycan, and RNA. MBcAMP stimulated [14C]AIB uptake, [3H]uridine transport, and UTP formation. MBcAMP inhibited 35SO4 and [3H]leucine incorporation into proteoglycan and stimulated [3H]uridine incorporation into RNA. MIX elevated endogenous cyclic AMP levels in the Swarm rat chondrosarcoma and mimicked the effects of MBcAMP on AIB transport and radiolabeled precursor incorporation into macromolecules. For comparative purposes, the effects of MBcAMP on AIB uptake and macromolecule synthesis in rat costal cartilage segments were investigated. MBcAMP and MIX stimulated AIB uptake by costal cartilage segments, inhibited [3H]leucine incorporation into total protein and 35SO4 incorporation into proteoglycan, and had no effect on [3H]uridine incorporation into RNA.
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PMID:Regulation of chondrosarcoma metabolism by cyclic adenosine 3'5'-monophosphate. 615 89

ATP pyrophosphohydrolase was partially purified from fetal bovine epiphyseal cartilage. The purification was about 10- and 100-fold over the enzyme activities of matrix vesicle fraction and cell homogenate, respectively. The pyrophosphohydrolase and alkaline phosphatase were separated by a sequential application of Sepharose CL-6B and DEAE-cellulose column chromatographies. The purified enzyme migrated as a single band corresponding to the molecular weight of 230,000 in sodium dodecyl sulfate-polyacrylamide disc gel by electrophoresis. The enzyme absolutely required Zn2+ for its activity and appeared to bind Zn2+ strongly with an apparent affinity of p[Zn2+]0.5 = 13.4. The apparent Km for ATP was 0.18 mM. The enzyme was also reactive toward various nucleoside triphosphates including GTP, CTP, and UTP. In contrast, various phosphodiesters including RNA, UDP-glucose, NAD, and bis-p-nitrophenylphosphate were 5% or less as reactive as the nucleoside triphosphates. The pyrophosphohydrolase was inactive toward adenosine 3':5'-monophosphate or various phosphonates. UDP-glucose (1 mM), NAD (1 mM), or RNA (1 mg/ml) failed to inhibit the ATP pyrophosphohydrolase activity. These observations suggest that the ATP pyrophosphohydrolase of the cartilage is probably not a phosphodiesterase I. The matrix vesicle fraction, which probably also included some plasma membrane vesiculated during collagenase digestion, contained the highest specific activity of the enzyme as compared to other subcellular fractions of either epiphyseal or articular cartilage.
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PMID:Purification and partial characterization of ATP pyrophosphohydrolase from fetal bovine epiphyseal cartilage. 621 90


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