Comparative study of 2',3'-O-isopropylidene adenosine and adenosine transformation in rat liver homogenates

Transformation of synthesized 2',3'-O-isopropylidene adenosine was studied in comparison with adenosine in rat liver homogenates. It is stated that 2',3'-O-isopropylidene adenosine is subjected to deamination similar to adenosine but less intensively. Due to deamination 2',3'-O-isopropylidene inosine is formed from 2',3'-O-isopropylidene adenosine. It is shown that under conditions of the conducted experiments enzymic splitting of the isopropylidene grouping from the preparation does not occur; this substrate contrary to adenosine does not split under the effect of purine nucleoside phosphorylase.     

Synthesis of the cyclic and acyclic acetal derivatives of 1-(3-C-ethynyl-beta-D-ribo-pentofuranosyl)cytosine,a potent antitumor nucleoside. Design of prodrugs to be selectively activated in tumor tissues via the bio-reduction-hydrolysis mechanism

We have designed and synthesized the acetal derivatives of 1-(3-C-ethynyl-beta-D-ribo-pentofuranosyl)cytosine (ECyd, 1), the 2',3'-O-nitrobenzylidene derivatives 2 and 3 and the 5'-O-(alkoxy)(nitrophenyl)methyl derivatives 6-10 as potential prodrugs of ECyd. These prodrugs can be selectively activated in tumor tissues via a bio-reduction-hydrolysis mechanism owing to the characteristic properties of tumor tissues, such as hypoxia and lower pH. Although the 2',3'-O-(4-nitrobenzylidene) derivatives 2 and 3 were converted bio-reductively into the corresponding 4-aminobenzylidene derivatives by rat S-9 mix, the reduction products, that is, the corresponding amino congeners 4 and 5, proved to be rather stable in an aqueous solution at pH 6.5 used as a pH model for acidic tumor tissues. In contrast, the 5'-O-(alkoxy)(4-nitropheny)methyl derivatives 6-8 were also reduced by rat S-9 mix to the corresponding amino congeners 11-13, which were hydrolyzed to release ECyd more effectively at pH 6.5 than at pH 7.4. Accordingly, the acyclic acetals 6-8 may be efficient prodrugs of ECyd, that are effectively reduced under physiological conditions releasing ECyd in acidic tumor tissues.     

Donor activation in the T4 RNA ligase reaction

T4 RNA ligase catalyzes the adenylation of donor oligonucleotide substrates. These activated intermediates react with an acceptor oligonucleotide which results in phosphodiester bond formation and the concomitant release of AMP. Adenylation of the four common nucleoside 3',5'-bisphosphates as catalyzed by T4 RNA ligase in the absence of an acceptor oligonucleotide has been examined. The extents of product formation indicate that pCp is the best substrate in the reaction and pGp is the poorest. Kinetic parameters for the joining reaction between the preadenylated nucleoside 3',5'-bisphosphates, A(5')pp(5')Cp or A(5')pp(5')Gp, and a good acceptor substrate (ApApA) or a poor acceptor substrate (UpUpU) have been determined. The apparent Km values for both preadenylated donors in the joining reaction are similar, and the reaction velocity is much faster than observed in the overall joining reaction. The nonnucleotide adenylated substrate P1-(5'-adenosyl) P2-(o-nitrobenzyl) diphosphate also exhibits a similar apparent Km but reacts with a velocity 80-fold slower than the adenylated nucleoside 3',5'-bisphosphates. By use of preadenylated donors, oligonucleotide substrates can be elongated more efficiently than occurs with the nucleoside 3',5'-bisphosphates.     

Absorption and transition of [8-14C]adenosine and 2',3'-O-isopropylidene adenosine by Zaidel hepatomas and their homogenates

Transformation and uptake of [8-14C]-adenosine and its synthetic analog 2',3'-O-isopropylideneadenosine was studied in Zajdel hepatoma cells and their homogenates. Uptake and deamination of adenosine and 2',3'-O-isopropylideneadenosine by Zajdel hepatoma cells proceed differently. A small part of adenosine is phosphorylated and then it is included into biosynthesis of polymer substances. The uptake and deamination of 2',3'-O-isopropylideneadenosine by hepatoma cells occurs more intensively than uptake and deamination of adenosine. The formed 2',3'-O-isopropylideneadenosine is not splitted by purine nucleoside phosphorylase and is accumulated in cells in the incubation medium that lead to cell death. The same rate of 2',3'-O-isopropylideneadenosine deamination in cells and their homogenates indicates its high penetrability through plasma membranes. The high uptake of 2',3'-O-isopropylideneadenosine contrary to adenosine leads to deaggregation of cells and their destruction.     

Catalytic mechanism of nucleoside diphosphate kinase investigated using nucleotide analogues, viscosity effects, and X-ray crystallography.

Nucleoside diphosphate (NDP) kinases display low specificity with respect to the base moiety of the nucleotides and to the 2'-position of the ribose, but the 3'-hydroxyl is found to be important for catalysis. We report in this paper the enzymatic analysis of a series of derivatives of thymidine diphosphate (TDP) where the 3'-OH group was removed or replaced by fluorine, azido, and amino groups. With Dictyostelium NDP kinase, kcat decreases 15-200-fold from 1100 s-1 with TDP, and (kcat/Km)NDP decreases from 12 x 10(6) to 10(3) to 5 x 10(4) M-1 s-1, depending on the substrate. The poorest substrates are 3'-deoxyTDP and 3'-azido-3'-deoxyTDP, while the best modified substrates are 2',3'-dehydro-3'-deoxyTDP and 3'-fluoro-3'-deoxyTDP. In a similar way, 3'-fluoro-2',3'-dideoxyUDP was found to be a better substrate than 2',3'-dideoxyUDP, but a much poorer substrate than 2'-deoxyUDP. (kcat/Km)NDP is sensitive to the viscosity of the solution with TDP as the substrate but not with the modified substrates. To understand the poor catalytic efficiency of the modified nucleotides at a structural level, we determined the crystal structure of Dictyostelium NDP kinase complexed to 3'-fluoro-2',3'-dideoxyUDP at 2.7 A resolution. Significant differences are noted as compared to the TDP complex. Substrate-assisted catalysis by the 3'-OH, which is effective in the NDP kinase reaction, cannot occur with the modified substrate. With TDP, the beta-phosphate, which is the leaving group when a gamma-phosphate is transferred to His122, hydrogen bonds to the 3'-hydroxyl group of the sugar; with 3'-fluoro-2',3'-dideoxyUDP, the beta-phosphate hydrogen bonds to Asn119 and moves away from the attacking Ndelta of the catalytic His122. Since all anti-AIDS nucleoside drugs are modified at the 3'-position, these results are relevant to the role of NDP kinase in their cellular metabolism.     

Activation of 5-lipoxygenase by guanosine 5'-O-(3-thiotriphosphate) and other nucleoside phosphorothioates: redox properties of thionucleotide analogs

The stable nucleotide analog guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) was found to be a very potent activator of 5-lipoxygenase in cell-free preparations from rat polymorphonuclear (PMN) leukocytes, causing a 10-fold stimulation of arachidonic acid oxidation at concentrations as low as 0.5-1 microM. The enhancement of enzyme activity was not directly related to G protein activation since the effect of GTP gamma S could not be abolished by GDP nor replaced by GTP or guanylyl-imidodiphosphate (up to 100 microM). Furthermore, other phosphorothioate analogs, such as guanosine 5'-O-(2-thiodiphosphate), adenosine 5'-O-(3-thiotriphosphate), adenosine 5'-O-(2-thiodiphosphate), and adenosine 5'-O-thiomonophosphate all stimulated 5-lipoxygenase activity at concentrations of 10 microM or lower. This effect could not be detected with any of the corresponding nucleoside phosphate derivatives. The stimulation of 5-lipoxygenase activity by nucleoside phosphorothioates was observed under conditions where the reaction is highly dependent on exogenous hydroperoxides, such as in the presence of beta-mercaptoethanol or using enzyme preparations pretreated with sodium borohydride or glutathione peroxidase. GTP gamma S stimulated arachidonic acid oxidation by 5-lipoxygenase to the same extent as the activating hydroperoxides but had no effect on the reaction measured in the presence of optimal concentrations of 13-hydroperoxyoctadecadienoic acid (1-5 microM). Finally, sodium thiophosphate, but not sodium phosphate, markedly stimulated 5-lipoxygenase activity with properties similar to those of GTP gamma S. These results indicate that GTP gamma S and other phosphorothioate derivatives have redox properties that can contribute to increase 5-lipoxygenase activity by replacing the effect of hydroperoxides.     

Expression of human malaria parasite purine nucleoside phosphorylase in host enzyme-deficient erythrocyte culture. Enzyme characterization and identification of novel inhibitors

The intraerythrocytic human malaria parasite, Plasmodium falciparum, requires a source of hypoxanthine for nucleic acid synthesis and energy metabolism. Adenosine has been implicated as a major source for intraerythrocytic hypoxanthine production via deamination and phosphorolysis, utilizing adenosine deaminase and purine nucleoside phosphorylase, respectively. To study the expression and characteristics of human malaria purine nucleoside phosphorylase, P. falciparum was successfully cultured in purine nucleoside phosphorylase-deficient human erythrocytes to an 8% parasitemia level. Purine nucleoside phosphorylase activity was undetectable in the uninfected enzyme-deficient host red cells but after parasite infection rose to 1.5% of normal erythrocyte levels. The parasite purine nucleoside phosphorylase was not cross-reactive with antibody against human enzyme, exhibited a calculated native molecular weight of 147,000, and showed a single major electrophoretic form of pI 5.4 and substrate specificity for inosine, guanosine and deoxyguanosine but not xanthosine or adenosine. The Km values for substrates, inosine and guanosine, were 4-fold lower than that for the human erythrocyte enzyme. In these studies we have identified two novel potent inhibitors of both human erythrocyte and parasite purine nucleoside phosphorylase, 8-amino-5'-deoxy-5'-chloroguanosine and 8-amino-9-benzylguanine. These enzyme inhibitors may have some antimalarial potential by limiting hypoxanthine production in the parasite-infected erythrocyte.     

Influence of dipeptidyl peptidase IV on enzymatic properties of adenosine deaminase

The importance of ADA (adenosine deaminase) in the immune system and the role of its interaction with an ADA-binding cell membrane protein dipeptidyl peptidase IV (DPPIV), identical to the activated immune cell antigen, CD26, has attracted the interest of researchers for many years. To investigate the specific properties in the structure-function relationship of the ADA/DPPIV-CD26 complex, its soluble form, identical to large ADA (LADA), was isolated from human blood serum, human pleural fluid and bovine kidney cortex. The kinetic constants (Km and Vmax) of LADA and of small ADA (SADA), purified from bovine lung and spleen, were compared using adenosine (Ado) and 2'-deoxyadenosine (2'-dAdo) as substrates. The Michaelis constant, Km, evidences a higher affinity of both substrates (in particular of more toxic 2'-dAdo) for LADA and proves the modulation of toxic nucleoside neutralization in the extracellular medium due to complex formation between ADA and DPPIV-CD26. The values of Vmax are significantly higher for SADA, but the efficiency, Vmax/Km, in LADA-catalyzed 2'-dAdo deamination is higher than that in Ado deamination. The interaction of all enzyme preparations with derivatives of adenosine and erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA) was studied. 1-DeazaEHNA and 3-deazaEHNA demonstrate stronger inhibiting activity towards LADA, the DPPIV-CD26-bound form of ADA. The observed differences between the properties of the two ADA isoforms may be considered as a consequence of SADA binding with DPPIV-CD26. Both SADA and LADA indicated a similar pH-profile of adenosine deamination reaction with the optimum at pHs 6.5-7.5, while the pH-profile of dipeptidyl peptidase activity of the ADA/DPPIV-CD26 complex appeared in a more alkaline region.     

Human erythrocytic purine nucleoside phosphorylase: reaction with sugar-modified nucleoside substrates

The kinetic parameters (Km and Vmax) of sugar-modified analogues of inosine and guanosine have been determined with human erythrocytic purine nucleoside phosphorylase (PNP). Steric alterations at the 2' and 3' positions greatly lessened or abolished substrate activity. However, the 5'-deoxy- and 2',5'-dideoxy-beta-D-ribofuranosyl and the alpha-L-lyxosyl analogues were good substrates, indicating that the 5'-hydroxyl and the orientation of the 5'-hydroxy-methyl group are not important for binding. The sugar phosphate analogue, 5-deoxyribose 1-phosphate, was synthesized from 5'-deoxyinosine with immobilized PNP, and its presence was verified by using it in the enzymic synthesis of 5'-deoxyguanosine. The adenosine versions of the 5'-modified analogues were also found to react with adenosine deaminase, albeit at less than 1% of Vmax.     

Introduction of a carboxyl group through an acetal as a new route to carboxylic acid derivatives of sugars.

A new class of carboxylic acid derivatives of sugars is described. Acetalation of mono- and disaccharides with a functionalized vinylic ether or a diethoxybutanoate afforded mono- and diacetals bearing an ester group. Their saponification led to the corresponding carboxylic acid acetals in which the length of the acetal side chain can be modulated.     

Incorporation of 1,N6-ethenoadenosine into the 3' terminus of tRNA using T4 RNA ligase. 1. Preparation of yeast tRNAPhe derivatives

The 3'-terminal A-C-C-A sequence of yeast tRNAPhe has been modified by replacing either adenosine 76 or 73 with the fluorescent analogues 1,N6-ethenoadenosine (epsilon A) or 2-aza-1,N6-ethenoadenosine (aza-epsilon A). T4 RNA ligase was used to join the nucleoside 3',5'-bisphosphates to the 3' end of the tRNA which was shortened by one [tRNAPhe(-A)] or four [tRNAPhe(-ACCA)] nucleotides. It was found that the base-paired 3'-terminal cytidine 72 in tRNAPhe(-ACCA) is a more efficient acceptor in the ligation reaction than the unpaired cytidine 75 at the A-C-C terminus of tRNAPhe(-A). This finding indicates that the mobility of the accepting nucleoside substantially influences the ligation reaction, the efficiency being higher the lower the mobility. This conclusion is corroborated by the observation that the ligation reaction with the double-stranded substrate exhibits a positive temperature dependence rather than a negative one as found for single-stranded acceptors. The replacement of the 3'-terminal adenosine 76 with epsilon A and aza-epsilon A leads to moderately fluorescent tRNAPhe derivatives, which are inactive in the aminoacylation reaction. A number of other tRNAs (Met, Ser, Glu, Lys and Leu-specific tRNAs both from yeast and Escherichia coli) are also inactivated by epsilon A incorporation. Replacement of adenosine 73 followed by repair of the C-C-A end using nucleotidyl transferase leads to tRNAPhe derivatives which are fully active in the aminoacylation reaction and in polyphenylalanine synthesis. The fluorescence of epsilon A and aza-epsilon A at position 73 is virtually completely quenched, suggesting a stacked arrangement of bases around this position. There is no fluorescence increase when the epsilon A-labeled tRNAPhe is complexed with phenylalanyl-tRNA synthetase, elongation factor Tu, or ribosomes. These observations indicate that the stacked conformation of the 3' terminus is not changed appreciably in these complexes.     

Cellular metabolism of 2',3'-dideoxycytidine, a compound active against human immunodeficiency virus in vitro

The nucleoside analog 2',3'-dideoxycytidine (ddCyd) has been shown to inhibit the infectivity and cytopathic effect of human immunodeficiency virus on human OKT4+ lymphocytes in vitro. Metabolism of ddCyd by human T-lymphoblastic cells (Molt 4) negative for human immunodeficiency virus and OKT4 was examined. Molt 4 cells accumulated ddCyd and its phosphorylated derivatives into acid-soluble and acid-insoluble material in a dose-dependent manner. For each concentration tested, 2',3'-dideoxycytidine triphosphate represented 40% of the total acid-soluble pool of ddCyd metabolites. Uptake of 5 microM ddCyd was linear for 4 h after addition of drug. Efflux of ddCyd metabolites from cells followed a biphasic course with an initial retention half-life of 2.6 h for 2',3'-dideoxycytidine triphosphate. DNA, but not RNA, of cells incubated with [3H]ddCyd became radiolabeled. Nuclease and phosphatase treatment of DNA followed by reverse-phase high pressure liquid chromatography showed that the nucleoside was incorporated into DNA in its original form. ddCyd was not susceptible to deamination by human Cyd-dCyd deaminase. It was a poor substrate for human cytoplasmic and mitochondrial dCyd kinases, with Km values of 180 +/- 30 and 120 +/- 20 microM, respectively. DNA polymerases alpha, beta, and gamma varied in their sensitivity to inhibition by ddCTP with Ki values of 110 +/- 40, 2.6 +/- 0.3, and 0.016 +/- 0.008 microM, respectively; however, inhibition was competitive with dCTP in each case.     

Biosynthesis of 9-beta-D-arabinofuranosyladenine: hydrogen exchange at C-2' and oxygen exchange at C-3' of adenosine

The data presented here describe new findings related to the bioconversion of adenosine to 9-beta-D-arabinofuranosyladenine (ara-A) by Streptomyces antibioticus by in vivo investigations and with a partially purified enzyme. First, in double label in vivo experiments with [2'-18O]- and [U-14C]adenosine, the 18O:14C ratio of the ara-A isolated does not change appreciably, indicating a stereospecific inversion of the C-2' hydroxyl of adenosine to ara-A with retention of the 18O at C-2'. In experiments with [3'-18O]- and [U-14C]-adenosine, [U-14C]ara-A was isolated; however, the 18O at C-3' is below detection. The adenosine isolated from the RNA from both double label experiments has essentially the same ratio of 18O:14C. Second, an enzyme has been isolated and partially purified from extracts of S. antibioticus that catalyzes the conversion of adenosine, but not AMP, ADP, ATP, inosine, guanosine, or D-ribose, to ara-A. In a single label enzyme-catalyzed experiment with [U-14C]adenosine, there was a 9.9% conversion to [U-14C]ara-A; with [2'-3H]-adenosine, there was a 8.9% release of the C-2' tritium from [2'-3H]adenosine which was recovered as 3H2O. Third, the release of 3H as 3H2O from [2'-3H]adenosine was confirmed by incubations of the enzyme with 3H2O and adenosine. Ninety percent of the tritium incorporated into the D-arabinose of the isolated ara-A was in C-2 and 8% was in C-3. The enzyme-catalyzed conversion of adenosine to ara-A occurs without added cofactors, displays saturation kinetics, a pH optimum of 6.8, a Km of 8 X 10(-4) M, and an inhibition by heavy metal cations. The enzyme also catalyzes the stereospecific inversion of the C-2' hydroxyl of the nucleoside antibiotic, tubercidin to form 7-beta-D-arabinofuranosyl-4-aminopyrrolo[2,3-d]pyrimidine. The nucleoside antibiotic, sangivamycin, in which the C-5 hydrogen is replaced with a carboxamide group, is not a substrate. On the basis of the single and double label experiments in vivo and the in vitro enzyme-catalyzed experiments, two mechanisms involving either a 3'-ketonucleoside intermediate or a radical cation are proposed to explain the observed data.     

Kinetic studies on degradation of nucleotides catalyzed by phosphodiesterase-phosphomonoesterase from Fusarium moniliforme

Degradation of the 2'-phosphates, 3'-phosphates, 5'-phosphates, 2':3'-cyclic phosphates, 3':5'-cyclic phosphates, and 5'-(p-nitrophenylphosphates) of adenosine, guanosine, cytidine, and uridine catalyzed by Fusarium phosphodiesterase-phosphomonoesterase was followed by means of high performance liquid chromatography. All the nucleotides were susceptible to the enzyme to a greater or lesser degree, and the kinetic constants, Km and kcat, were determined at pH 5.3 and 37 degrees C. These constants were affected by both the nucleoside moiety and the position of the phosphate. Judged from kcat/Km, the 3'-phosphates, 2':3'-cyclic phosphates, and 5'-(p-nitrophenylphosphates) were good substrates, whereas the 2'-phosphates, 5'-phosphates, and 3':5'-cyclic phosphates were poor substrates except for adenosine 2'-phosphate, adenosine 5'-phosphate, and cytidine 5'-phosphate, which were hydrolyzed relatively easily. Among the phosphodiesters, the 2':3'-cyclic phosphates of adenosine, guanosine, and cytidine; and the 3':5'-cyclic phosphates of adenosine and cytidine were degraded into nucleoside and inorganic phosphate without release of intermediary phosphomonoester into the medium. Other phosphodiesters were degraded stepwise releasing definite intermediates.     

A rationale for the shift in colour towards blue in transgenic carnation flowers expressing the flavonoid 3',5'-hydroxylase gene

Recently marketed genetically modified violet carnations cv. Moondust and Moonshadow (Dianthus caryophyllus) produce a delphinidin type anthocyanin that native carnations cannot produce and this was achieved by heterologous flavonoid 3',5'-hydroxylase gene expression. Since wild type carnations lack a flavonoid 3',5'-hydroxylase gene, they cannot produce delphinidin, and instead accumulate pelargonidin or cyanidin type anthocyanins, such as pelargonidin or cyanidin 3,5-diglucoside-6"-O-4, 6"'-O-1-cyclic-malyl diester. On the other hand, the anthocyanins in the transgenic flowers were revealed to be delphinidin 3,5-diglucoside-6"-O-4, 6"'-O-1-cyclic-malyl diester (main pigment), delphinidin 3,5-diglucoside-6"-malyl ester, and delphinidin 3,5-diglucoside-6",6"'- dimalyl ester. These are delphinidin derivatives analogous to the natural carnation anthocyanins. This observation indicates that carnation anthocyanin biosynthetic enzymes are versatile enough to modify delphinidin. Additionally, the petals contained flavonol and flavone glycosides. Three of them were identified by spectroscopic methods to be kaempferol 3-(6"'-rhamnosyl-2"'-glucosyl-glucoside), kaempferol 3-(6"'-rhamnosyl-2"'-(6-malyl-glucosyl)-glucoside), and apigenin 6-C-glucosyl-7-O-glucoside-6"'-malyl ester. Among these flavonoids, the apigenin derivative exhibited the strongest co-pigment effect. When two equivalents of the apigenin derivative were added to 1 mM of the main pigment (delphinidin 3,5-diglucoside-6"-O-4,6"'-O-1-cyclic-malyl diester) dissolved in pH 5.0 buffer solution, the lambda(max) shifted to a wavelength 28 nm longer. The vacuolar pH of the Moonshadow flower was estimated to be around 5.5 by measuring the pH of petal. We conclude that the following reasons account for the bluish hue of the transgenic carnation flowers: (1). accumulation of the delphinidin type anthocyanins as a result of flavonoid 3',5'-hydroxylase gene expression, (2). the presence of the flavone derivative strong co-pigment, and (3). an estimated relatively high vacuolar pH of 5.5.     

Streospecific substitution of oxygen-18 for sulfur in nucleoside phosphorothioates

Reaction of nucleoside phosphorothioates with N-bromosuccinimide in dioxane and H218O leads to the exchange of sulfur for oxygen-18. Using the Sp-isomers of adenosine 5'-O-(1-thiodiphosphate) and adenosine 3',5'-cyclic phosphorothioate, it can be shown by 31P NMR spectroscopy that this reaction proceeds with inversion of configuration yielding the Rp-isomers of [alpha-18O]ADP and [18O]cAMP, respectively. Adenosine 5'-O-(2-thiotriphosphate) and adenosine 5'-O-(3-thiotriphosphate) are likewise converted to [beta-18O]ATP and [gamma-18O]ATP although the stereochemistry of the former reaction has yet to be evaluated. With very slight modifications this reaction is applicable to all the common bases.     

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.     

Adenosine transport and metabolism in mouse leukemia cells and in canine thymocytes and peripheral blood leukocytes.

The intracellular accumulation of free [³H] adenosine was measured by rapid kinetic techniques in P388 murine leukemia cells in which adenosine metabolism (phosphorylation and deamination) was completely prevented by depletion of cellular ATP and by treatment with deoxycoformycin. Nonlinear regression of integrated rate equations on the data demonstrate that the time courses of labeled adenosine accumulation at various extracellular adenosine concentrations in zero-trans and equilibrium exchange protocols are well described by a simple, completely symmetrical, transport model with a carrier:substrate affinity constant of about 150 μM. Adenosine transport was not affected by 1 mM deoxycoformycin indicating that this analog has a low affinity for the nucleoside transport system. The transport capacity of dog thymocytes and peripheral leukocytes was similar to that of P388 cells. Transport was not inhibited by deoxycoformycin and remained constant during the first two hours after mitogenic stimulation with concanavalin A. In untreated, metabolizing P388 cells transport was found to be the major determinant of the rate of intracellular metabolism, regardless of the extracellular adenosine concentration (up to at least 160 μM), but the long-term accumulation (longer than 30–60 seconds) of radioactivity from extracellular adenosine strictly reflected the rate of formation of nucleotides (mainly ATP). The metabolism of adenosine by whole cells was entirely consistent with the kinetic properties of the transport system and those of the metabolic enzymes. At low exogenous adenosine concentrations (1 μM and below) transport was slow enough to allow direct phosphorylation of most of the entering adenosine. The remainder was deaminated and rapidly converted to nucleotides via inosine, hypoxanthine, and IMP. At concentrations of 100 μM or higher, on the other hand, influx exceeded the maximum velocity of adenosine kinase about 100 times so that most of the entering adenosine was deaminated. But since the maximum velocity of adenosine deaminase exceeded those of nucleoside phosphorylase and hypoxanthine/guanine phosphoribosyltransferase about 5 and 100 times, respectively, hypoxanthine and inosine rapidly exited from the cells and accumulated in the medium. A 98% reduction of adenosine transport (at 100 μM), caused by the transport inhibitor Persantin, inhibited adenosine deamination by whole cells to about the same extent as transport, whereas adenosine phosphorylation was relatively little affected; thus in the presence of Persantin, transport and metabolism resembled that occurring at the low adenosine concentration. These and other results indicate that adenosine deamination is an event distinct from transport, which occurs only subsequent to adenosine's transport into the cell.