Mass transfer process and separation mechanism of four 5'-ribonucleotides on a strong acid cation exchange resin.

5'-ribonucleotides including adenosine 5'-monophosphate (AMP), cytidine 5'-monophsphate (CMP), guanosine 5'-monophosphate (GMP) and uridine 5'-monophosphate (UMP) have been widely used in the food and pharmaceutical industries. This work focused on the assessment of mass transfer process and separation mechanism of four 5'-ribonucleotides and counter-ion Na+ on the strong cation exchange resin NH-1. The intraparticle diffusion was determined as the rate-limiting step for the mass transfer of AMP, CMP, GMP, and Na+ on the resin NH-1 through the Boyd model. Meanwhile, a homogeneous surface diffusion model (HSDM) combing ion exchange and physical adsorption was proposed and tested against adsorption kinetic data in the batch adsorption systems. The fixed-bed film-surface diffusion model based on the HSDM was then developed and successfully predicted the concentration profiles of 5'-ribonucleotides and the change of pH at the outlet of the fixed-bed in the dynamic adsorption and separation process. Finally, the separation mechanism of 5'-ribonucleotides was presented combining model prediction and experimental results. The separation of UMP, GMP and CMP were mainly based on their differences in isoelectric points, while that of AMP and CMP were lied with the discrepancy of their physical adsorption binding capacity with the resin NH-1.

A new ribonucleotide from Cordyceps militaris.

One new ribonucleotide, 5'-(3''-deoxy-ß-D-ribofuranosyl)-3'-deoxyadenosine (1), and 14 known compounds (2-15) were isolated from an ethanol extract of Cordyceps militaris. The chemical structures of these compounds were determined from 1D and 2D NMR (1H-1H COSY, HMBC, HMQC and NOESY) and HR-ESI-MS spectra, and results were compared with data from the literature. The effects of all isolated compounds were measured on NF-κB activation, with compound 2 exhibiting significant inhibitory activity against TNF-α-induced NF-κB reporter gene expression in HeLa cells from 3 to 100 µM.

Applications of reversible covalent chemistry in analytical sample preparation.

Reversible covalent chemistry (RCC) adds another dimension to commonly used sample preparation techniques like solid-phase extraction (SPE), solid-phase microextraction (SPME), molecular imprinted polymers (MIPs) or immuno-affinity cleanup (IAC): chemical selectivity. By selecting analytes according to their covalent reactivity, sample complexity can be reduced significantly, resulting in enhanced analytical performance for low-abundance target analytes. This review gives a comprehensive overview of the applications of RCC in analytical sample preparation. The major reactions covered include reversible boronic ester formation, thiol-disulfide exchange and reversible hydrazone formation, targeting analyte groups like diols (sugars, glycoproteins and glycopeptides, catechols), thiols (cysteinyl-proteins and cysteinyl-peptides) and carbonyls (carbonylated proteins, mycotoxins). Their applications range from low abundance proteomics to reversible protein/peptide labelling to antibody chromatography to quantitative and qualitative food analysis. In discussing the potential of RCC, a special focus is on the conditions and restrictions of the utilized reaction chemistry.

Preparation of 5-amino-4-imidazole-N-succinocarboxamide ribotide, 5-amino-4-imidazole-N-succinocarboxamide riboside and succinyladenosine, compounds usable in diagnosis and research of adenylosuccinate lyase deficiency.

The enzyme adenylosuccinate lyase (ADSL) intervenes twice in the biosynthesis of adenine nucleotides. ADSL deficiency is an inherited metabolic disease characterized by various degrees of psychomotor retardation and accumulation of dephosphorylated enzyme substrates 5-amino-4-imidazole-N-succinocarboxamide riboside (SAICAr) and succinyladenosine (SAdo) in body fluids. Severity of symptoms seems to correlate with residual activity of mutant enzyme and with SAdo/SAICAr concentration ratio in cerebrospinal fluid. To better understand the pathogenetic mechanisms of the disease symptoms, studies of catalytic properties of mutant enzymes together with in vitro and in vivo experiments utilizing SAICAr and SAdo must be performed. Such studies require availability of both ADSL substrates, 5-amino-4-imidazole-N-succinocarboxamide ribotide (SAICAR) and succinyladenosine 5'-monophosphate (SAMP) and their dephosphorylated products in sufficient amounts and purity. Except for SAMP, none of these compounds is commercially available and they must therefore be synthesized. SAICAR was prepared by recombinant human ADSL-catalysed reaction of AICAR (5-aminoimidazole-4-carboxamide) with fumarate and isolated by thin-layer chromatography. SAICAr and SAdo were prepared by calf intestine alkaline phosphatase-catalysed dephosphorylation of SAICAR and SAMP and isolated on cation- and anion-exchange resin columns. The procedures described are easily scalable and provide high yields of sufficiently pure products for use in experiments related to studies of pathogenetic mechanisms in ADSL deficiency.

RNA aptamers selected against the receptor activator of NF-kappaB acquire general affinity to proteins of the tumor necrosis factor receptor family.

The receptor activator of NF-kappaB (RANK) is a member of the tumor necrosis factor (TNF) receptor family and acts to cause osteoclastgenesis through the interaction with its ligand, RANKL. We isolated RNA aptamers with high affinity to human RANK by SELEX. Sequence and mutational analysis revealed that the selected RNAs form a G-quartet conformation that is crucial for binding to RANK. When the aptamer binding to RANK was challenged by RANKL, there was no competition between the aptamer and RANKL. Instead, the formation of a ternary complex, aptamer-RANK-RANKL, was detected by a spin down assay and by BIAcore surface plasmon resonance analysis. Moreover, the selected aptamer efficiently bound to other TNF receptor family proteins, such as TRAIL-R2, CD30, NGFR as well as osteoprotegerin, a decoy receptor for RANK. These results suggest that the selected aptamer recognizes not the ligand-binding site, but rather a common structure conserved in the TNF receptor family proteins.

Simple and rapid chromatographic method for the separation of major classes of ribosomal ribomononucleotides.

We have described a simple and rapid chromatographic method for the analytical and preparative separation of major types of ribosomal ribomononucleotides with Dowex 1-X10 (HCOO-, 37-74 microns) and Dowex 2-X10 (HCOO-, 37-74 microns) columns, by desorption with formiate solutions in 1-2 h. The separation has been achieved for Cp, Ap, Up and Gp, while a mixture of 2'-, and 3'-nucleoside phosphates desorbs as a single peak; with both resins, a successful separation was achieved with a load from 25 micrograms to 1 mg of ribomononucleotide mixture per ml of packed resin. A complete separation was achieved with Dowex 1, while the separation with Dowex 2 resin was even better. The resins cannot separate unusual nucleosides; therefore, our method is suitable for studies of ribonucleic acids with a low content of unusual nucleosides. Our method has been applied for the quantitative determination of the ribomononucleotide composition of 18S and 28S rRNAs, isolated from mammalian tissues: rat liver, mouse kidney and Ehrlich ascites cells. Dowex 1 and Dowex 2 resins afforded similar or identical ribomononucleotide compositions in all cases; analytical data were in agreement with the literature data. Our method is competitive, in several respects, with modern HPLC techniques for the separation of ribomononucleotides.

Regulation of T4 phage aerobic ribonucleotide reductase. Simultaneous assay of the four activities.

We have devised an assay procedure that permits simultaneous monitoring of the four activities of ribonucleotide reductase. Using this assay, we have compared the reduction of all four substrates by the T4 bacteriophage aerobic ribonucleotide reductase within different allosteric environments. Specifically, we compared the relative turnover rates by the enzyme when activated with "in vivo" concentrations of the known allosteric effectors versus activation by ATP alone. Consistent with the known allosteric properties of this enzyme, our results show that ATP does act as a general activator, although the rate of purine nucleotide reduction was approximately 5% of the rate for the pyrimidine nucleotides. However, addition of the allosteric effectors at their estimated physiological concentrations dramatically changed the relative rates of substrate reduction, creating a more "balanced" pool of products. Addition of the substrates at their respective in vivo concentrations further pushed rates of product formation toward a ratio similar to the base composition of the T4 genome. The similarity of the product profile produced under in vivo conditions to the genomic composition of T4 phage is discussed.

Enzymatic synthesis of ATP analogs and their purification by reverse-phase high-performance liquid chromatography.

The enzymatic syntheses of ATP analogs, such as tubercidin 5'-triphosphate, formycin A 5'-triphosphate, and etheno-ATP, from their respective mono- and diphosphate are described. The reaction products were purified by reverse-phase HPLC using a C-18 matrix and a volatile mobile phase at pH 7, with tributylamine as the ion-pairing agent. Each of the analogs required a buffer of somewhat different composition for the baseline separation of reaction product and reactants. The elutions were isocratic and allowed several successive runs without any intermediate equilibration of the column. After freeze-drying of the pooled fractions, the yield of the synthesized nucleoside triphosphate was approximately 70%. The described procedures are applicable either for analytical investigations or for semi-preparative purposes.

Measurement of nucleoside diphosphate kinase-Nm23 activity by anion-exchange high-performance liquid chromatography.

A first-order assay to detect the activity of nucleoside diphosphate kinase (NDP-kinase; EC 2.7.4.6) was developed. In this assay, the activity of NDP-kinase is measured using various deoxy- and ribonucleotide triphosphates as phosphate donors and dADP as phosphate acceptor. The enzyme activity is determined by quantifying, after anion-exchange HPLC, the amount of newly synthesized dATP. Contrary to the most common coupled enzymic assays or isotopic assays the use of different donor-acceptor pairs is not restricted. The resolution of the procedure described is limited only by the chromatographic separation of substrate and product pairs participating in the reaction.

Tryptophan fluorescence provides a direct probe of nucleotide binding in the noncatalytic sites of Escherichia coli F1-ATPase.

Tryptophan fluorescence was investigated as a tool to study the noncatalytic nucleotide-binding sites of Escherichia coli F1-ATPase. Site-directed mutagenesis, affinity labeling, and lin-benzo-ATP binding studies had shown that residues alpha R365 and beta Y354 are located close to the base moiety of bound nucleotide; here, we mutagenized each to tryptophan. The new tryptophans gave a fluorescence signal indicating an environment of high (alpha W365) or intermediate (beta W354) polarity in unoccupied sites. alpha W365 fluorescence was completely quenched by binding of ATP or ADP, providing a direct, specific probe of noncatalytic site nucleotide occupancy. Using this signal, we measured binding parameters for ATP and ADP, showed that nucleotide binding was magnesium-dependent, and showed that GTP and ITP did bind to some extent, but AMP, GDP, and IDP did not. It was possible to follow initial rates of MgATP hydrolysis and noncatalytic site binding under identical conditions; the results indicated that occupancy of noncatalytic sites was not required for catalysis. Fluorescence from beta W354 was quenched completely by lin-benzo-ATP, but only slightly by ATP or ADP. Probably, residue beta 354 is not as closely juxtaposed to the adenine ring of bound ATP and ADP as is residue alpha 365. With either alpha W365 or beta W354 as donor and catalytic site-bound lin-benzo-ADP as acceptor, no fluorescence resonance energy transfer was detected, indicating that the distance between non-catalytic and catalytic sites is > or = 27 A.

N5-carboxyaminoimidazole ribonucleotide: evidence for a new intermediate and two new enzymatic activities in the de novo purine biosynthetic pathway of Escherichia coli.

Conversion of aminoimidazole ribonucleotide (AIR) to 4-carboxyaminoimidazole ribonucleotide (CAIR) in Escherichia coli requires two proteins, PurE and PurK, previously thought to be subunits of a single enzyme, AIR carboxylase. Past studies revealing an ATP requirement for this reaction (Meyer et al., 1992), in conjunction with present studies, reveal that PurE and PurK possess independent catalytic activities. PurK is shown, by NMR spectroscopy, to catalyze the conversion of AIR in the presence of HCO3- and ATP to ADP, P(i), and the carbamate of AIR (designated N5-CAIR). PurE has been shown by NMR spectroscopy and kinetic analysis, to catalyze the reversible conversion of N5-CAIR and CAIR. N5-CAIR has a half-life of 0.9 min at pH 7.8 and 30 degrees C. Thus, two new enzymatic activities and a new intermediate have been discovered in the de novo purine biosynthetic pathway of E. coli.

Formation of ribonucleotides in DNA modified by oxidative damage in vitro and in vivo. Characterization by 32P-postlabeling.

Oxygen free radicals generated by the interaction of Fe2+ and H2O2 (Fenton reaction) are capable of reacting with DNA bases, which may induce premutagenic and precarcinogenic lesions. Products formed in DNA by such reactions have been characterized as hydroxylated derivatives of cytosine, thymine, adenine, and guanine and imidazole ring-opened derivatives of adenine and guanine. As shown here by 32P-postlabeling, incubation of DNA under Fenton reaction conditions gave rise to additional oxidation products in DNA that were characterized as putative ribonucleosides by enzymatic hydrolysis of the oxidized DNA, 32P-postlabeling, and co-chromatography in multiple systems with authentic markers. Formation of these products in DNA was enhanced by the presence of L-ascorbic acid in the reaction mixtures and their total amounts were similar to those of the major DNA oxidation product, 8-hydroxy-2'-deoxyguanosine. The ribonucleoside guanosine was also formed in kidney DNA of male rats treated with ferric nitrilotriacetate, a renal carcinogen. It is postulated that ribonucleotides alter conformation and function of DNA and thus their presence in DNA may lead to adverse health effects.

Cellular elimination of 2',2'-difluorodeoxycytidine 5'-triphosphate: a mechanism of self-potentiation.

2',2'-Difluorodeoxycytidine (dFdC, Gemcitabine) is a deoxycytidine analogue which, after phosphorylation to the 5'-di- and 5'-triphosphate (dFdCTP), induces inhibition of DNA synthesis and cell death. We examined the values for elimination kinetics of cellular dFdCTP and found they were dependent on cellular concentration after incubation of CCRF-CEM cells with dFdC and washing into drug-free medium. When the drug was washed out at low cellular dFdCTP levels (less than 50 microM), dFdCTP elimination was linear (t1/2 = 3.3 h), but it became biphasic at intracellular dFdCTP levels greater than 100 microM. Although the initial elimination rate was similar at all concentrations, at higher concentrations the terminal elimination rate increased with increasing cellular dFdCTP concentration, with a nearly complete inhibition of dFdCTP elimination at 300 microM. The deamination product 2',2'-difluorodeoxyuridine was the predominant extracellular catabolite at low cellular dFdCTP concentrations, whereas at high dFdCTP concentrations dFdC was the major excretion product. The dCMP deaminase inhibitor 3,4,5,6-tetrahydrodeoxyuridine transformed the monophasic dFdCTP degradation seen at low dFdCTP levels into a biphasic process, whereas the deoxycytidine deaminase inhibitor 3,4,5,6-tetrahydrouridine had no effect on dFdCTP elimination. An in situ assay indicated that dCMP deaminase activity was inhibited in whole cells, an action that was associated with a decreased dCTP:dTTP value. In addition, dFdCTP inhibited partially purified dCMP deaminase with a 50% inhibitory concentration of 0.46 mM. We conclude that dFdC-induced inhibition of dCMP deaminase resulted in a decrease of dFdCTP catabolism, contributing to the concentration-dependent elimination kinetics. This action constitutes a self-potentiation of dFdC activity.

Changes of nucleotide patterns in liver, muscle and blood during the growth of Ehrlich ascites cells: application of the reversed-phase and ion-pair reversed-phase high-performance liquid chromatography with radial compression column.

The pool of purine compounds was analysed in liver, skeletal muscle and blood of mice during the growth of Ehrlich ascites tumour cells. Three fast isocratic high-performance liquid chromatographic methods were used. (1) Determination of nucleotides by an isocratic ion-pair reversed-phase chromatography with a 10 mM ammonium phosphate buffer containing acetonitrile and tetrabutylammonium phosphate. (2) Separation of nucleosides and nucleobases in cell extracts by a reversed-phase system with methanol and 50 mM potassium phosphate buffer as eluent. (3) Nucleosides and nucleobases in body fluids were analysed by a reversed-phase system with 10 mM potassium phosphate containing methanol. These methods allow the rapid determination of purine compounds in small biological samples from various cell types and body fluids, with high accuracy and sensitivity. The pool of cellular nucleotides increased during the exponential phase of tumour growth. Adenosine accumulated significantly in all tissues in the stationary phase of tumour growth.

Metabolic activation of 2,6-diaminopurine and 2,6-diaminopurine-2'-deoxyriboside to antitumor agents.

2,6-Diaminopurine (DAP) and 2,6-diaminopurine 2'-deoxyriboside (DAPdR) are analogs of adenine and deoxyadenosine, respectively. It was the purpose of this study to compare these analogs under identical conditions in order to define their inhibitory properties and the underlying mechanism in L1210 mouse leukemia cells. In a 5-day cell growth experiment, DAP exerted a significantly stronger antiproliferative effect than DAPdR. Correspondingly, colony formation of L1210 cells in soft agarose was inhibited by DAP to a greater extent than by DAPdR. A differential distribution of L1210 cells in the cell cycle resulted from an exposure to DAP and DAPdR. While DAPdR arrested cells in the G1/G0 phase of the cell cycle, DAP appeared to lead to an accumulation of G2/M cells. The diaminopurines were combined with modulatory agents to test the antiproliferative action of the combinations. Deoxycytidine partially rescued the cells from the growth inhibitory action of DAPdR without affecting the growth of DAP-treated cells. When adenine was used, the antiproliferative effect of DAPdR was slightly enhanced while the effect of DAP was completely abolished. 8-Aminoguanosine, a specific inhibitor of purine nucleoside phosphorylase, synergistically potentiated the cytostatic effect of DAPdR. However, this inhibitor did not alter DAP effects. At the biochemical level, the target of DAPdR was ribonucleotide reductase which was in line with a drastic expansion of the dGTP pool in DAPdR-treated cells. In cells exposed to DAP, high levels of DAP riboside triphosphate were measured; concomitantly, the ATP level dropped markedly. Enzymological studies revealed that DAPdR is an excellent substrate of adenosine deaminase giving rise to the formation of deoxyguanosine. DAP was found to be activated in the purine nucleoside phosphorylase reaction and in a phosphoribosyl-pyrophosphate-dependent reaction. The data from this comparative study suggest that DAPdR and DAP possess different toxicity mechanisms. DAPdR and DAP possess different toxicity mechanisms. DAPdR acts as a precursor of deoxyguanosine, and DAP is metabolically activated to DAP-containing ribonucleotide analogs. These different metabolic routes seem to account for the different effects of DAP and DAPdR at the cellular level.

Periodate-methylamine degradation of ribonucleotides in rat liver extracts in comparison to extracts of cultured cells.

Periodate-methylamine degradation of ribonucleotides, which enables separation of deoxyribonucleotides in cell extracts by high-performance liquid chromatography, was tested on the acid-soluble fraction of L1210 cells, Ehrlich ascites tumor (ELD) cells, liver tissue, and liver cell nuclei. It was shown that dTTP, dCTP, and dATP could be clearly separated in L1210 and ELD extracts. In samples from liver tissue, however, dTTP coeluted with another compound, the dCTP peak often eluted very close to another peak, and only dATP separated quite satisfactorily. Furthermore, extracts from liver cell nuclei, isolated in glycerol, were not directly susceptible to periodate oxidation. This problem can be avoided by use of a different procedure for the isolation of cell nuclei.

Isotachophoretic and HPLC determination of nucleotides in rat liver cell nuclei isolated by non-aqueous technique.

Rat liver whole cells and cell nuclei were prepared by a non-aqueous technique (glycerol). The nuclear preparations were of different purity as determined by RNA/DNA ratios (0.17-1.60) and accordingly were divided into 3 subgroups (mean values 0.29, 1.04 and 1.48). RNA nucleotides were separated by isotachophoresis and HPLC and calculated per mg DNA. Two of the nuclear subgroups (RNA/DNA = 1.04 and 1.48) had significantly elevated nucleotide values in relation to RNA/DNA. UDP-N-acetylhexosamine/DNA, on the contrary, was reduced in conformity with RNA in the preparations. Our findings may indicate different nucleotide concentrations in different parts of the cell.

A set of procedures for resolving purine compounds by reversed-phase high performance liquid chromatography: application to the study of purine nucleotide and nucleic acid metabolism.

A set of simple procedures for the separation of major purine 5'-ribonucleotides including diguanosine polyphosphates, purine and pyrimidine bases, and 2'- and 3'-nucleotide monophosphates using reversed-phase high-performance liquid chromatography and isocratic elution study of purine nucleotide and nucleic acid biosynthesis in Artemia is presented.