Robert J. Suhadolnik

Analysis of inorganic pyrophosphate at the picomole level.

Abstract A radiometric determination of inorganic pyrophosphate is described in this communication. Excess uridine 5′-diphospho[ 14 C]glucose is used to react with inorganic pyrophosphate in a reaction catalyzed by uridine 5′-diphosphoglucose pyrophosphorylase. This reaction liberates [ 14 C]glucose 1-phosphate, which is then further converted to 6-phospho[ 14 C]gluconic acid in the presence of phosphoglucose mutase and glucose 6-phosphate dehydrogenase. The reaction product, 6-phospho[ 14 C]gluconic acid, is separated from uridine diphospho[ 14 C]glucose by the addition of activated charcoal, which adsorbs uridine diphospho[ 14 C]glucose preferentially. The sensitivity of the assay for inorganic pyrophosphate is 20 pmol.

Human leukocyte interferon and the antiviral factor (AVF) from virus-infected plants stimulate plant tissues to produce nucleotides with antiviral activity

Leaves of Nicotiana glutinosa and tobacco, as well as tobacco callus cultures, were treated with the plant antiviral factor AVF or with a purified subspecies of human leukocyte interferon. After incubation, a fraction containing short oligonucleotides was extracted directly from the plant tissue. In addition, a synthetase preparation was fractionated from treated tissues, which polymerized ATP in the presence of polyinosinic, polycytidylic acid (poly(I:C)). The various plant nucleotides were applied to tobacco mosaic virus (TMV)-infected leaf disks, the TMV content of which were determined by an enzyme-linked immunosorbent assay. The nucleotide fraction extracted directly from TMV-infected leaves exhibited a considerable antiviral activity, whereas similar fractions from AVF- or interferon-treated leaves did not, even though an antiviral state was induced in the tissue by these agents. However, when the synthetase fraction from TMV-infected, AVF-treated, or interferon-treated tissues was incubated with ATP and poly(I:C), the resultant, heat-stable, acid-soluble, polymerized ATP markedly inhibited TMV multiplication. It is concluded that the presence of double-stranded RNA is obligatory for the formation of the antiviral nucleotides. The analogy to interferon-induced resistance in animal tissues is discussed.

Naturally occurring nucleoside and nucleotide antibiotics.

Publisher Summary This chapter reviews that three of the naturally occurring nucleoside antibiotics—that is, tubercidin, 5-azacytidine, and ara-A—have been successfully used in humans as antineoplastic and antiviral agents. A fourth antibiotic, pyrazofurin, is undergoing clinical testing as an antineoplastic agent for carcinoma of the breast. In addition to the health-related application of these naturally occurring nucleoside and nucleotide antibiotics, some have found application as antifungal agents. The chapter discusses that once the structures of some of the nucleoside antibiotics establish, it is possible in some cases to predict the cellular reaction which would be affected by them. The pyrimidine nucleoside antibiotics, which resemble the -CCA terminus of tRNA, bind to ribosomal peptidyltransferase and inhibit protein synthesis in prokaryotes and eukaryotes. Two adenosine analogs, agrocin 84 and thuringiensin, have been isolated as nucleotides. Agrocin 84 prevents cell growth, most probably by interacting with receptor sites on bacterial cell surfaces. Thuringiensin, as a nucleotide, is taken up by mammalian cells and acts as an ATP analog. The aim of this chapter is to evaluate the progress that has been made in the nucleoside antibiotics. Two nucleosides, eritadenine and clitidine, although not antibiotics, are also discussed because of their biological properties.

IDENTIFICATION OF THE ATP BINDING DOMAIN OF RECOMBINANT HUMAN 40-KDA 2',5'-OLIGOADENYLATE SYNTHETASE BY PHOTOAFFINITY LABELING WITH 8-AZIDO-ALPHA -32P ATP

Three isoforms of the interferon-inducible 2′,5′-oligoadenylate (2-5A) synthetase that require double-stranded RNA have been isolated and cloned. However, identification of the amino acid(s) of 2-5A synthetase directly interacting with ATP is crucial to the elucidation of the mechanism of the enzymatic conversion of ATP to 2′,5′-oligoadenylates by 2-5A synthetase. Recombinant human 40-kDa 2-5A synthetase has been expressed as a glutathione S-transferase fusion protein in E. coli and purified to near homogeneity in milligram quantities. The azido photoprobe, 8-azido-[α-32P]ATP, has been used to identify the ATP binding domain of the recombinant human 40-kDa 2-5A synthetase. Specific covalent photoincorporation of 8-azido-[α-32P]ATP into the 2-5A synthetase, tryptic digestion of the covalently 32P-labeled enzyme, isolation of the photolabeled phosphopeptide by metal (Al3+) chelate chromatography, and high pressure liquid chromatography identified a 32P-pentapeptide, which has been assigned to the ATP binding domain of 2-5A synthetase. The radioactive pentapeptide has the sequence D196FLKQ200 in which the photoprobe, 8-azido-[α-32P]ATP, chemically modified the amino acid lysine 199. The catalytic importance of Lys199 was further established by mutation of lysine 199 to arginine 199 and histidine 199 using site-directed mutagenesis. The K199R and K199H recombinant human 40-kDa 2-5A synthetase mutants bind 8-azido-ATP and the allosteric activator, poly(I)·poly(C) but are enzymatically inactive. These photoaffinity labeling and mutation data strongly suggest that lysine 199 is essential for the formation of a productive 2-5A synthetase-ATP-double-stranded RNA complex for the enzymatic conversion of ATP to 2-5A.

Inhibition of Epstein-Barr virus-associated nuclear antigen (EBNA) induction by (2′,5′)oligoadenylate and the cordycepin analog: Mechanism of action for inhibition of EBV-induced transformation

Abstract We have previously shown that interferon inhibits Epstein-Barr virus-induced host cell DNA synthesis and morphologic transformation of human adult peripheral blood leukocytes and that core (2′,5′)oligoadenylate and the cordycepin analog can replace interferon with respect to the blockage of EBV-induced functions. The present communication describes the inhibition of Epstein-Barr virus-associated nuclear antigen (EBNA) in EBV-infected human adult peripheral blood leukocytes and the inhibition of EBV-induced morphologic transformation of human umbilical cord leukocytes by interferon, core (2′,5′)oligoadenylate and the core cordycepin analog. These data suggest that the antiviral mechanism of interferon, core (2′,5′)oligoadenylate and the core cordycepin analog is through the inhibition of EBV early functions, possibly transcription or translation of EBNA.

The biosynthesis of natural and unnatural polyoxins by Streptomyces cacaoi.

Abstract The biosynthesis of the pyrimidine moiety and the uronic acid moiety of the polyoxins and the formation of unnatural polyoxins has been studied in Streptomyces cacaoi. Experimental evidence is provided for the biosynthesis of thymine via a pathway that is independent of thymidylate synthetase. This new thymine pathway is based on two experimental approaches. First, two known inhibitors of DNA synthesis (1-formylisoquinoline thiosemicarbazide and 5-fluoro-2′-deoxyuridine), when added to polyoxin-producing cultures of S. cacaoi, inhibit the synthesis of TMP from exogenously supplied uracil but do not inhibit the synthesis of the thymine or hydroxymethyluracil in the polyoxin complex. Second, exogenously supplied thymine and hydroxymethyluracil are taken up by S. cacaoi but are not incorporated into the thymine or hydroxymethyluracil of the polyoxin complex. The thymine is incorporated into the DNA. The uracil in polyoxin L could be the parent pyrimidine chromophore with C-1 additions occurring at carbon-5 to form thymine and hydroxymethyluracil. Carbon-3 of serine but not the methyl group of methionine is a one-carbon source for the formation of the thymine and hydroxymethyluracil in the polyoxin complex. S. cacaoi can synthesize unnatural polyoxins, as evidenced by the incorporation of 5-fluoro, 5-bromo, and 6-azauracil into the polyoxins; 5-iodo-, 2-thio-, or 4-thiouracil is not a substrate. Two new polyoxin analogs synthesized and characterized when 5-fluorouracil is added to the cultures are 5-fluoropolyoxin L and 5-fluoropolyoxin M. There is a marked change in the molar ratio of the uracil:thymine:hydroxymethyluracil chromophores in the polyoxin complex following the incorporation of 5-fluoro-, 5-bromo-, or 6-azauracil. Apparently, the unnatural polyoxins inhibit the addition of the C-1 unit to carbon-5 of uracil in the polyoxin complex. Polyoxin L and polyoxin C do not inhibit Escherichia coli and Streptococcus faecalis, but 5-fluoropolyoxin L and 5-fluoropolyoxin C inhibit both these organisms. There is little or no difference in the inhibition of the fluorinated and natural polyoxins against leukemia L-1210 cells. The fluoro group on carbon-5 of the uracil ring does not affect the enzyme-inhibition complex with chitin synthetase since the inhibition constant of fluoropolyoxins L is the same as has been reported for polyoxins A, D, and L. The 14C-labeling pattern in the 5′-amino-5′-deoxy- d -allofuranosyluronic acid moiety of the polyoxins from 14C-labeled glucose, allose, and glycerol suggests that the formation of this unique C-6 uronic acid in the polyoxins does not proceed via the direct oxidation of either d -glucose or d -allose to the -onic or -uronic acids. Glucose is converted to two three-carbon trioses, followed by either (i) the oxidation of one of the trioses to a threecarbon acid and subsequent condensation with another three-carbon sugar to form the C-6 uronic or (ii) an 80:20 equilibrium of the two trioses followed by condensation to a hexose which is then oxidized to the C-6 uronic acid.

Enzymatic synthesis of the 2′,5′-A4 tetramer analog, 2′,5′-ppp3′dA(p3′dA)3, by rabbit reticulocyte lysates: Binding and activation of the 2′,5′-An dependent nuclease, hydrolysis of mRNA, and inhibition of protein synthesis

Summary The structurally modified 2′,5′-pppA(pA) 3 (tetramer) analog, 2′,5′-ppp3′ dA(p3′dA) 3 (referred to as p 3 3′dA 4 ), synthesized by 2′,5′-A n synthetase from cordycepin 5′-triphosphate (3′dATP) in lysates from rabbit reticulocytes has the same structure as chemically synthesized p 3 3′dA. Under our assay conditions, when the 14 C-amino acid concentration is ≥ 50 μM, p 3 3′dA 4 (enzymatically or chemically synthesized) is the most potent inhibitor of protein synthesis in lysates from rabbit reticulocytes reported to date (61% inhibition at 6.7 × 10 −10 M). p 3 3′dA 4 binds to and activates the 2′,5′-A n dependent of nuclease to hydrolyze VSV [ 3 H]mRNA. The 3′-hydroxyl groups of the adenylate of the p 3 A 4 are not required to activate the 2′,5′-A n dependent nuclease in lysates from rabbit reticulocytes.

A rapid quantitative method for measuring UTP, UDP, and UMP in the picomole range.

Abstract A procedure for the determination of picomole amounts of uracil nucleotides is described. The key reaction is the condensation of UTP and [ 14 C]glucose 1-phosphate catalyzed by uridine 5′-diphosphoglucose pyrophosphorylase yielding UDP-[ 14 C]glucose. The product is determined by selective adsorption onto charcoal in the presence of 0.8 m Trizma Base. UDP is measured as UTP after its conversion in an incubation with excess ATP and nucleoside diphosphate kinase. Similarly, UMP is analyzed after it is converted to UDP by nucleoside monophosphate kinase. The uracil nucleotide content of germinated wheat embryos had been determined with this method.

Characterization of a 2′,5′-Oligoadenylate (2–5A)-dependent 37-kDa RNase L AZIDO PHOTOAFFINITY LABELING AND 2–5A-DEPENDENT ACTIVATION

Upregulation of key components of the 2′,5′-oligoadenylate (2–5A) synthetase/RNase L pathway have been identified in extracts of peripheral blood mononuclear cells from individuals with chronic syndrome, including the presence of a low molecular weight form of RNase L. In this study, analysis of 2′,5′-Oligoadenylate (2–5A) binding and activation of the 80- and 37-kDa forms of RNase L has been completed utilizing photolabeling/immunoprecipitation and affinity assays, respectively. Saturation of photolabeling of the 80- and the 37-kDa RNase L with the 2–5A azido photoprobe, [32P]pApAp(8-azidoA), was achieved. Half-maximal photoinsertion of [32P]pApAp(8-azidoA) occurred at 3.7 × 10−8 m for the 80-kDa RNase L and at 6.3 × 10−8 m for the 37-kDa RNase L. Competition experiments using 100-fold excess unlabeled 2–5A photoaffinity probe, pApAp(8-azidoA), and authentic 2–5A (p3A3) resulted in complete protection against photolabeling, demonstrating that [32P]pApAp(8-azidoA) binds specifically to the 2–5A-binding site of the 80- and 37-kDa RNase L. The rate of RNA hydrolysis by the 37-kDa RNase L was three times faster than the 80-kDa RNase L. The data obtained from these 2–5A binding and 2–5A-dependent activation studies demonstrate the utility of [32P]pApAp(8-azidoA) for the detection of the 37-kDa RNase L in peripheral blood mononuclear cell extracts.

Differential antiproliferative actions of 2',5' oligo a trimer core and its cordycepin analogue on human tumor cells

The antiproliferative effect of 2′, 5′A3 core and 2′5′‐3′dA3 (cordycepin trimer) core was measured in 8 human tumor cell lines. Cells were treated in a dose‐response manner for 72 hr and the concentration of drug necessary to inhibit cell growth 50% (Gl50) was determined. A wide range of sensitivities to these drugs was found, even among tumors of the same histological type. The cell lines showed different sensitivities and dose‐response curves to the 2′, 5′A3 and 2′, 5′‐3′dA3 cores. Uptake studies of the 2′,5′A3 and 2′,5′‐3′ dA3 cores, using high‐pressure liquid chromatography , demonstrated that both cores were rapidly degraded in the tissue culture medium and taken up as adenosine or cordycepin, respectively. There was a direct correlation between the uptake of cordycepin and the antiproliferative effect. In contrast, there was no correlation between cell sensitivity and the uptake of the 2′,5′A3 core degradation products. Analysis of intracellular nucleosides and nucleotides indicated that differences in intracellular metabolism of adenosine might explain the different sensitivities of the various cell lines to 2′,5′A3 core. Molar equivalent concentrations of adenosine and cordycepin inhibited cell growth; however, equimolar concentrations of these nucleosides were not effective. In addition, the anti‐proliferative effect of both core compounds and their corresponding nucleosides could be potentiated by the addition of the adenosine deaminase inhibitor, deoxycoformycin . The results indicate that these cores act prodrugs and that the active metabolites are their corresponding nucleosides.