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)

Glutamine synthetase from a Gram-positive acid-fast bacterium, Mycobacterium smegmatis, was purified to homogeneity from cells grown with glycerol-bouillon medium. Electron micrographs of the enzyme revealed a dodecameric arrangement of its subunits in two superimposed hexagonal rings, similar to the structure of glutamine synthetase of Escherichia coli. Disc electrophoresis in the presence of sodium dodecyl sulfate indicated a subunit molecular weight of 56,000. The sedimentation coefficient of the native enzyme was estimated to be 19.4S by ultracentrifugation in a sucrose gradient. Like the E. coli enzyme, the glutamine synthetase from M. smegmatis is regulated by adenylylation/deadenylylation. This conclusion was based on studies of the effect of snake venom phosphodiesterase treatment on the catalytic and spectral properties of the isolated enzyme. The AMP released from the enzyme by the phosphodiesterase was identified by thin-layer chromatography. Despite the structural similarity of both enzymes, striking differences were found between the catalytic properties of M. smegmatis and E. coli glutamine synthetases. The divalent cation specificity of the M. smegmatis enzyme was not altered by adenylylation of the enzyme, and deadenylylation of the enzyme caused a significant increase in the specific activities for both biosynthetic and transfer reactions with either Mg2+ or Mn2+.
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PMID:Regulation of Mycobacterium smegmatis glutamine synthetase by adenylylation. 614 40

Following the parenteral administration of tiazofurin, 2-beta D-ribofuranosylthiazole-4-carboxamide (thiazole nucleoside, TR), a potent but reversible inhibitor of IMP dehydrogenase is generated in subcutaneous nodules of the P388 leukemia. The compound responsible for this effect has been isolated from homogenates of the tumor by ion-exchange HPLC, and its presence monitored by enzyme-inhibition assay. The inhibitor has also been prepared by incubation of tiazofurin with P388 cells in culture. Chromatographically, the inhibitory principle exhibits a moderately strong set negative charge at pH 3, and elutes in the general vicinity of the nucleoside-5'-diphosphates; its absorption maximum in aqueous solution (pH 7) lies at 252 nm. Exposure of the molecule to snake-venom phosphodiesterase or to nucleotide pyrophosphatase destroys its inhibitory potency, whereas other phosphodiesterases are either less effective or inert. Since these results suggested that the anabolite might be a dinucleotide with a phosphodiester linkage of the kind found in NAD, attempts were made to synthesize such an analogue from the 5'-monophosphate of thiazole nucleoside and ATP-Mg2+, using a purified preparation of NAD pyrophosphorylase; modest yields were obtained of a compound with chromatographic, spectral and enzyme-inhibitory properties identical to those of the material isolated from P388 tumor nodules. This enzyme-synthesized material was radioactive when [3H]ATP was used as cosubstrate, and yielded both AMP and thiazole nucleoside-5'-monophosphate on treatment with phosphodiesterase. It resisted attack by NAD glycohydrolase. An apparently identical dinucleotide was also synthesized chemically by means of the Khorana condensation. Mass spectral analysis and nuclear magnetic resonance studies with homogeneous preparations of both the enzymically and chemically synthesized compound were compatible with its being a dinucleotide in which the nicotinamide of NAD has been replaced by thiazole-4-carboxamide. Versus IMP dehydrogenase, the dinucleotide exhibited a K1 of approximately 2 X 10(-7) M and was non-competitive with NAD as the variable substrate. Other NAD utilizing enzymes, including representative dehydrogenases and poly ADP ribose polymerase, were, by comparison to mammalian IMPD, resistant to inhibition by TAD. The properties of this novel dinucleotide are compared and contrasted with those of analogs of NAD containing modifications in the pyridine, adenine or ribofuranose rings, as well as in the pyrophosphate bridge.
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PMID:Studies on the mechanism of action of tiazofurin metabolism to an analog of NAD with potent IMP dehydrogenase-inhibitory activity. 615 29

The immunoelectrophoretic purity of the exonuclease preparation, isolated from Crotalus adamanteus venom according to a procedure previously published (Dolapchiev, L.B., Sulkowski, E. and Laskowski, M., Sr. (1974) Biochem. Biophys. Res. Commun. 61, 271-281), is reported. The enzyme showed one precipitin line against antibody prepared against partially purified venom. The exonuclease is unstable in dilute (1.25 microgram/ml and below) solutions. Bovine serum albumin stabilized the enzyme nonspecifically whereas the homologous antibody demonstrated a specific stabilizing effect under the same conditions. The binding of the anti-enzyme with the enzyme caused inhibition of both its activities--phosphodiesterase and pyrophosphatase. The inhibition of the exonuclease when attacking high molecular weight substrates is similar to the above and is of the same noncompetitive type. The thermal stability of venom exonuclease is reported.
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PMID:Venom exonuclease. III. Immunochemical characterization and modification by specific antibody. 616 75

The chemical synthesis of the tital bridged trinucleoside diphosphates 3e and 3f along with the corresponding dinucleoside phosphates 3c and 3d is described. Bridged nucleosides 3a and 3b gave on treatment with triethyl orthoformate in the presence of p-toluenesulfonic acid in dimethylformamide the cyclic orthoesters 2a and 2b. Condensation of 2a and 2b with N,2',5'-O-triacetylcytidine 3'-phosphate (1) using dicyclohexylcarbodiimide in pyridine afforded after deblocking and chromatographic separation products 3c-f. The latter were readily degraded with pancreatic RNase, but 3c and 3e were completely resistant toward snake venom phosphodiesterase whereas 3d and 3f were digested to the extent of 65 and 43%, respectively. The major product of degradation of 3f with phosphodiesterase was compound 3d resulting from the combined action of phosphodiesterase and contaminating phosphomonoesterase. The results are explained in terms of stacking of terminal bridge nucleoside units in 3c-f. The implications of these findings for the function of snake venom phosphodiesterase are discussed.
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PMID:Synthesis of dicytidylyl-(3'-5')-1,2-di(adenosin-N6-yl)ethane and dicytidylyl-(3'-5')-1,4-di(adenosin-N6-yl)butane: covalently joined terminals of two transfer ribonucleic acids and their behavior toward snake venom phosphodiesterase. 624 71

Snake venom phosphodiesterase (SVP) catalyzes the alcoholysis of ATP by primary R-CH2OH alcohols with uncharged R residues, yielding AMP-O-CH2R esterification products. The alcohols compete with water for an SVP-bound adenylyl intermediate. In this study, it has been shown that SVP also catalyzes the reactions of glycerol 2-phosphate and sn-glycerol 3-phosphate with ATP to yield AMP-O-glycerophosphoryl esters. The products were identified by HPLC, the dependency of the reactions on glycerol phosphates, ultraviolet spectroscopy, and conversion to AMP by phosphodiesterase, or to AMP-O-glyceryl esters by alkaline phosphatase. The results demonstrated that R-CH2OH alcohols with negatively charged R residues, as well as secondary alcohols, act as adenylyl acceptors in SVP reactions, thus extending the usefulness of SVP as a tool to produce 5'-nucleotide derivatives. The efficiencies (EA) of glycerol phosphates as adenylyl acceptors were very high at low, millimolar concentrations, but decreased abruptly when the acceptor concentration was increased and, for glycerol 2-phosphate, when Pi or NaCl was present. In contrast, glycerol EA was independent of its own concentration, Pi, and NaCl. The responses of glycerol phosphates indicate that they act as adenylyl acceptors via a mechanism different from uncharged R-CH2OH alcohols. The occurrence of an acceptor-binding enzyme site, specific for negatively charged R residues, and its potential relevance to the in vivo role of 5'-nucleotide phosphodiesterases as 5'-nucleotidyl transferases are discussed.
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PMID:High efficiency of glycerol 2-phosphate and sn-glycerol 3-phosphate as nucleotidyl acceptors in snake venom phosphodiesterase esterifications. Formation of primary and secondary AMP-O-glyceryl and AMP-O-glycerophosphoryl esters and evidence for an acceptor-binding enzyme site. 758 86

Mechanical stimulation of one mammary tumor cell in culture induced an increase in its intracellular calcium concentration which spread to surrounding cells. The increase in calcium can also be induced by addition of a solution in which cultured mammary tumor cells were stimulated by repeated pipetting (solution after pipetting cells, SAPC). The activity of the SAPC was completely abolished by treatment with snake venom phosphodiesterase or pyrophosphatase. Uridine triphosphate (UTP), uridine diphosphate (UDP) and ATP (1 microM each) were detected in the SAPC, whereas 5'-UMP and 5'-AMP were produced by phosphodiesterase digestion. A mixture of UTP, UDP and ATP (1 microM each) elicited a calcium response which was comparable to that induced by SAPC, while UTP, UDP or ATP alone at 1 microM elicited a small increase in calcium concentration in mammary tumor cells. Suramin, a competitive antagonist of P2 purinoceptors, diminished the spreading of the calcium wave induced by mechanical stimulation. It also blocked the responses to SAPC, UTP, UDP and ATP. These findings suggest that the mechanical stimulation results in the release of UTP, UDP and ATP into the extracellular space which mediates induction of the spreading calcium response via P2U-type purinoceptors.
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PMID:The increase in the intracellular Ca2+ concentration induced by mechanical stimulation is propagated via release of pyrophosphorylated nucleotides in mammary epithelial cells. 797 Nov 52

The deoxyribooligonucleotide 5'-d(CTCACATGTACACTCT) was reacted separately with the chiral diol epoxide isomers 7 beta, 8 alpha-dihydroxy-9 alpha, 10 alpha- epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene [(+)-anti-BPDE)] and 7 alpha, 8 beta-dihydroxy-9 beta, 10 beta-epoxy-7,8,9,10- tetrahydrobenzo[a]pyrene [(-)-anti-BPDE)], to produce the modified oligonucleotides 5'-d(CTCACATGBPDETACACTCT). Adducts in which either (+)-anti-BPDE or (-)-anti-BPDE are covalently bound via their C10 positions by trans addition to the exocyclic amino group of the single G residues were isolated and purified by HPLC methods. Snake venom phosphodiesterase (SVPD, phosphodiesterase I), which hydrolyzes DNA from the 3'-OH terminus to the 5'-end, digests the (+)-trans-anti-BPDE-oligonucleotide adducts at a significantly faster rate than that of the sterically different (-)-trans-anti-BPDE-oligonucleotide adducts. However, using spleen phosphodiesterase (SPD, phosphodiesterase II), which hydrolyzes DNA in the 5'-->3' direction, the opposite stereoselective resistance to digestion is observed. Using shorter BPDE-modified oligonucleotides as standards, the enzyme stall sites have been defined by gel electrophoresis methods; the most digestion-resistant phosphodiester linkage is the 5'-d(...T-G*...)-3' bond in the case of (+)-trans-BPDE-modified oligonucleotide adducts for both enzymes, SVPD and SPD (the starred G denotes the site of BPDE modification). In the case of the (-)-trans-BPDE-modified oligonucleotide adducts, the phosphodiester bond on the 3'-side of the modified G [5'-d(...G*-T...)-3'] is most resistant to digestion by both enzymes.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Opposite stereoselective resistance to digestion by phosphodiesterases I and II of benzo[a]pyrene diol epoxide-modified oligonucleotide adducts. 821 49

In order to stabilize 2-5A-antisense chimeras to exonucleases, we have synthesized chimeric oligonucleotides in which the last phosphodiester bond at the 3'-terminus of the antisense domain was inverted from the usual 3',5'-linkage to a 3',3'-linkage. The preparation of such analogues was accomplished through standard phosphoramidite chemistry with the use of a controlled pore glass solid support with a nucleoside attached through its 5'-hydroxyl, thereby permitting elongation at the 3'-hydroxyl. The structures of such terminally inverted linkage chimeras of the general formula pA4-[pBu]2-(pdNn3'-3'dN) were corroborated by a combination of snake venom phosphodiesterase digestion in the presence or absence of bacterial alkaline phosphatase. Most characteristically, the presence of the 3'-terminal-inverted phosphodiester linkage produced an unnatural dinucleotide of general composition dN3'p3'dM. These structures could be confirmed by independent synthesis and fast atom bombardment mass spectroscopy (FAB). 2-5A-Antisense chimeras of this structural class, pA4-[pBu]2-(pdNn'3-3'dN), were 5-6-fold more stable than their unmodified congeners, pA4-[pBu]2-(pdN)n, to degradation by a representative phosphodiesterase from snake venom. In 10% human serum, the new 2-5A-antisense chimeras, pA4-[pBu]2-(pdNn3'-3'dN), possessed a half-life that was 28-fold longer than that of the unmodified chimeras. These results provide entry to a second generation of 2-5A-antisense chimeras.
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PMID:Synthesis and properties of second-generation 2-5A-antisense chimeras with enhanced resistance to exonucleases. 928 79

We describe physicochemical and enzymatic properties of 5' bridging phosphorothioester linkages at specific sites in DNA oligonucleotides. The susceptibility to hydrolysis at various pH values is examined and no measurable hydrolysis is observed at pH 5-9 after 4 days at 25 degrees C. The abilities of three 3'- and 5'-exonuclease enzymes to hydrolyze the DNA past this linkage are examined and it is found that the linkage causes significant pauses at the sulfur linkage for T4 DNA polymerase and calf spleen phosphodiesterase, but not for snake venom phosphodiesterase. Restriction endonuclease (Nsi I) cleavage is also attempted at a 5'-thioester junction and strong resistance to cleavage is observed. Also tested is the ability of polymerase enzymes to utilize templates containing single 5'-S-thioester linkages; both Klenow DNA polymerase and T7 RNA polymerase are found to synthesize complementary strands successfully without any apparent pause at the sulfur linkage. Finally, the thermal stabilities of duplexes containing such linkages are measured; results show that T m values are lowered by a small amount (2 degrees C) when one or two thioester linkages are present in an otherwise unmodified duplex. The chemical stability and surprisingly small perturbation by the 5' bridging sulfur make it a good candidate as a physical and mechanistic probe for specific protein or metal interactions involving this position in DNA.
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PMID:Chemical and enzymatic properties of bridging 5'-S-phosphorothioester linkages in DNA. 962 13

Lysophospholipids are liberated during venomous action. In this study we demonstrated that lysophosphatidyl choline (LPC) of various acyl chains enhances considerably the activity of snake venom phosphodiesterase (PDE). Lysophosphatidic acid (LPA) and its cyclic form (cLPA), on the other hand, were found to inhibit this enzyme in a non-competitive (LPA) or competitive (cLPA) manner. Both of these activities may contribute to the progression and subsidence of the poisoning profile. PDE from cellular origin was not substantially affected by any of the above lysophospholipids.
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PMID:Enhancement and inhibition of snake venom phosphodiesterase activity by lysophospholipids. 978 90


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