inosinic-acid and deoxyinosine-monophosphate

Nucleotide quality surveillance enzymes play important roles in human health, by detecting damaged molecules in the nucleotide pool and deactivating them before they are incorporated into chromosomal DNA or adversely affect metabolism. In particular, deamination of adenine moiety in (deoxy)nucleoside triphosphates, resulting in formation of (d)ITP, can be deleterious, leading to DNA damage, mutagenesis and other harmful cellular effects. The 21.5 kDa human enzyme that mitigates this damage by conversion of (d)ITP to monophosphate, ITPA, has been proposed as a possible therapeutic and diagnostic target for multiple diseases. Measuring the activity of this enzyme is useful both in basic research and in clinical applications involving this pathway, but current methods are nonselective and are not applicable to measurement of the enzyme from cells or tissues. Here, we describe the design and synthesis of an ITPA-specific chimeric dinucleotide (DIAL) that replaces the pyrophosphate leaving group of the native substrate with adenosine triphosphate, enabling sensitive detection via luciferase luminescence signaling. The probe is shown to function sensitively and selectively to quantify enzyme activity in vitro, and can be used to measure the activity of ITPA in bacterial, yeast and human cell lysates.

Topics: Adenosine Triphosphate; Cell Extracts; Cell Line, Tumor; Deamination; DNA; DNA Damage; Enzyme Assays; Fluorescent Dyes; HeLa Cells; Humans; Inosine Monophosphate; Luminescent Measurements; Pyrophosphatases; RNA Interference; RNA, Small Interfering

DNA is the ultimate target of platinum-based anticancer therapy. Since the N7 of guanine is known to be the major binding site of cisplatin and its analogues, adduct formation with model nucleotides, especially 2'-deoxyguanosine 5'-monophosphate (dGMP), has been studied in detail. During the last few years a coupled capillary eletrophoresis/electrospray-ionization mass spectrometry (CE/ESI-MS) method has been advantageously used in order to separate and identify platinum adducts with nucleotides in submillimolar concentrations in aqueous solutions. Beside the bisadduct, [Pt(NH(3))(2)(dNMP)(2)](2-) (NMP=2'-deoxynucleoside 5'-monophosphate), and the well-known monochloro and monohydroxo adducts, [Pt(NH(3))(2)Cl(dNMP)](-) and [Pt(NH(3))(2)(dNMP)OH](-), respectively, a third kind of monoadduct species with a composition of [Pt(NH(3))(2)(dNMP)](-) can be separated by CE and detected through the m/z values measured with ESI-MS. Different experimental setups indicate the existence of an O(6)-N7 chelate, whereas the formation of N7-alphaPO(4) macrochelates or dinuclear species is unlikely. Additionally, offline MS experiments with 2'-deoxyguanosine (dG) and stabilization of the controversially discussed O(6)-N7 chelate by oxidation with hydrogen peroxide support the assumption of the existence of O(6)-N7 chelation.

Topics: Cisplatin; Deoxyguanine Nucleotides; DNA Adducts; Electrophoresis, Capillary; Guanosine Monophosphate; Inosine Monophosphate; Molecular Structure; Nucleosides; Nucleotides; Organoplatinum Compounds; Spectrometry, Mass, Electrospray Ionization

In DNA, the deamination of dAMP generates 2'-deoxy-inosine 5'-monophosphate (dIMP). Hypoxanthine (HX) residues are mutagenic since they give rise to A.T-->G.C transition. They are excised, although with different efficiencies, by an activity of the 3-methyl-adenine (3-meAde)-DNA glycosylases from Escherichia coli (AlkA protein), human cells (ANPG protein), rat cells (APDG protein) and yeast (MAG protein). Comparison of the kinetic constants for the excision of HX residues by the four enzymes shows that the E.coli and yeast enzymes are quite inefficient, whereas for the ANPG and the APDG proteins they repair the HX residues with an efficiency comparable to that of alkylated bases, which are believed to be the primary substrates of these DNA glycosylases. Since the use of various substrates to monitor the activity of HX-DNA glycosylases has generated conflicting results, the efficacy of the four 3-meAde-DNA glycosylases of different origin was compared using three different substrates. Moreover, using oligo-nucleotides containing a single dIMP residue, we investigated a putative sequence specificity of the enzymes involving the bases next to the HX residue. We found up to 2-5-fold difference in the rates of HX excision between the various sequences of the oligonucleotides studied. When the dIMP residue was placed opposite to each of the four bases, a preferential recognition of dI:T over dI:dG, dI:dC and dI:dA mismatches was observed for both human (ANPG) and E.coli (AlkA) proteins. At variance, the yeast MAG protein removed more efficiently HX from a dI:dG over dI:dC, dI:T and dI:dA mismatches.

Topics: Animals; Bacterial Proteins; Base Pair Mismatch; Base Sequence; DNA; DNA Glycosylases; Escherichia coli; Fungal Proteins; Humans; Hypoxanthine; Inosine Monophosphate; Kinetics; N-Glycosyl Hydrolases; Oligodeoxyribonucleotides; Piperidines; Rats; Saccharomyces cerevisiae; Substrate Specificity; Thermodynamics

IMP dehydrogenase (IMPDH) catalyzes the oxidation of IMP to XMP with the concomitant reduction of NAD(+). This reaction involves the formation of a covalent adduct with an active site Cys. This intermediate, E-XMP, hydrolyzes to produce XMP. The mutation of Asp338 to Ala severely impairs the activity of Escherichia coli IMPDH, decreasing the value of k(cat) by 650-fold. No (D)V(m) or (D)V/K(m) isotope effects are observed when 2-(2)H-IMP is the substrate for wild-type IMPDH. Values of (D)V(m) = 2.6 and (D)V/K(m) (IMP) = 3.4 are observed for Asp338Ala. Moreover, while a burst of NADH production is observed for wild-type IMPDH, no burst is observed for Asp338Ala. These observations indicate that the mutation has decreased the rate of hydride transfer by at least 5 x 10(3)-fold. In contrast, k(cat) for the hydrolysis of 2-chloroinosine-5'-monophosphate is decreased by only 8-fold. In addition, the rate constant for inactivation by 6-chloropurine riboside 5'-monophosphate is increased by 3-fold. These observations suggest that the mutation has little effect on the nucleophilicity of the active site Cys residue. These results are consistent with a recent crystal structure that shows a hydrogen bonding network between Asp338, the 2'-OH of IMP, and the amide group of NAD(+) [Colby, T. D., Vanderveen, K., Strickler, M. D., Markham, G. D., and Goldstein, B. M. (1999) Proc. Natl. Acad. Sci. U.S.A. 96, 3531-3536].

Topics: Alkylation; Aspartic Acid; Catalysis; Catalytic Domain; Deuterium; Escherichia coli; Hydrogen; IMP Dehydrogenase; Inosine Monophosphate; Kinetics; Models, Chemical; Mutation

Fab fragments from Jel 103, an antibody which specifically binds to single-stranded poly(rl), were prepared by papain digestion, separated into eight isoforms and characterized by mass spectrometry. One of the purified isoforms yielded crystals suitable for structural studies by X-ray diffraction and its crystal structure was determined to 2.4 A resolution. Soaking the crystals in solutions containing either of the mononucleotides inosine-5'-diphosphate, guanosine-5'-diphosphate or deoxyinosine-5'-monophosphate resulted in binding of the nucleotide in a single binding site. However, adenosine-5'-diphosphate does not bind to this antibody. The recognition of the base is achieved through hydrogen bonds to the C6 carbonyl oxygen and the imino NH group of the purine in a pattern similar to that of the base-base interactions in a double-stranded nucleic acid. Additional binding energy is provided by stacking of the base and the Tyr32L side-chain and by interaction of the alpha-phosphate with the antibody in an anionic binding site. Most of the side-chains interacting with the nucleotide come from the light chain. Surprisingly, this antibody shares the VL sequence with another nucleic acid-binding antibody, BV04-1. The latter binds to a single stranded DNA with a high preference for thymine bases. The structures of the unliganded and complexed Jel 103 Fab are compared to those of BV-04-1 Fab and while they show similarity in recognition of the base of the immunodominant nucleotide, their 5' phosphates occupy different positions, suggesting different orientation of the nucleic acid bound to these two antibodies. Differences in the conformations of the L1 loops between the two Fabs have been noted.

Topics: Amino Acid Sequence; Antibody Specificity; Base Sequence; Binding Sites, Antibody; Crystallization; Crystallography, X-Ray; Guanosine Diphosphate; Immunoglobulin Fab Fragments; Immunoglobulin Variable Region; Inosine Diphosphate; Inosine Monophosphate; Isoelectric Point; Mass Spectrometry; Models, Molecular; Molecular Sequence Data; Protein Conformation; Ribose; RNA

Hypoxanthine-DNA glycosylase from Escherichia coli was partially purified by ammonium sulfate fractionation and by chromatography on Sephacryl S-200, DEAE-cellulose, and phosphocellulose P-11 columns. Analysis of the enzymatic reaction products was carried out on a minicolumn of DEAE-cellulose and/or by paper chromatography, by following the release of the free base [3H]hypoxanthine from [3H]dIMP-containing phi X174 DNA. In native conditions, the enzyme has a molecular mass of 60 +/- 4 kDa, as determined by gel filtration on Sephadex G-150 and Sephacryl S-200 columns. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis revealed a major polypeptide band of an apparent molecular mass of 56 kDa, and glycerol gradient centrifugation indicated a sedimentation coefficient of 4.0 S. Hypoxanthine-DNA glycosylase from E. coli has an obligatory requirement for Mg2+ and is totally inhibited in the presence of EDTA. Co2+ can only partially replace Mg2+. The enzyme is inhibited by hypoxanthine which at 4 mM causes 85% inhibition. The optimal pH range of the enzymatic activity is 5.5-7.8, and the apparent Km value is 2.5 x 10(-7) M.

Topics: Ammonium Sulfate; Bacteriophage phi X 174; Cations, Divalent; Centrifugation, Density Gradient; Chromatography; Chromatography, Paper; DNA, Viral; Edetic Acid; Escherichia coli; Fractional Precipitation; Glycoside Hydrolases; Hydrogen-Ion Concentration; Hypoxanthine; Hypoxanthines; Inosine Monophosphate; Magnesium; Molecular Weight; Streptomycin; Substrate Specificity; Temperature

A DNA glycosylase that releases free hypoxanthine by selective cleavage of dIMP residues in polydeoxynucleotides and DNA containing this nonconventional nucleotide is present in Escherichia coli cell extracts. The partly purified enzyme, termed hypoxanthine-DNA glycosylase, does not require divalent cations or phosphate for activity, and it acts more efficiently on double-stranded than on single-stranded substrates. The enzyme has properties different from either uracil-DNA glycosylase or 3-methyladenine-DNA glycosylase and is present at normal levels in E. coli mutants deficient in either of the latter two DNA glycosylases.

Topics: Bacillus subtilis; Deoxyribonucleotides; DNA; DNA, Bacterial; Escherichia coli; Glycoside Hydrolases; Hypoxanthines; Inosine Monophosphate; Inosine Nucleotides; Polydeoxyribonucleotides; Species Specificity