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:3.1.30.2 (endonuclease)
18,621 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Analogs of the 2',5'-linked adenylate trimer diphosphate (pp5'A2'p5'A2'p5'A or 2-5A) containing 8-bromoadenosine in the first, second, third, first and third, or second and third nucleotide positions (from the 5' terminus) were synthesized and found to vary dramatically in their ability to bind to and activate the RNase L of mouse L cells. Whenever the 8-bromoadenosine residue was substituted for adenosine in the first or 5'-terminal residue, there resulted a marked decrease in ability to bind to the 2-5A-dependent endonuclease. A similar result was obtained when the second adenosine nucleotide was replaced by 8-bromoadenosine. To the contrary, all analogs that bore an 8-bromoadenosine (br8A) in the third or 2'-terminal position were bound about as well as parent 2-5A to RNase L. Additionally, the 5'-diphosphate pp5'A2'p5'A2'p5' (br8A) was 10 times more effective than 2-5A as an inhibitor of translation. An increase in stability could not explain this significantly enhanced ability since the 2'-terminally brominated analog showed a similar half-life to 2-5A itself. Finally of particular interest was the analog monophosphate p5'A2'p5'(br8A)2'p5'(br8A) which possessed nearly 10% of the translational inhibitory activity of 2-5A triphosphate itself. These results suggest that changes in the base-sugar torsion angles of 2-5A may modulate both binding to and activation of mouse L cell RNase L.
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PMID:Purine 8-bromination modulates the ribonuclease L binding and activation abilities of 2',5'-oligoadenylates. Possible influence of glycosyl torsion angle. 381 84

Recently developed chemical synthetic methodology for facile preparation of sequence-specific 2'5'-oligonucleotides has allowed more exacting questions to be asked regarding the biological role of each of the nucleotide residues of 2-5A. We have found, employing analogs in which each adenosine residue has been sequentially replaced by adenosine (viz, ppp5'I2'p5'A2'p5'I, ppp5'A2'p5'I2'p5'A, ppp5'A2'p5'A2'p5'I) that the N6 amino group of the first (or 5'-terminal) adenosine residue of 2-5A trimer is critical in RNase L binding whereas the N6 amino moiety of the third (or 2'-terminal) adenosine residue of 2-5A is crucial for the activation of RNase L. The second or middle adenosine unit of 2-5A does not seem to be critical for either binding or activation. Similarly, in studies on sequence-specific tubercidin analogs of 2-5A, activation of but not binding to mouse RNase L was dependent on the presence of the purine N7 atoms of the first and third adenosine residues of 2-5A, but, as with the N6 amino group, the N7 moiety of the second adenosine residue of 2-5A was not essential for either binding to or activation of the 2-5A-dependent endonuclease. Finally, sequence-specific purine 8-bromination provided analogs of dramatically varying biological properties, and provided a 5'-monophosphate, p5'A2'p5'(br8A)2'p5'(br8A), which possessed 8% of the translational inhibitory action of 2-5A itself. This latter result may represent an important impetus toward obtaining a 2-5A derivative with biological activity in an intact cell.
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PMID:Sequence-specific 2'5'-oligonucleotides in the molecular dissection of the biological activity of 2-5A. 383 77

c-myc gene mRNA is reduced by greater than 75% in the human lymphoblastoid cell line Daudi when growth is inhibited by treatment with human interferon beta (IFN-beta). In the present communication, we describe the effect of IFN-beta treatment on transcription of the c-myc gene and on the steady-state level of c-myc mRNA in the cytoplasm of Daudi cells. The results show that, although the rate of c-myc transcription is not significantly different in nuclei isolated either from untreated cells or from those treated with IFN-beta for 3 or 24 hr, the level of c-myc mRNA in the cytoplasm is reduced by 60% within 3 hr of IFN-beta treatment. These results suggest that IFN-beta regulates the c-myc mRNA at a post-transcriptional level. These results are in contrast to the regulation of two IFN-beta-induced genes that under identical conditions are regulated in these cells at the transcriptional level. We have also detected induction of the (2'-5')oligoadenylate synthetase (2-5A synthetase) gene in IFN-beta-treated Daudi cells. Since certain c-myc transcripts have the capacity to form double-stranded RNA regions, we propose that one mechanism by which c-myc could be regulated post-transcriptionally in IFN-beta-treated cells is by activating, through its own double-strandedness, the 2-5A synthetase/RNase L endonuclease system, which would cause selective degradation of the c-myc RNA.
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PMID:Interferon regulates c-myc gene expression in Daudi cells at the post-transcriptional level. 385 53

pppA(2'p5'A)n-1 ((2'-5')(A)n) synthetase is one of the mediators of interferon action. On activation by double-stranded RNA, it converts ATP into (2'-5')(A)n; in turn, (2'-5')(A)n activates an endonuclease (RNase L) which cleaves single-stranded RNA. We report a simple procedure for the isolation of pure (2'-5')(A)n synthetase from interferon-treated Ehrlich ascites tumor cells. The procedure involves differential precipitation of the ribosomal salt wash fraction with ammonium sulfate and chromatography on DEAE-cellulose and CM-cellulose. The apparent molecular weight of the enzyme is 105,000 as determined by gel electrophoresis in sodium dodecyl sulfate and about 85,000 when determined by centrifugation through a glycerol gradient. The size range of the (2'-5')(A)n produced by the enzyme extends from the dimer to at least the pentadecamer.
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PMID:Interferon, double-stranded RNA, and RNA degradation. Isolation of homogeneous pppA(2'p5'A)n-1 synthetase from Ehrlich ascites tumor cells. 615 3

Extracts from interferon-treated, not virus-infected Ehrlich ascites tumor cells differ in various biochemical characteristics from extracts of control cells. We studied three enzymes whose level is enhanced in cells upon treatment with IF and which are causing some of the differences. (2'-5')(A)n synthetase, an enzyme converting ATP into a series of (2'-5') linked oligoadenylates ((2'-5')(An)) in the presence of dsRNA was purified to homogeneity and characterized. The second enzyme, RNase L, a latent endonuclease, which can be activated by (2'-5')(A)n to cleave single-stranded RNAs, was purified several hundredfold. The activation of this enzyme is reversible and is lost upon removal of (2'-5')(A)n. The activation is not accompanied by a large change in shape of conformation of the enzyme. The third enzyme is a protein kinase which if activated by dsRNA can phosphorylate the peptide chain initiation factor eIF-2 and a protein designated P1 of 67,000 daltons. This enzyme was purified several thousandfold. The most highly purified preparation consists of three proteins with P1 as the most abundant component.
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PMID:Double-stranded RNA and the enzymology of interferon action. 616 95

One of the mediators of interferon action is an endonuclease system. This consists of (2'-5')(A)n synthetase, which, if activated by double-stranded RNA, converts ATP into (2'-5')(A)n and RNase L, a latent endoribonuclease, which binds (2'-5')(A)n and is thereby activated. We report here that a derivative of (2'-5')(A)n (i.e. (2'-5')pppApApA[32P]pCp) can be covalently cross-linked by UV irradiation to a protein in cytoplasmic extracts from mouse Ehrlich ascites tumor cells. This protein has an apparent molecular weight of 77,000 as determined by gel electrophoresis in sodium dodecyl sulfate. It appears to be identical with RNase L according to the following criteria: co-chromatography on DEAE-cellulose and Sephacryl S300. The gel filtration in Sephacryl S300 reveals that the apparent molecular weight of the protein is between 70,000 and 90,000, indicating that it is a monomer. The cross-linking is oligonucleotide specific. It is inhibited by 10 nM (2'-5')pppApApA or 1 microM (2'-5')ApApA, i.e, compounds known to block, even at low concentration, the binding of (2'-5')pppApApApCp to RNase L. (3'-5')ApApA inhibits only at a 0.1-1 mM concentration, and 1 mM ATP, 2'-AMP, or 5', 3'-pCp have no effect. (2'-5')pppApApApCp was also cross-linked to a protein with a molecular weight of about 78,000 (as determined by gel electrophoresis in sodium dodecyl sulfate) in cytoplasmic extracts from human (HeLa) cells and to protein(s) with molecular weight(s) of 75,000-77,000 (as determined by gel electrophoresis in sodium dodecyl sulfate) in nuclear extracts from Ehrlich ascites tumor cells.
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PMID:Interferon action. Covalent linkage of (2'-5')pppApApA(32P)pCp to (2'-5')(A)n-dependent ribonucleases in cell extracts by ultraviolet irradiation. 617 33

Treatment with interferon protected HeLa cells from infection with reovirus. This virus apparently activated an antiviral mechanism that was detected by the presence of (2'-5')oligoadenylate [(2'-5')An] in intact cells. The (2'-5')An was previously shown to activate an endoribonuclease, RNase L. We measured (2'-5')An by a sensitive competition-binding assay in cells infected at different multiplicities and for different lengths of time. Nanomolar concentrations of (2'-5')An were detected in cells infected at a multiplicity of greater than 5 after 2 h of infection, the time at which the infecting virions were uncoated. The level of (2'-5')An increased up to 6 h postinfection but declined afterward. To establish whether viral mRNAs were cleaved by RNase L, we analyzed the RNA extracted from infected cells by a highly specific hybridization assay on Northern blots. Full-sized reovirus mRNAs were detected in control infected cells, but not in interferon-treated infected cells, at 6 h postinfection. At this time, a nuclease activity could be detected in these cells by demonstration of cleavage of rRNA, degradation of cellular mRNA, and polysome breakdown in the presence of emetine. Since this inhibitor freezes ribosomes, cleavage of mRNA between ribosomes could only be accounted for by an endonuclease, presumably RNase L.
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PMID:Synthesis of (2'-5')oligoadenylate and activation of an endoribonuclease in interferon-treated HeLa cells infected with reovirus. 617 44

To determine the relative importance of the 2',5'-phosphodiester bond of 2-5A in its binding to and activation of the 2-5A-dependent ribonuclease (RNase L, RNase F), a number of phosphodiester linkage isomers of 2-5A were prepared. These isomers were obtained either by lead ion-catalyzed polymerization of adenosine 5'-phosphorimidazolidate or by T4 polynucleotide kinase-catalyzed 5'-phosphorylation of adenylyl(3' leads to 5')adenylyl(3' leads to 5')adenosine followed by reaction of the corresponding phosphorimidazolidates with tri(n-butylammonium)pyrophosphate. The following 2-5A isomers thus were prepared: ppp5'A2'p5'A3'p5'A, ppp5'A3'p5'A2'p5'A, ppp5'A3'p5'A3'p5'A("3-5A"), ppp5'A2'p5'A3'p5'A2'p5'A,and ppp5'A3'p5'A2'p5'-A2'p5'A. The ability of these isomeric 2-5As to interact with the 2-5A-dependent endonuclease was ascertained by three different criteria: (i) ability to prevent the protein synthesis inhibitory effects of 2-5A, (ii) activity as an inhibitor of translation in encephalomyocarditis RNA-programmed L cell extracts, and (iii) ability to prevent binding of the radiolabeled probe, ppp5'A2'p5'A2'p5'A2'p5'A3'[32P]p5'Cp, to the endonuclease of L cell extracts. In certain experiments, degradation of oligonucleotide was minimized or eliminated by altering assay conditions, providing alternate phosphodiesterase substrates, or by using purified endoribonuclease of Ehrlich ascites cells. By all criteria, replacement of 2',5'-bond by a 3',5'-bond led to a substantial decrease in biological activity. Generally, replacement of just one 2',5'-phosphodiester bond with a 3',5'-linkage led to at least a one order of magnitude loss of activity. In accord with this trend, ppp5'A3'p5'A3'p5'A(3-5A) was greater than 10,000 less active than 2-5A in binding to the endonuclease or as an inhibitor of protein synthesis.
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PMID:Biological activities of phosphodiester linkage isomers of 2-5A. 663 Feb 22

Interferon induces the synthesis of an enzyme which synthesizes 2',5'-oligoadenylate [2',5'-oligo(A)] when activated by double-stranded RNA. The 2',5'-oligo(A) in turn activates an endonuclease (RNase L). Concentrations of 2',5'-oligo(A) sufficient to activate RNase L are formed in interferon-treated HeLa cells infected with reovirus, and a large fraction of cellular mRNA is degraded (T. W. Nilsen, P. A. Maroney, and C. Baglioni, J. Virol. 42:1039-1045, 1982). We report here that in spite of this mRNA degradation, protein synthesis was not significantly inhibited in these cells. When mRNA synthesis was inhibited with 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole, protein synthesis was markedly decreased, as shown by reduced incorporation of labeled amino acids and a decrease in polyribosomes. This suggested that the turnover of mRNA could be compensated for by increased production of mRNA. The relative concentration of specific mRNAs was measured with cloned cDNA probes. The amount of these mRNAs present in control cells was comparable to that in interferon-treated cells infected with reovirus, whereas it was decreased in the latter cells treated with 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole.
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PMID:Maintenance of protein synthesis in spite of mRNA breakdown in interferon-treated HeLa cells infected with reovirus. 682 30

We have measured the binding of highly radioactive (2'-5')pppA4-5[32P]cytidine 3',5'-diphosphate to human, mouse, and rabbit cell and tissue extracts. A binding activity for this oligonucleotide is present in all extracts examined and high levels of this activity are found in some cells of lymphoid origin. In particular, the mouse thymoma cell line W7 has about 10 times higher binding activity than other cell lines. The oligonucleotide is bound with a single high affinity constant, as shown by Scatchard plot analyses. Cell extracts fractionated by centrifugation on glycerol gradients show a single peak of binding activity, which co-sediments with (2'-5')oligo(A)-dependent endoribonuclease (RNase L). This enzyme apparently binds the oligonucleotide, as shown by experiments with an analog of (2'-5')oligo(A) which competes in the binding and inhibits the RNase L. This endonuclease cannot be directly assayed in unfractionated cell or tissue extracts with high levels of other nuclease activities. The binding assay for the oligonucleotide may provide an estimate of RNase L levels in such cell and tissue extracts.
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PMID:2',5'-Oligo(A)-activated endoribonuclease. Tissue distribution and characterization with a binding assay. 728 31


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