Assay of 2',5'-oligoadenylate phosphodiesterase activity in mouse L-cell extracts

A rapid and convenient assay for adenylyl(2' leads to 5')adenosine(A2'p5'A) or adenylyl(3' leads to 5')adenosine(A3'p5'A) phosphodiesterase activities is described. The dinucleotides A3'p5'A and A2'p5'A were labeled to a high specific activity by means of a catalytic-exchange procedure. Degradation studies of each of these labeled dinucleotides showed an asymmetrical distribution of label between the two adenine bases. Enzymatic degradation of [3H]A3'p5'A or [3H]A2'p5'A could be quantitated by first digesting the reaction products with bacterial alkaline phosphatase and then adding a slurry of DEAE-Sephadex. Under conditions described, adenosine did not adsorb to the resin, whereas dinucleotides as well as AMP did adsorb. As a consequence, when liquid scintillation fluid was added to the DEAE-Sephadex reaction mixture slurry, the radioactivity of the dinucleotides and AMP was severely quenched. This permitted a direct estimation of the amount of adenosine liberated during the phosphodiesterase degradation and subsequent alkaline phosphatase digestion. This method was applied to the measurement of A2'p5'A degrading activities in extracts of mouse L cells. Extracts from control mouse L cells were as active in degrading A2'p5'A as extracts from interferon pretreated cells.     

Induction and function of 2',5'-oligoadenylate synthetase in differentiation of mouse myeloid leukemia cells

The activity of 2′,5′-oligoadenylate synthetase (2-5A synthetase), known to be induced by interferon, was detected in mouse myeloid leukemic M1 cells only when they differentiated to phagocytic cells after incubation with conditioned medium (CM) from rat embryo cells. However, no interferon activity occurred in culture fluids of CM-treated M1 cells, although some activity was detected in the cell extracts. When anti-interferon serum was added to M1 cell cultures, the induction of 2-5A synthetase by CM was suppressed. These results suggest that CM stimulated the M1 cells to produce a minute amount of interferon, which was reponsible for induction of the 2-5A synthetase activity. On the other hand, development of the phagocytic activity of M1 cells could not be influenced by addition of antiserum. Interferon added exogenously per se neither induced phagocytic activity of M1 cells, nor did it enhance the CM-induced differentiation of the cells. Moreover, dexamethasone, which induced differentiation of M1 cells, was not capable of inducing 2-5A synthetase. These results indicate that interferon and/or 2-5A synthetase plays no essential part in the differentiation of M1 cells.     

2',5'-Oligoadenylate and 2',5'-oligoadenylate phosphodiesterase in human plasma

2',5'-Oligoadenylate and 2',5'-oligoadenylate phosphodiesterase activity were detected in the human plasma and serum by sensitive radioimmuno assays. The phosphodiesterase in the serum degraded 20 nM of added 2',5'-oligoadenylate in less than 1 hr. Addition of EDTA in the blood sample inhibited the phosphodiesterase activity completely and allowed the measurement of low levels of 2',5'-oligoadenylate. The concentration in the plasma from healty people was in the range of 0.03 to 0.3 nM.     

Spectrophotometric pyrophosphate assay of 2',5'-oligoadenylate synthetase.

A nonradioactive multiwell spectrophotometric assay for the interferon-induced enzyme 2',5'-oligoadenylate synthetase measuring the inorganic pyrophosphate produced during oligoadenylate synthesis has been developed. A coupled enzymatic reaction results in a mole to mole formation of NADPH compared to the inorganic pyrophosphate through the use of the three enzymes UDP-Glc pyrophosphorylase (EC2.7.7.9), phosphoglucomutase (EC5.4.2.2), and glucose-6-phosphate dehydrogenase (EC1.1.1.49). The assay is at least as sensitive for measurements of 2',5'-oligoadenylate synthetase activity as the conventional assays using radioactive nucleotides as substrates. Even higher sensitivity of the assay can be obtained by taking advantage of the strong fluorescence of NADPH.     

Phenobarbital enhances 2',5'-oligoadenylate synthesis in rat liver nuclei

Treatment of rats with phenobarbital for three days greatly increases the activity of 2,5 oligoadenylate synthetase in liver nuclei. Analysis of 2',5'-oligoadenylates synthesized in vitro showed that nuclei from both phenobarbital-treated and control rats synthesized 2',5'-oligoadenylates ranging from di- to hexamers. However, nuclei from drug treated rats showed a two fold increase in trimer and tetramer synthesis and a three-four fold increase in longer chained oligoadenylates. There was no change in the nuclear 2'-phosphodiesterase activity as the result of phenobarbital treatment, This activity remained low in nuclei from either the treated or the control rats. To our knowledge, this is the first report on phenobarbital affecting the liver 2',5'-oligoadenylate system.     

An improved method for purifying 2',5'-oligoadenylate synthetases

We describe a new, rapid, and convenient procedure for purifying 2',5'-oligoadenylate synthetases, employing precipitation with ammonium sulfate, fractionation by gel filtration, rapid binding to poly(I) X poly(C) cellulose, and elution with 0.35 M KCl. Unlike previously published methods, the procedure does not require sedimentation of the enzyme at 200,000 X g. Therefore, it is more general and more likely to succeed with synthetases extracted from a variety of cells or tissues, or from different subcellular fractions. We have purified the enzymes from two sources to apparent homogeneity, about 2500-fold from the cytoplasm of HeLa cells in 40% yield and more than 400,000-fold from the cytoplasm of rabbit reticulocytes in 25% yield. The specific activity of the HeLa enzyme is about 4 times higher than reported previously. The physical and functional properties of the pure enzymes are very similar to those reported by others for preparations of 2',5'-oligoadenylate synthetase from rabbit reticulocytes, mouse L cells, and human HeLa cells. A new affinity matrix was prepared by linking periodate-oxidized poly(I) X poly(C) to a hydrazide derivative of finely divided cellulose. Poly(I) X poly(C) cellulose binds about twice as much synthetase as the corresponding amount of poly(I) X poly(C) paper and activates the bound enzyme about three times better.     

Alternative function of a protein kinase homology domain in 2', 5'-oligoadenylate dependent RNase L.

RNase L is the 2',5'-oligoadenylate (2-5A)-dependent endoribonuclease that functions in interferon action and apoptosis. One of the intriguing, albeit unexplained, features of RNase L is its significant homology to protein kinases. Despite the homology, however, no protein kinase activity was detected during activation and RNA cleavage reactions with human RNase L. Similarly, the kinase plus ribonuclease domains of RNase L produced no detectable protein kinase activity in contrast to the phosphorylation obtained with homologous domains of the related kinase and endoribonuclease, yeast IRE1p. In addition, neither ATP nor pA(2'p5'A)3was hydrolyzed by RNase L. To further investigate the function of the kinase homology in RNase L, the conserved lysine at residue 392 in protein kinase-like domain II was replaced with an arginine residue. The resulting mutant, RNase LK392R, showed >100-fold decreases in 2-5A-dependent ribonuclease activity without reducing 2-5A- or RNA-binding activities. The greatly reduced activity of RNase LK392Rwas correlated to a defect in the ability of RNase L to dimerize. These results demonstrate a critical role for lysine 392 in the activation and dimerization of RNase L, thus suggesting that these two activities are intimately linked.     

Induction of poly(I):poly(C)-binding 50 K protein and 2',5'-oligoadenylate synthetase in interferon-treated mouse L929 cells

A new protein retained by poly(I):poly(C)-Sepharose was induced together with dsRNA-dependent enzymatic activities, a protein kinase and 2',5'-oligoadenylate synthetase (2,5A synthetase), in interferon-treated mouse L929 cells; it had an apparent molecular weight of 50,000 (50 K) and was not phosphorylated by the protein kinase. The kinetics of the induction of the poly(I):poly(C)-binding 50 K protein were similar to those of dsRNA-dependent protein kinase and 2',5'-oligoadenylate synthetase, and their inductions were all dependent on the interferon dose added, though a relatively higher dose was required for the 50 K protein. When the interferon preparation was heated to 100 degrees C in the presence of sodium dodecyl sulfate, its effect on cells of inducing the activity of 2',5'-oligoadenylate synthetase was preserved completely, indicating that the interferon molecule itself is responsible for the induction of the synthetase. Since the induction of the enzymatic activity was inhibited by addition of either actinomycin D or cycloheximide, it may not be an activation of a latent enzyme but a de novo synthesis of the enzyme.     

Synthesis, properties and use of beta-alanyltyrosine derivatives of 2',5'-oligoadenylate 5'-triphosphate

beta-Alanyltyrosine methyl ester derivatives of 2-5 A, ppp-(A2'p5') A-beta-Ala-Tyr, were prepared by coupling of periodate oxidizedn2-5 A with beta-alanyltyrosine methyl ester, followed by reduction with sodium cyanoborohydride. The compounds were resistant to the hydrolysis by 2',5'-phosphodiesterase in the mouse L cells extract. They bound to the 2-5 A dependent RNAse (RNAse L) in the mouse L cells extract and in the rabbit reticulocyte lysate, and displaced by addition of 2-5 A. The compound, pppA2'p5'A2'p5'A2'p5'A-beta-Ala-Tyr, after iodination with 125I, was proved to be useful as a radio-labeled probe for the radiobinding assay for 2-5 A.     

Cloning of a novel 2',5'-oligoadenylate synthetase-like molecule, Oasl5 in mice.

The 2',5'-oligoadenylate synthetase (2-5OAS) is a enzyme that catalyzes synthesis of 2',5'-oligoadenylates (2-5A) in a dsRNA-dependent manner, and known as a major component of the IFN-induced host defense mechanisms against microbial infections. Here, we report the presence of a novel 2-5OAS-like molecule, termed Oasl5, in mice. The size of Oasl5 cDNA was about 2 kb and encoded a protein consisting of 362 aa. The amino acid sequence showed 76% similarity to the mouse 2-5OAS, however, several motifs being important for the enzyme activity were not conserved. The Oasl5 mRNA was most significantly expressed in the brain, and relatively weak expression was found in other organs such as the spleen, kidney, ovary and testis. It was also expressed in embryonic stem (ES) cells. The Oasl5 mRNA expression in ES cells was elevated 5-fold after treatment with IFN and about 2-fold in the brain when stimulated with IFN inducer, polyinosinic-polycytidylic acid (poly[I:C]). In situ hybridization analysis revealed that Oasl5 is expressed in neurons in the central nervous system in adult mice. When Oasl5 was expressed in E. coli, it yielded 42 kDa protein that binds to dsRNA, but it did not show oligoadenylate synthetase activity. These findings suggest a novel function of Oasl5, which are independent of oligoadenylate synthetase activity, in the brain and developing embryos.     

Synthesis of 2',5'-oligoadenylate by rat liver nuclear matrix protein

Nuclear matrix was prepared from unstimulated rat liver by treatment of nuclei with DNAse and 0.4 M NaCl and was further extracted with 2.0 M NaCl. Proteins were bound to poly(rI):(rC)-agarose, incubated with (alpha-32P) adenosine 5'-triphosphate and 2',5'-linked oligoadenylate was isolated from the supernatant. The substance inhibited amino acid incorporation in a reticulocyte translation system and was identified after enzymatic treatment followed by thin-layer chromatography on PEI-cellulose. The possible function of 2',5'-oligo(A) synthetase in the maturation of pre-mRNA associated with nuclear matrix is discussed.     

Chloroquine impairs the interferon-induced antiviral state without affecting the 2',5'-oligoadenylate synthetase

Chloroquine, a weak base which raises the pH in acidic cellular compartments such as lysosomes and endosomes, counteracts the induction by interferon of the antiviral state but not that of the 2',5'-oligoadenylate synthetase in three different types of cell lines (MDBK, WISH, and L929). Active interferon is recovered in crude extracts of cells which have been treated with interferon and chloroquine together, but not in extracts of cells treated with interferon alone, indicating that chloroquine has inhibited the intralysosomal proteolysis of interferon. A low pH-dependent event in the intracellular fate of interferon (perhaps its intralysosomal degradation) is, therefore, necessary for the establishment of the antiviral state but not for the induction of the 2',5'-oligoadenylate synthetase.     

Effects of dephosphorylated core of 2',5'-oligoadenylate (2',5'-ApApA) on the electric activity of snail neurons

On nervous ganglion of snail Helix pomatia was shown the effect of dephosphorylated core of 2',5'-oligoadenylate (2',5'-ApApA) on pacemaker electrical activity of RPa2 snail neurons. In experiments, solution of 2',5'-ApApA in concentration 5 x 10(-5) mol/l effected snail nervous ganglion in the course of 2 min then it was washed in the course of 2 hours.     

Interferon-induced 2',5'-oligoadenylate system: key components and biological functions

The current data about the 2',5'-oligoadenylate system is reviewed. Its role in interferon signaling and cell metabolism regulation is discussed. The interferon system is known to be characterized by a wide range of biological functions such as antiviral defense, control of cell growth and differentiation, oncogenic stability, apoptosis, immune activation, etc. The biological role of interferon that is the multifunctional cytokine is discussed more in detail. The structure of main components of interferon signal transduction cascade (2',5'-oligoadenylate, 2',5'-oligoadenylate-synthetase and ribonuclease L) is reviewed. The interferon-induced 2',5'-oligoadenylate system is considered as the component of common regulatory system coordinating cell metabolism.     

2',5'-Oligoadenylates and related 2',5'-oligonucleotide analogues. 1. Substrate specificity of the interferon-induced murine 2',5'-oligoadenylate synthetase and enzymatic synthesis of oligomers

The substrate specificity of the interferon-induced mouse L-cell enzyme, 2',5'-oligoadenylate synthetase, was determined with a number of nucleoside 5'-triphosphate analogues. Selected nucleoside 5'-triphosphates were converted to 2',5'-oligonucleotides with the following order of efficiency for the nucleoside: 8-azaadenosine greater than adenosine = 2-chloroadenosine greater than sangivamycin greater than toyocamycin greater than formycin greater than 3-ribosyladenine greater than ribavirin greater than tubercidin greater than adenosine 1-oxide greater than 2-beta-D-ribofuranosylthiazole-4-carboxamide greater than inosine = 1,N6-ethenoadenosine greater than guanosine greater than 8-bromoadenosine = uridine greater than cytidine. Adenosine 5'-((beta, gamma-imidotriphosphate) did not seem to be a recognizable substrate since no detectable product resulted. Either the 2',5'-oligoadenylate synthetase is not as specific as had been previously thought, or there may be more than one 2',5'-oligonucleotide synthetase. The 2',5'-oligonucleotide analogue products in which the adenosine of ppp(A2'P5')nA was replaced by the various nucleoside analogues were separated by DEAE-cellulose column chromatography and the chain length and number of 5'-phosphate residues analyzed by a rapid, efficient high-performance liquid chromatographic (HPLC) system involving ion-pairing C18 reversed-phase column chromatography. Separation of the 5'-mono-, 5'-di-, and 5'-triphosphorylated forms of the 2',5'-oligonucleotide analogue dimers, trimers, tetramers, and pentamers was readily achieved by this useful HPLC system. No 5'-nonphosphorylated forms were detected for any of the 2',5'-oligonucleotide analogue products.     

Synthesis and biological activities of beta-alanyltyrosine derivative of 2',5'-oligoadenylate, and its use in radiobinding assay for 2',5-oligoadenylate

beta-Alanyltyrosine derivative of 2',5'-tetraadenylate 5'-triphosphate, pppA2'p5'A2'-p5'A2'p5'A-beta-Ala-Tyr was prepared by coupling of periodate-oxidized pppA2'p5'-A2'p5'A2'p5'A with beta-alanyltyrosine methyl ester, followed by reduction with sodium cyanoborohydride. Its stability to 2',5'-phosphodiesterase and phosphatase was investigated in mouse L cell extract. The 5'-triphosphate of the compound was cleaved gradually to form the 5'-dephosphorylated derivative, A2'p5'A2'p5'A2'p5'A-beta-Ala-Tyr, followed by slow degradation of the 2',5'-phosphodiester bond. On the other hand, pppA2'p5'A2'p5'A2'p5'A was hydrolyzed very quickly under the same conditions. The tetramer derivative bound tightly to the 2',5'-oligoadenylate-dependent endoribonuclease in rabbit reticulocyte lysate or mouse L cell extract and inhibited protein synthesis of mouse L cells more effectively than the unmodified 2',5'-tetraadenylate 5'-triphosphate. The corresponding trimer derivative had slightly weaker activities than the unmodified trimer for binding to the endoribonuclease and for inhibition of protein synthesis. The compound, pppA2'p5'A2'p5'-A2'p5'A-beta-Ala-Tyr, was iodinated easily at the tyrosine residue with 125I, giving a high-specific-radioactivity derivative which was used as a radio-labeled probe in a radiobinding assay for 2',5'-oligoadenylate.     

Protein factors that bind to the murine 2',5'-oligoadenylate synthetase ME-12 gene 5' upstream regulatory region

The murine 2',5'-oligoadenylate synthetase ME-12 gene regulatory region AB forms six complexes with protein factors in murine BALB/c 3T3 cells as demonstrated by the mobility shift electrophoresis assay under the reaction conditions used. The complexes, designated C1-C6 in order of their decreasing electrophoretic mobility, showed three distinctive specificities with regulatory region AB, element A, and element B as probes or competing DNA: 1) C1 is region AB-specific (this complex did not form with either element A or B used alone or as a mixture); 2) C5 formed both with element A and element B; 3) C2, C3, C4, and C6 formed with element B, but not A. The protein factors that give rise to these complexes show differential DNA binding activities in various buffer solutions at different pH values. The C4-forming protein factor is the interferon (IFN)-alpha/beta-stimulated response factor (ISRF) which shows element B specificity. It preexists in the cytoplasm. ISRF appears to be complexed to an inhibitor (ISRFI) in the cytoplasm and to dissociate from the inhibitor and to translocate into the nucleus upon treatment of cells with IFN-alpha/beta. We propose that IFN-alpha/beta treatment of BALB/c 3T3 can trigger at least two events: 1) loosening of a tight inhibitor-ISRF complex with the release of free ISRF; this may be mediated via phosphorylation of ISRF or ISRFI; 2) translocation of ISRF into the nucleus and binding to the enhancer element B, which results in the activation of 2',5'-oligoadenylate synthetase gene expression.     

Phosphorothioate analogues of 2',5'-oligoadenylate. Enzymatically synthesized 2',5'-phosphorothioate dimer and trimer: unequivocal structural assignment and activation of 2',5'-oligoadenylate-dependent endoribonuclease

In continued studies to elucidate the requirements for binding to and activation of the 2',5'-oligoadenylate-dependent endoribonuclease (RNase L), chirality has been introduced into the 2',5'-oligoadenylate (2-5A, p3An) molecule to give the Rp configuration in the 2',5'-internucleotide backbone and the Sp configuration in the alpha-phosphorus of the pyrophosphoryl moiety of the 5'-terminus. This was accomplished by the enzymatic conversion of (Sp)-ATP alpha S to the 2',5'-phosphorothioate dimer and trimer by the 2-5A synthetase from lysed rabbit reticulocytes. The most striking finding reported here is the ability of the 2',5'-phosphorothioate dimer 5'-triphosphate (i.e., p3A2 alpha S) to bind to and activate RNase L. p3A2 alpha S displaces the p3A4[32P]pCp probe from RNase L with an IC50 of 5 X 10(-7) M, compared to an IC50 of 5 X 10(-9) M for authentic p3A3. Further, p3A2 alpha S activates RNase L to hydrolyze poly(U)-3'-[32P]pCp (20% at 2 X 10(-7) M), whereas authentic p3A2 is unable to activate the enzyme. Similarly, the enzymatically synthesized p3A2 alpha S at 10(-6) M activated RNase L to degrade 18S and 28S rRNA, whereas authentic p3A2 was devoid of activity. p3A3 alpha S was as active as authentic p3A3 in the core--cellulose and rRNA cleavage assays. The absolute structural and configurational assignment of the enzymatically synthesized p3A2 alpha S and p3A3 alpha S was accomplished by high-performance liquid chromatography, charge separation, enzymatic hydrolyses, and comparison to fully characterized chemically synthesized (Rp)- and (Sp)-2', 5'-phosphorothioate dimer and trimer cores.(ABSTRACT TRUNCATED AT 250 WORDS)