Heterogeneous nuclear RNA promotes synthesis of (2'',5'')oligoadenylate and is cleaved by the (2'',5'')oligoadenylate-activated endoribonuclease.

Heterogeneous nuclear RNA contains double-stranded regions that are not found in mRNA and that may serve as recognition elements for processing enzymes. The double-stranded regions of heterogeneous nuclear RNA prepared from HeLa cells promoted the synthesis of (2',5')oligoadenylate [(2',5')oligo(A) or (2'5')An] when incubated with (2',5')An polymerase. This enzyme is present in elevated levels in interferon-treated cells, and labeled heterogeneous nuclear RNA incubated with extracts of these cells is preferentially cleaved, since mRNA included in the same incubations is not appreciably degraded. The cleavage of heterogenous nuclear RNA is caused by the synthesis of (2'5')An and by a "localized" activation of the (2',5')An-dependent endonuclease, since it was enhanced by ATP, the substrate of the (2',5')An polymerase, and inhibited by 2'-dATP and ethidium bromide. Both of these compounds suppress the synthesis of (2',5')An, the first by competitive inhibition and the latter by intercalating into double-stranded RNA. The possible role of double-stranded regions and of the (2',5')An polymerase-endonuclease system in the processing of heterogeneous nuclear RNA is discussed.     

2',5' A synthetase: allosteric activation by fructose 1,6-bisphosphate

Fructose 1,6-bisphosphate (fru-1,6-P2), but not other glycolytic intermediates, activates highly purified 2',5' A synthetases from rabbit reticulocyte lysates and from 2',5'-ADP-agarose purified extracts of interferon-treated HeLa cells without the addition of dsRNA. The 2',5' A was structurally and biologically identical to authentic 2',5' A. Micrococcal nuclease inhibited the activation of 2',5' A synthetase by poly(I)-poly(C), but did not affect activation by fru-1,6-P2. Addition of fru-1,6-P2 aldolase prevented the activation of 2',5' A synthetase by fru-1,6-P2.     

Synthesis and separation of diastereomers of uridine 2',3'-cyclic boranophosphate

The first boron-containing 2',3'-cyclic phosphate-modified analogue, uridine 2',3'-cyclic boranophosphate (2',3'-cyclic-UMPB), was synthesized. 5'-O-Protected uridine was cyclophosphorylated by diphenyl H-phosphonate to yield uridine 2',3'-cyclic H-phosphonate, which upon silylation followed by boronation and subsequent acid treatment gave 2',3'-cyclic-UMPB in high yield. The two diastereomers of 2',3'-cyclic-UMPB were separated by HPLC. An alternative method for synthesis of uridine 2',3'-cyclic phosphorothioate (2',3'-cyclic-UMPS) via H-phosphonate was also described.     

Interaction of muscle glycogen phosphorylase b with riboflavin, its tetraacetyl derivative and their analogs

The interaction of rabbit skeletal muscle glycogen phosphorylase b with riboflavin, 2',3',4',5'-tetraacetylriboflavin and their analogues, containing different substituents in the positions 6, 8 and 8 alpha, has been studied. Dissociation constant for the complex of the enzyme and riboflavin was determined to be 12.5 microM (pH 6.8; 20 degrees C) by sedimentation velocity method. Riboflavin and its analogues have been found to inhibit glycogen phosphorylase b. The inhibitor half-saturation concentration values increase in the following order: riboflavin (18 microM), 8-methoxy(nor)rifoblavin (23 microM), 8 alpha-bromo-2',3',4',5'-tetraacetylriboflavin (40 microM), 6-bromoriboflavin (40 microM), 8 alpha-hydroxyriboflavin (60 microM), 8-hydroxy(nor)riboflavin (90 microM), 8 alpha-(gamma-carboxypropylamino-2',3',4',5'-tetraacetylriboflav in (90 microM), 8 alpha-[p-(5-ethyl-1,3,4-thiodiazol-2-ylsulfamido)phenylamino ]- 2',3',4',5'-tetraacetylriboflavin (100 microM), 8 alpha-(L-methionyno)-2',3',4',5'-tetraacetylriboflavin (120 microM), 8 alpha-[p-(thiazol-2-ylsulfamido)phenylamino]- 2',3',4',5'-tetraacetylriboflavin (140 microM), 8 alpha-(p-sulfamidophenylamino)-2',3',4',5'-tetraacetylriboflavi n (180 microM), 8 alpha-(p-carboxyphenylamino)-2',3',4',5'-tetraacetylriboflavin+ ++ (210 microM), 2',3',4',5'-tetraacetylriboflavin (250 microM), 8 alpha-(L-homoserino)-2',3',4',5'-tetraacetylriboflavin (340 microM), 8 alpha-(L-glutamo)-2',3',4',5'-tetraacetylriboflavin (360 microM). The existence of glycogen phosphorylase b complexes with riboflavin and its analogues has been proved by methods of absolute and difference spectrophotometry.     

Conformational properties of 2',5' linked Rp- and Sp-phosphorothioate oligoadenylates studied by circular dichroism and NMR

2',5'-Linked oligoadenylates (2-5A) are involved in the antiviral action of interferon. The 2-5A binds and activates 2-5A dependent RNase (RNase L), which degrades viral mRNA, resulting in the inhibition of protein synthesis. 2',5'-Linked phosphorothioate oligoadenylates with an Rp configuration bind to and activate the RNase L. On the other hand, 2',5' phosphorothioate oligoadenylate with an Sp configuration weakly binds to the RNase L and is devoid of the RNase L activation ability. Comparative circular dichroism (CD) and NMR studies are carried out to characterize the difference in properties between the two configurations of the 2',5' phosphorothioate oligoadenylates. 2',5' Rp-Phosphorothioate oligoadenylates showed CD spectra similar to those of the corresponding native 2',5' oligoadenylates, while the 2',5' Sp-phosphorothioate oligoadenylates exhibited a weaker CD band compared to the former two, indicating the weaker base-stacking interaction of the 2',5' Sp-phosphorothioate oligoadenylates. The temperature-dependent change in the CD revealed that 2',5' phosphorothioate oligoadenylates showed larger DeltaH(0) and DeltaS(0) values for the thermal transition of the conformation than the corresponding native 2',5' oligoadenylates. The NMR spectral assignment was accomplished by several NMR measuring techniques. The 2'-H of the ribose ring linked to the 2',5' Sp-phosphorothioate showed a higher field chemical shift of the proton NMR than that linked to the corresponding 2',5' Rp-phosphorothioate. 2',5' Rp- and Sp-phosphorothioate oligoadenylates possess a sugar conformation similar to that of the corresponding native 2',5' oligoadenylates.     

Mechanisms of degradation of 2′-5′ oligoadenylates

We have studied the mechanisms of breakdown of 2'-5' oligoadenylates. We monitored the time-courses of degradation of ppp(A2'p5')nA (dimer to tetramer) and of 5'OH-(A2'p5')nA (dimer to pentamer) in unfractionated L1210 cell extract. The 5' triphosphorylated 2'-5' oligoadenylates are converted by a phosphatase activity. However, 2'-5' oligoadenylates are degraded mainly by phosphodiesterase activity which splits the 2'-5' phosphodiester bond sequentially at the 2' end to yield 5' AMP and one-unit-shorter oligomers. The nonlinear least-squares curve-fitting program CONSAM was used to fit these kinetics and to determine the degradation rate constant of each oligomer. Trimers and tetramers, whether 5' triphosphorylated or not, are degraded at the same rate, whereas 5' triphosphorylated dimer is rapidly hydrolyzed and 5'-OH dimer is the most stable oligomer. The interaction between degradation enzymes and the substrate strongly depends on the presence of a 5' phosphate group in the vicinity of the phosphodiester bond to be hydrolyzed; indeed, when this 5' phosphate group is present, as in pp/pA2'p5'A/or A2'/p5'A2'p5'A/, affinity is high and maximal velocity is low. Such a degradation pattern can control the concentration of 2'-5' oligoadenylates active on RNAse L either by limiting their synthesis (5' triphosphorylated dimer is the primer necessary for the formation of longer oligomers) and/or by converting them into inhibitory (e.g., monophosphorylated trimer) or inactive (e.g., nonphosphorylated oligomers) molecules.     

2',3'-cyclic nucleotide 2'-phosphodiesterase from Fusarium culmorum

We describe the properties of a 2',3'-cyclic nucleotide 2'-phosphodiesterase (EC 3.1.4.16), found in Fusarium culmorum, which hydrolyzes nucleoside 2',3'-cyclic monophosphates to nucleoside 3'-phosphates. In contrast with a similar enzyme found in bacteria, the Fusarium enzyme does not exhibit nucleotidase activity and does not show a requirement for metal ions, but is inhibited by micromolar concentrations of Cu++ and Zn++, and is very stable to heat. This cyclic phosphodiesterase hydrolyzes the four major nucleoside 2',3'-cyclic monophosphates and has greater affinity for purine (Kms for Ado-2',3'-P = 0.3 mM and for Guo-2',3'-P = 0.1 mM) than for pyrimidine nucleotides (Kms for Cyd-2',3'-P = 0.6 mM and for Urd-2',3'-P = 2 mM). The respective Vmax for Urd-2',3'-P; Cyd-2',3'-P; Ado-2',3'-P; and Guo-2',3' are 100:45:16:5. The efficacy of the phosphodiesterase to hydrolyze the four major 2',3' cyclic nucleotides (based on the relative values of Vmax/Km) is not significantly different. The Fusarium enzyme differs from a previously described 2',3' cyclic phosphodiesterase from Neurospora, in that it is inactive on 3',5'-nucleoside monophosphates and nucleoside 2' or 3' phosphates.     

Degradation by the 2',5'-phosphodiesterase activity of mouse cells requires the presence of a ribo hydroxyl group in the penultimate position of the oligonucleotide substrate

A series of 9-beta-D-xylofuranosyladenine (xyloA or xyloadenosine) substituted analogs of 2-5A core trimer and tetramer were examined for their ability to be degraded by the 2',5'-phosphodiesterase activity of cytoplasmic extracts of mouse L cells. Two distinct groups of xyloA-substituted analogs could be readily discriminated. The first group contained xyloadenosine at the 2'-termini and included A2'p5'A2'p5'(xyloA) and A2'p5'A2'p5'A2'p5'(xyloA). These oligomers behaved as did their parent oligoadenylates in that they were equally sensitive to degradation by the 2',5'-phosphodiesterase activity. The second group of oligonucleotides bore a xyloadenosine residue in the penultimate nucleotide residues of the oligomers and included A2'p5'(xyloA)2'p5'(xyloA), (xyloA)2'p5'(xyloA)2'p5'(xyloA), A2'p5'A2'p5'(xyloA)2'p5'(xyloA) and (xyloA)2'p5' (xyloA)2'p5'(xyloA)2'p5'(xyloA). This group was quite resistant to 2',5'-phosphodiesterase activity. In all, the findings demonstrate that the ribo configuration 3'-hydroxyl group in the penultimate nucleotide of the oligonucleotide substrate is a prerequisite for the 2',5'-phosphodiesterase activity.     

Metabolism and anti-human immunodeficiency virus-1 activity of 2-halo-2',3'-dideoxyadenosine derivatives

Both 2',3'-dideoxyadenosine and 2',3'-dideoxyinosine have been shown (Mitsuya, H., and Broder, S. (1987) Nature 325, 773-778) to have in vitro activity against the human immunodeficiency virus-1 (HIV). However, these dideoxynucleosides may be catabolized by human T cells, even when adenosine deaminase is inhibited by deoxycoformycin. To overcome this problem, we have synthesized the 2-fluoro-, 2-chloro-, and 2-bromo-derivatives of 2',3'-dideoxyadenosine. The metabolism and anti-HIV activity of the 2-halo-2',3'-dideoxyadenosine derivatives and of 2',3'-dideoxyadenosine were compared. The 2-halo-2',3'-dideoxyadenosine derivatives were not deaminated significantly by cultured CEM T lymphoblasts. Experiments with 2-chloro-2',3'-dideoxyadenosine showed that the T cells converted the dideoxynucleoside to the 5'-monophosphate, 5'-diphosphate, and 5'-triphosphate metabolites. At concentrations lower than those producing cytotoxicity in uninfected cells (3-10 microM), the 2-halo-2',3-dideoxyadenosine derivatives inhibited the cytopathic effects of HIV toward MT-2 T lymphoblasts, and retarded viral replication in CEM T lymphoblasts. Experiments with a deoxycytidine kinase-deficient mutant CEM T cell line showed that this enzyme was necessary for the phosphorylation and anti-HIV activity of the 2-chloro-2',3'-dideoxyadenosine. In contrast, 2',3'-dideoxyadenosine was phosphorylated by the deoxycytidine kinase-deficient mutant and retained anti-HIV activity in this cell line. Thus, the 2-halo derivatives of 2',3'-dideoxyadenosine, in contrast to 2',3'-dideoxyadenosine itself, are not catabolized by T cells. Their anti-HIV and anti-proliferative activities are manifest only in cells expressing deoxycytidine kinase. The in vivo implications of these results for anti-HIV chemotherapy are discussed.     

3' processing of human pre-U2 small nuclear RNA: a base-pairing interaction between the 3' extension of the precursor and an internal region.

The spliceosomal small nuclear RNAs U1, U2, U4, and U5 are transcribed by RNA polymerase II as precursors with extensions at their 3' ends. The 3' processing of these pre-snRNAs is not understood in detail. Two pathways of pre-U2 RNA 3' processing in vitro were revealed in the present investigation by using a series of human wild-type and mutant pre-U2 RNAs. Substrates with wild-type 3' ends were initially shortened by three or four nucleotides (which is the first step in vivo), and the correct mature 3' end was then rapidly generated. In contrast, certain mutant pre-U2 RNAs displayed an aberrant 3' processing pathway typified by the persistence of intermediates representing cleavage at each internucleoside bond in the precursor 3' extension. Comparison of the wild-type and mutant pre-U2 RNAs revealed a potential base-pairing interaction between nucleotides in the precursor 3' extension and a sequence located between the Sm domain and stem-loop III of U2 RNA. Substrate processing competition experiments using a highly purified pre-U2 RNA 3' processing activity suggested that only RNAs capable of this base-pairing interaction had high affinity for the pre-U2 RNA 3' processing enzyme. The importance of this postulated base-pairing interaction between the precursor 3' extension and the internal region between the Sm domain and stem-loop III was confirmed by the results obtained with a compensatory substitution that restores the base pairing, which displayed the normal 3' processing reaction. These results implicate a precursor-specific base-paired structure involving sequences on both sides of the mature cleavage site in the 3' processing of human U2 RNA.     

2''5'' oligoadenylate synthetase, an interferon induced enzyme: direct assay methods for the products, 2''5'' oligoadenylates and 2''5'' co-oligonucleotides.

The interferon induced enzyme 2'5' oligoadenylate synthetase produces 2'5' pppA(pA)n the first discovered natural nucleotide with a 2'5' linkage. We describe a direct assay of this enzyme based on separation by thin layer chromatography (TLC) of the substrate ATP and the products 2'5' pppA(pA)n (n larger than or equal to 1). This technique presents obvious advantages compared to the currently used methods. Moreover the enzyme uses other nucleotides as substrates forming co-oligonucleotides 2'5 pppA(pA)n pN (N = U,G,C,dA,dG,dT and dC). Additional procedures are described using different developing solvent systems for the separation of the core-2'5' oligonucleotides (2'5' A(pA)npN) containing AMP-residues entirely and those with another nucleotide at the 2' end.     

Phosphorylated and dephosphorylated myosin light chains of Drosophila fly and larva

1. The present study confirmed that light chains of Drosophila adult fibrillar (flight) muscle myosin consist of Lf1, Lf2, Lf2' and Lf3, and tubular muscle myosin light chains contain Lt1, Lt2, Lt2' and Lt3, as revealed by two-dimensional (isoelectric focusing and SDS-gel electrophoresis) gel electrophoresis. 2. Larva myosin light chains were of all the tubular type. However, it was found that Lt1 and Lt2' are produced by phosphorylation of Lt2, and Lf1 is produced by phosphorylation of Lf2'. 3. Injection of radioactive phosphate into Drosophila fly resulted in phosphorylations of Lf1 and Lt1. When larva or late pupa myosin was incubated with myosin light chain kinase from chicken gizzard or adult flies, phosphorylation of Lt1, Lf2' and Lt2' occurred. Drosophila myosin light chain kinase phosphorylated Lf1 in addition to Lt1 and L2' (Lf2' + Lt2') of adult myosin. 4. Dephosphorylation of adult myosin by potato acid and calf intestine alkaline phosphatases led to the shift of Lf1 (34,000), Lt1 (31,000) and L2' (Lf2' + Lt2') (30,000) to L2 (Lf2 + Lt2) positions (30,000). 5. Peptide mapping analyses revealed that larva Lt1, Lt2', Lt2 and adult Lt1 were all the same; therefore, it is thought that a single species of Lt2 specific to the tubular type of myosin and its phosphorylated isoforms (Lt1, Lt2') exist. 6. The peptide map of Lf1 was slightly different from that of Lt1, but very similar to that of L2' in adult myosin. L2 and L2' of adult myosin showed very similar peptide maps, but there were several different peptide fragments.(ABSTRACT TRUNCATED AT 250 WORDS)     

Inhibition of protein synthesis by the cordycepin analog of (2'-5')ppp(Ap)nA, (2'-5')ppp(3'dAp)n3'dA, in intact mammalian cells

The introduction of the cordycepin analog of (2'-5')An, (2'-5')ppp(3'dAp)n3'dA [referred to as (2'-5')p33'dAn], into mouse L929 cells and cultured human fibroblasts resulted in a dose-dependent inhibition of protein synthesis which was comparable to the inhibition observed by (2'-5')ppp(Ap)nA [referred to as (2'-5')p3An]. The inhibition of protein synthesis by (2'-5')p33'dAn was much more persistent than that of the naturally occurring (2'-5')p3An following prolonged incubation of cells. Furthermore, the (2'-5')p3An was cytotoxic to mammalian cells in culture, whereas the (2'-5')p33'dAn was not.     

Chemical synthesis and biological characterization of phosphorothioate analogs of 2', 5'-3'-deoxyadenylate trimer.

In continued studies to elucidate the requirements for binding to and activation of the 2',5'-oligoadenylate (2-5A) dependent endoribonuclease (RNase L), four 2-5A trimer analogs were examined to evaluate the effect of chirality of phosphorothioate substitution on biological activity. The chemical syntheses and purification of the four isomers of P-thio-3'-deoxyadenylyl-(2'-5')-P-thio-3'- deoxyadenylyl-(2'-5')-3'-deoxyadenosine, by the phosphoramidite approach, is described. The isolated intermediates were characterized by elemental and spectral analyses. The fully deblocked compounds were characterized by 1H and 31P NMR and HPLC analyses. The 2',5'-(3'dA)3 cores with either Rp or Sp chirality in the 2',5'-internucleotide linkages will bind to but will not activate RNase L. This is in contrast to 2',5'-A3 core analogs with either RpRp or SpRp phosphorothioate substitution in the 2',5'-internucleotide linkages which can bind to and activate RNase L. There are also marked differences in the ability of the 2',5'-A3 analogs to activate RNase L following introduction of the 5'-monophosphate. For example, the 5'monophosphates of 2',5'-(3'dA)3-RpRp and 2',5'-(3'dA)3-SpRp can bind to and activate RNase L, whereas the 5'-monophosphates of 2',5'-(3'dA)3-RpSp and 2',5'-(3'dA)3-SpSp can bind to but can not activate RNase L.     

2' Derivatives of guanosine and inosine cyclic 3',5'-phosphates. Synthesis, enzymic activity, and the effect of 8-substituents.

A series of representative derivatives of guanosine cyclic 3',5'-phosphate (cGMP) and inosine cyclic 3',5'-phosphate (cIMP) which contained modifications in either the 2' position or the 8 and 2' positions were synthesized. Three types of derivatives were investigated: (1) derivatives in which the 2' position has been altered to produce a 2'-deoxynucleoside cyclic 3',5'-phosphate or a 9-beta-D-arabinofuranosylpurine cyclic 3',5'-phosphate; (2) 2'-omicron-acyl derivatives; and (3) doubly modified derivatives containing a 2' modification [as in (1) and (2)] and an 8-substitution. 2'-Deoxyinosine cyclic 3',5'-phosphate and 9-beta-D-arabinofuranosylhypoxanthine cyclic 3',5'-phosphate were obtained by HNO2 deamination of 2'-deoxyadenosine cyclic 3',5'-phosphate and 9-beta-D-arabinofuranosyladenine cyclic 3',5'-phosphate (ara-cAMP), respectively. Treatment of 8-bromo-2'-omicron-(p-toluenesulfonyl) adenosine cyclic 3',5'-phosphate with NaSH yielded the intermediate 8,2'-anhydro-9-beta-D-arabinofuranosyl-8-mercaptoadenine cyclic 3',5-phosphate, which was converted directly to 2'-deoxyadenosine cyclic 3',5'-phosphate (dcAMP) by treatment with Raney nickel. 8-Bromo-2'-omicron-(p-toluenesulfonyl) guanosine cyclic 3',5'-phosphate was converted to 8,2'-anhydro-9-beta-D-arabinofuranosyl-8-mercaptoguanine cyclic 3',5'-phosphate, and the latter was desulfurized with Raney nickel to give 2-deoxyguanosine cyclic 3',5'-phosphate. Ara-cAMP, 9-beta-D-arabinofuranosylguanine cyclic 3',5'-phosphate, and 9-beta-D-arabinofuranosyl-8-mercaptoguanine cyclic 3',5'-phosphate have been previously reported (Mian et al. (1974), J. Med. Chem. 17, 259). 8-Bromo-2'-omicron-acetylinosine cyclic 3',5'-phosphate and 8-[(p-chlorophenyl)thio]-2'-omicron-acetylinosine cyclic 3',5'-phosphate were produced by acylation of 8-bromoinosine cyclic 3',5'-phosphate and 8-[(p-chlorophenyl)thio]inosine cyclic 3',5'-phosphate, respectively; while 8-bromo-2'-omicron-butyrylguanosine cyclic 3',5'-phosphate was synthesized by bromination of 2'-omicron-butyrylguanosine cyclic 3',5'-phosphate.     

5'-modified agonist and antagonist (2'-5')(A)n analogues: synthesis and biological activity

Two 5'-modified (2'-5')(A)4 oligomers with an increased resistance to phosphatase degradation were synthesized and evaluated for their ability to develop an antiviral response when introduced into intact cells by microinjection or by chemical conjugation to poly(L-lysine). The enzymatic synthesis of 5'-gamma-phosphorothioate and beta,gamma-difluoromethylene (2'-5')(A)4 from adenosine 5'-O-(3-thiotriphosphate) and adenosine beta,gamma-difluoromethylenetriphosphate by (2'-5')-oligoadenylate synthetase is described. The isolation and characterization of these (2'-5')(A)4 analogues were achieved by high-performance liquid chromatography. The structures of 5'-modified tetramers were corroborated by enzyme digestion. These two 5'-modified tetramers compete as efficiently as natural (2'-5')(A)4 for the binding of a radiolabeled (2'-5')(A)4 probe to ribonuclease (RNase) L. Nevertheless, at the opposite to 5'-gamma-phosphorothioate (2'-5')(A)4, beta,gamma-difluoromethylene (2'-5')(A)4 failed to induce an antiviral response after microinjection in HeLa cells. In addition, it behaves as an antagonist of RNase L as demonstrated by its ability to inhibit the antiviral properties of 5'-gamma-phosphorothioate (2'-5')(A)4 when both are microinjected in HeLa cells. The increased metabolic stability of 5'-gamma-phosphorothioate (2'-5')(A)4 as compared to that of (2'-5')(A)4 was first demonstrated in cell-free extracts and then confirmed in intact cells after introduction in the form of a conjugate to poly(L-lysine). Indeed, 5'-gamma-phosphorothioate (2'-5')(A)4-poly(L-lysine) conjugate induces protein synthesis inhibition and characteristic ribosomal RNA cleavages for longer times than unmodified (2'-5')(A)4-poly(L-lysine) in the same cell system.(ABSTRACT TRUNCATED AT 250 WORDS)     

Chalconoids from Fissistigma bracteolatum

Phytochemical studies on the leaves of Fissistigma bracteolatum yielded besides the two known compounds 2-hydroxy-3,4,6-trimethoxychalcone (1) and 5,7,8-trimethoxyflav-3-ene (2), five new chalconoids 2-hydroxy-3,4,6-trimethoxychalcene (3), 2-hydroxy-3,4,6-trimethoxydihydrochalcone (4), 2'-hydroxy-3',4',6'-trimethoxydihydrochalcone (5), 2'-hydroxy-3',4',6'-trimethoxy-beta'-methoxychalcane (6) and 2'-hydroxy-3',4',6'-trimethoxy-beta'-ethoxychalcane (7). The structures of these compounds were determined by mass and NMR spectroscopic methods.     

Purification and characterization of a 2'-phosphodiesterase from bovine spleen

Interferon-induced 2',5'-oligoadenylates are transiently produced during viral infection and are believed to play a role in the interferon-mediated inhibition of replication of at least some viruses. 2',5'-Oligoadenylates must be catabolized but are resistant to degradation by most known ribonucleases. A 2'-phosphodiesterase that degrades 2',5'-oligoadenylates was purified 1500-fold from a low speed homogenate of bovine spleen by precipitation at pH 5.2, ammonium sulfate fractionation, differential ultrafiltration, and successive chromatography on DEAE-Sephacel, hydroxylapatite, and a fast protein liquid chromatography Mono P column. No other 2-5A-degrading activity was observed during the purification procedure. The molecular mass of the enzyme estimated by gel filtration and sodium dodecyl sulfate-polyacrylamide gel electrophoresis is 65,000. The enzyme is distinct from bovine spleen phosphodiesterase II. The 2'-phosphodiesterase cleaves 2',5'- and 3',5'-linked oligonucleotides, as well as branched oligoadenylate, A(2'pA)(3'pA), but appears to be most active on 3',5'-oligoribonucleotides. The enzyme cleaves 5'-AMP from the 2' terminus of 2',5'-oligoadenylates and appears to require a free 2' terminus and a 3'-oxygen on the penultimate nucleotide. Substrate length, 5'-phosphorylation, and base composition do not appear to be critical factors in determining enzyme activity. The effects of pH, Mg2+, Mn2+, EDTA, phosphate, 2-mercaptoethanol, and N-ethylmaleimide are also described. This enzyme may be involved in the catabolism of the interferon-induced 2',5'-oligoadenylates and other 2',5'-linked RNAs in the cell.     

Guanosine tetraphyosphate and its analogues. Chemical synthesis of guanosine 3',5'-dipyrophosphate, deoxyguanosine 3',5'-dipyrophosphate, guanosine 2',5'-bis(methylenediphosphonate), and guanosine 3',5'-bis(methylenediphosphonate).

A new procedure for the synthesis of the pyrophosphate bond has been employed in the preparation of nucleoside dipyrophosphates from nucleoside 3',5'-diphosphates. The method makes use of a powerful phosphorylating agent generated in a mixture of cyanoethyl phosphate, dicyclohexylcarbodiimide, and mesitylenesulfonyl chloride in order to avoid possible intramolecular reactions between the two phosphate groups on the sugar ring. That such reactions can readily occur was shown by the facile cyclization of deoxyguanosine 3',5'-diphosphate to P1,P2-deoxyguanosine 3',5'-cyclic pyrophosphate in the presence of dicyclohexylcarbodiimide alone. The phosphorylation reagent was initially tested in the conversion of deoxyguanosine 3',5'-diphosphate to the corresponding 3',5'-dipyrophosphate and was then used to phosphorylate 2'-O-(alpha-methoxyethyl)guanosine 3',5'-diphosphate, which had been prepared from 2'-O-(alpha-methoxyethyl)guanosine. In the latter case, the addition of the two beta phosphate groups was accomplished in 40% yield. Removal of the methoxyethyl group from the phosphorylated product gave guanosine 3',5'-dipyrophosphate, which was shown to be identical with guanosine tetraphosphate prepared enzymatically from a mixture of GDP and ATP. A modification of published procedures was also necessary to effect the synthesis of guanosine bis(methylenediphosphonate). Guanosine was treated with methylenediphosphonic acid and dicyclohexylcarbodiimide in the absence of added base. The product consisted of a mixture of guanosine 2',5' - and 3',5'-bis(methylenediphosphonate), which was resolved by anion-exchange chromatography. The 2',5' and 3',5' isomers are interconvertible at low pH, with the ultimate formation of an equilibrium mixture having a composition ratio of 2:3. The predominant constituent of this mixture has been unequivocally identified as the 3',5' isomer by synthesis from 2'-O-tetrahydropyranylguanosine.     

Inhibition of adenylate cyclase by the 2',3'-dialdehyde of adenosine triphosphate

The periodate-oxidized analog of ATP, 2',3'-dialATP, competitively inhibited bovine brain and rat liver adenylate cyclase. The apparent Ki for inhibition of brain adenylate cyclase by 2',3'-dialATP was 196 microM in the presence of Mg2+ and 37 microM in the presence of Mn2+. The Ki values for inhibition of rat liver adenylate cyclase by 2',3'-dialATP were 48 and 30 microM in the presence of Mg2+; and Mn2+, respectively. Adenylate cyclase activity was irreversibly inactivated by 2'3'-dialATP in the presence of NaCNBH3 and the kinetics for loss in enzyme activity were pseudo-first order. Both ATP and Tris protected adenylate cyclase from irreversible inhibition by 2',3'-dialATP and NaCNBH3. It is proposed that 2',3'-dialATP forms a Schiff's base with an amino group at the active site of the enzyme and that Na-CNBH3 reduction of this Schiff's base causes irreversible modification of the catalytic subunit. The Km for 2',3'-dialATP inactivation, the maximal rate constant of inactivation, and protection of the enzyme by ATP were not affected by the presence or absence of free Mg2+. These data indicate that a divalent cation is not required for binding of 2',3'-dialATP to the active site of adenylate cyclase.