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At least 73 records · Page 4Linked to original sources
PubMed2000

Evidence for a second nucleotide binding site in rat elongation factor eEF-2 specific for adenylic nucleotides.

The rat elongation factor eEF-2 catalyzes the translocation step of protein synthesis. Besides its well-characterized GTP/GDP binding properties, we have previously shown that ATP and ADP bind to eEF-2 [Sontag, B., Reboud, A. M., Divita, G., Di Pietro, A., Guillot, D., and Reboud, J. P. (1993) Biochemistry 32, 1976-1980]. However, whether the adenylic and guanylic nucleotide binding sites were different or not remained unclear. To further characterize these sites, eEF-2 was incubated in the presence of N-methylanthraniloyl (Mant) fluorescent derivatives of GTP, GDP, ATP, and ADP. This led to an increase in the probe fluorescence and to a partial quenching of eEF-2 tryptophans in each case. The Mant-derivatives and the unmodified corresponding nucleotides were shown to bind to eEF-2 with a similar affinity. Competition experiments between Mant-labeled and unmodified nucleotides suggested the presence of two different sites binding either guanylic or adenylic nucleotides. A Förster's transfer between tryptophan residues and the Mant-probe is obtained with both the adenylic and the guanylic Mant-nucleotides, and comparison of the transfer efficiencies confirmed the presence of a second binding site specific for adenylic nucleotides. A sequence alignment of EF-Gs with eEF-2s from different species suggests the presence of potential Walker A and B motifs in an insert of the G-domain of eEF-2s from higher eukaryotes. Our results raise the possibility that a site specific for adenylic nucleotides and located in this insert has appeared in the course of evolution although its physiological function is still unknown.

Adenine Nucleotides↗
PubMed2002

Probing the nucleotide binding domain of the osmoregulator EnvZ using fluorescent nucleotide derivatives.

EnvZ is a histidine protein kinase important for osmoregulation in bacteria. While structural data are available for this enzyme, the nucleotide binding pocket is not well characterized. The ATP binding domain (EnvZB) was expressed, and its ability to bind nucleotide derivatives was assessed using equilbrium and stopped-flow fluorescence spectroscopy. The fluorescence emission of the trinitrophenyl derivatives, TNP-ATP and TNP-ADP, increase upon binding to EnvZB. The fluorescence enhancements were quantitatively abolished in the presence of excess ADP, indicating that the fluorescent probes occupy the nucleotide binding pocket. Both TNP-ATP and TNP-ADP bind to EnvZB with high affinity (K(d) = 2-3 microM). The TNP moiety attached to the ribose ring does not impede access of the fluorescent nucleotide into the binding pocket. The association rate constant for TNP-ADP is 7 microM(-1) s(-1), a value consistent with those for natural nucleotides and the eucaryotic protein kinases. Using competition experiments, it was found that ATP and ADP bind 30- and 150-fold more poorly, respectively, than the corresponding TNP-derivatized forms. Surprisingly, the physiological metal Mg(2+) is not required for ADP binding and only enhances ATP affinity by 3-fold. Although portions of the nucleotide pocket are disordered, the recombinant enzyme is highly stable, unfolding only at temperatures in excess of 70 degrees C. The unusually high affinity of the TNP derivatives compared to the natural nucleotides suggests that hydrophobic substitutions on the ribose ring enforce an altered binding mode that may be exploited for drug design strategies.

Amino Acid Sequence↗
PubMed1976

Hamster brown-adipose-tissue mitochondria. Purine nucleotide control of the ion conductance of the inner membrane, the nature of the nucleotide binding site.

The inner membrane of hamster brown adipose tissue mitochondria possesses a mechanism for the conductance of protons (or hydroxyl ions) and halide anions which may be specifically inhibited by exogenous purine nucleoside di- or triphosphates. The mechanism of the nucleotide interaction is examined. The added nucleotides can inhibit the ion conductances without equilibrating with the matrix pools of purine nucleotides. ADP translocation is completely sensitive to atractylate, and no mechanism for GDP translocation could be detected. The nucleotides act on the conductance mechanism without covalent modification. A purine nucleotide binding site is described which is distinct from the adenine nucleotide translocase, does not bind atractylate, has a capacity of 0.7 nmol - mg-1, and affinities, specificities and a pH dependency closely corresponding to the conditions required for the inhibition of the ion conductances. The binding site is not apparent in rat liver mitochondria. A causal relationship is suggested between the occupation of this site by added purine nucleotides, and the inhibition of the ion conductance pathway. The role of the pathway in the physiological control of non-shivering thermogenesis by the tissue is discussed.

Adenosine Diphosphate↗
PubMed1994

Evidence that the recently discovered high-energy nucleotide derivative, oligo(phosphoglyceroyl-ATP) forms the end chains of purinogen, a complex polymeric major storage form of adenine nucleotide and phosphate in heart.

Previous work in our laboratory has shown that free adenine nucleotides in heart, liver and kidney exchange rapidly with a major acid-insoluble species which we have characterised as oligo[3-phospho-glyceroyl-gamma- triphospho(5')adenosine(3')], abbreviated to (PG-ATP)n. This has been found in liver and in heart to be complexed to a specific 3'-phosphodiesterase which releases PG-ATP monomers and is located in mitochondrial inter-membrane space. In [14C]adenosine-perfused hearts, (PG-ATP)n has been shown to reach specific radioactivity equilibrium with free ATP within 30 min. Attempts to estimate the quantities of nucleotide in (PG-ATP)n and free nucleotides in response to a variety of stimuli using previously labelled hearts showed by contrast that the free and sequestered nucleotide pools were not at equilibrium. Re-examination of the rapidly labelled acid-insoluble species led to the recognition of radioactive inhomogeneity and of three additional nucleotide components. Perfusion of hearts with phosphate-free medium increased the proportion of 14C incorporated into the sequestered from by 70% and caused the net transfer of 0.9 mumol.g-1 of purine from free adenine nucleotides to the sequestered form. The findings point to the existence of a more complex polymer whose rapidly exchanging end chains we had previously isolated and characterised as (PG-ATP)n; we suggest the name purinogen for this polymer and show that it can contain between 25% and 55% of the tissue nucleotide pool in rat heart.

Adenine Nucleotides↗
PubMed2002

Association between severity of type A hepatitis and nucleotide variations in the 5' non-translated region of hepatitis A virus RNA: strains from fulminant hepatitis have fewer nucleotide substitutions.

BACKGROUND: Type A hepatitis is still a considerable problem in both underdeveloped and developed countries. Why some patients progress to fulminant type A hepatitis and others do not is unclear. AIMS: To determine if nucleotide differences in the genome of hepatitis A virus (HAV) are responsible for the range of clinical severities, we analysed the 5' non-translated region (5'NTR) of the HAV genome, which has an internal ribosomal entry site and is important for cap independent translation of the viral message. METHODS: Serum samples from 84 Japanese patients with sporadic type A hepatitis from five distant regions of Japan, comprising 12 patients with fulminant hepatitis (FH), 13 with severe acute hepatitis (AHs), and 59 with acute hepatitis (AH), were examined for HAV RNA. The fragment between nucleotides 75 and 638 of the 5'NTR was amplified by reverse transcription-polymerase chain reaction, and the nucleotide sequence was determined by direct sequencing. RESULTS: Comparison of sequences of the 5'NTR revealed relatively fewer nucleotide substitutions in FH and AHs patients compared with the considerable sequence variations found in strains of AH. This tendency was most prominent between nucleotides 200 and 500. Strains from FH and AHs cases had fewer nucleotide substitutions (p<0.001) in this region. CONCLUSIONS: Nucleotide variations in the central portion of the 5'NTR of HAV may influence the severity of type A hepatitis.

5' Untranslated Regions↗
PubMed2005

The nucleotide-binding site of the Escherichia coli DnaC protein: molecular topography of DnaC protein-nucleotide cofactor complexes.

The structure of the nucleotide-binding site of the Escherichia coli replication factor DnaC protein and the effect of the nucleotide cofactor on the protein structure have been examined using ultraviolet, steady-state, and time-dependent fluorescence spectroscopy. Emission spectra and quenching studies of the fluorescent nucleotide analogs, 3'-O-(N-methylantraniloyl)-5'-triphosphate (MANT-ATP) and 3'-O-(N-methylantraniloyl)-5'-diphosphate (MANT-ADP), bound to the DnaC protein indicate that the nucleotide-binding site forms a hydrophobic cleft on the surface of the protein. Fluorescence decays of free and bound MANT-ATP and MANT-ADP indicate that cofactors exist in two different conformations both, free and bound to the protein. However, the two conformations of the bound nucleotides differ in their solvent accessibilities. Moreover, there are significant differences in the solvent accessibility between ATP and ADP complexes. Specific binding of magnesium to the protein controls the structure of the binding site, particularly, in the case of the ATP complex, leading to additional opening of the binding site cleft. Both tyrosine and tryptophan residues are located on the surface of the protein. The tryptophans are clustered at a large distance from the nucleotide-binding site. However, in spite of a large spatial separation, binding of both cofactors induces significant and different changes in the structure of the environment of tryptophans, indicating long-range structural effects through the DnaC molecule. Moreover, only ATP induces changes in the distribution of the tyrosine residues on the surface of the protein. The data reveal that the nucleotide-DnaC protein complex is a sophisticated allosteric system, responding differently to the ATP and ADP binding.

Adenosine Diphosphate↗
PubMed1994

Cdc25p, the guanine nucleotide exchange factor for the Ras proteins of Saccharomyces cerevisiae, promotes exchange by stabilizing Ras in a nucleotide-free state.

In Saccharomyces cerevisiae, adenylate cyclase activity is controlled by Ras1p and Ras2p. Activation of the Ras proteins is in turn controlled by the GTPase-activating proteins (GAPs), Ira1p and Ira2p, and the guanine nucleotide exchange factor (GNEF), Cdc25p. We have characterized Cdc25p enzymologically in order to gain information about the mechanism of Cdc25p-mediated guanine nucleotide exchange and to appreciate how the activity of a GNEF is integrated as a part of a basic molecular switch module consisting of Ras, GNEF, and GAP. Using Ras2p and a catalytic fragment of Cdc25p, both expressed in and purified from Escherichia coli, we determined that Cdc25p has a Km for Ras2p-GDP of 160 nM and a maximal rate of 0.20 s-1. The Km of Cdc25p for Ras2p complexed to GTP is 3-fold greater than that for Ras2p complexed to GDP. The Km of free GDP is about 2-fold higher than the Km of free GTP. This suggests that Cdc25p activates Ras2p primarily by equilibrating Ras2p with the pool of free guanine nucleotides in the cell rather than by driving Ras2p inexorably into the activated state. This renders Ras activation potentially subject to energy charge fluctuations in the cell. The free guanine nucleotide affects kcat, indicating that the rate-limiting step is nucleotide association. Finally, we demonstrated that dominant negative alleles of Ras2p are potent competitive inhibitors of Cdc25p. These data, in conjunction with the kinetic data, are consistent with the hypothesis that Cdc25p catalyzes guanine nucleotide exchange by stabilizing a nucleotide-free intermediate of Ras.

Cell Cycle Proteins↗
PubMed1993

Labeling of adenine and guanine nucleotide-binding proteins in permeabilized cells with in situ periodate-oxidized nucleotides.

A novel approach for identification of adenine and guanine nucleotide-binding proteins in permeabilized cells is described. Cells were incubated for various periods with alpha-32P-labeled nucleotides and then briefly treated with periodate. Condensation products formed in situ between the protein bound alpha-32P-labeled oxidized nucleotide (NTPoxi) and a lysine residue near the nucleotide-binding sites were rapidly stabilized by the addition of cyanoborohydride. Analysis by two-dimensional isoelectric focusing/sodium dodecylsulfate-poly-acrylamide gel electrophoresis showed that in the human leukemic T-cell line Jurkat a number of distinct intracellular proteins could be labeled with ATPoxi (M(r) 40,000-200,000) or GTPoxi (M(r) 19,000-80,000). Competition with deoxyribonucleotides confirmed the selectivity of these affinity labeling reactions. To test this method two classical GTP-binding proteins were further examined. First the alpha-subunits of the Gs and Gi-2 proteins were specifically labeled with [alpha-32P]GTPoxi but not with [alpha-32P]ATPoxi. Second, p21ras was crosslinked specifically to [alpha-32P]GTPoxi or to its bound endogenous ligand. Surprisingly, under optimized conditions 60% of the ras protein was specifically modified, demonstrating the high efficiency and sensitivity of the method. As a first step toward isolation of hitherto unidentified nucleotide-binding proteins, rabbit antisera specific for the modified amino acid residues were raised. The presented labeling method can be applied for identification of nucleotide-binding proteins in all eukaryotic cells.

Adenine Nucleotides↗
PubMed2000

Mutating three residues in the bovine rod cyclic nucleotide-activated channel can switch a nucleotide from inactive to active.

Cyclic nucleotide-gated (CNG) channels, which were initially studied in retina and olfactory neurons, are activated by cytoplasmic cGMP or cAMP. Detailed comparisons of nucleotide-activated currents using nucleotide analogs and mutagenesis revealed channel-specific residues in the nucleotide-binding domain that regulate the binding and channel-activation properties. Of particular interest are N(1)-oxide cAMP, which does not activate bovine rod channels, and Rp-cGMPS, which activates bovine rod, but not catfish, olfactory channels. Previously, we showed that four residues coordinate the purine interactions in the binding domain and that three of these residues vary in the alpha subunits of the bovine rod, catfish, and rat olfactory channels. Here we show that both N(1)-oxide cAMP and Rp-cGMPS activate rat olfactory channels. A mutant of the bovine rod alpha subunit, substituted with residues from the rat olfactory channel at the three variable positions, was weakly activated by N(1)-oxide cAMP, and a catfish olfactory-like bovine rod mutant lost activation by Rp-cGMPS. These experiments underscore the functional importance of purine contacts with three residues in the cyclic nucleotide-binding domain. Molecular models of nucleotide analogs in the binding domains, constructed with AMMP, showed differences in the purine contacts among the channels that might account for activation differences.

Animals↗
PubMed2004

pH-dependent perturbation of Ras-guanine nucleotide interactions and Ras guanine nucleotide exchange.

p21Ras (Ras) proteins cycle between active GTP-bound and inactive GDP-bound states to mediate signal transduction pathways that promote cell growth, differentiation, and apoptosis. To better understand how cellular regulatory factors, such as guanine nucleotide exchange factors (GEFs) and nitric oxide (NO), modulate Ras-guanine nucleotide binding interactions, we have conducted NMR and kinetic studies to investigate the pH dependence of Ras-GDP interactions and Ras-guanine nucleotide exchange (GNE). pH-sensitive amide protons were identified and found to be associated with residues in the switch I (Phe28-Asp30) and switch II (Asp57 and Thr58) regions of Ras. Furthermore, most of the residues that interact with Mg2+ exhibit pH-sensitive amide proton chemical shifts which appear to be coupled to pH-dependent Ras Mg2+ binding and guanine nucleotide binding affinity. These results suggest that perturbation of Mg2+ interactions within the Ras-guanine nucleotide complex is critical for pH-dependent dissociation of guanine nucleotide ligands from Ras. Notably, these same regions undergo conformational changes upon association with the Ras GEF, SOS. In addition, although we have recently shown that addition of NO to Ras in the presence of oxygen produces a Ras thiyl radical intermediate that promotes Ras GNE, we have also postulated that another byproduct of this reaction, a H+, may contribute to NO-mediated GNE. However, the results presented herein suggest that the H+ byproduct of the reaction is unlikely to be involved in the NO-mediated Ras GNE.

Amino Acid Sequence↗
PubMed1999

Thermodynamics of nucleotide binding to the chaperonin GroEL studied by isothermal titration calorimetry: evidence for noncooperative nucleotide binding.

We characterized the thermodynamics of binding reactions of nucleotides ADP and ATPgammaS (a nonhydrolyzable analog of ATP) to GroEL in a temperature range of 5 degrees C to 35 degrees C by isothermal titration calorimetry. Analysis with a noncooperative binding model has shown that the bindings of nucleotides are driven enthalpically with binding constants of 7x103 M-1 and 4x104 M-1 for ADP and ATPgammaS, respectively, at 26 degrees C and that the heat capacity change DeltaCp is about 100 cal/mol.K for both the nucleotides. The stoichiometries of binding were about 8 and 9 molecules for ADP and ATPgammaS, respectively, per GroEL tetradecamer at 5 degrees C, and both increased with temperature to reach about 14 (ADP) and 12 (ATPgammaS) for both nucleotides at 35 degrees C. The absence of initial increase of binding heat as well as Hill coefficient less than 1.2, which were obtained from the fitting to the model curve by assuming positive cooperativity, showed that there was virtually no positive cooperativity in the nucleotide bindings. Incorporating a difference in affinity for the nucleotide (ADP and ATPgammaS) between the two rings of GroEL into the noncooperative binding model improved the goodness of fitting and the difference in the affinity increased with decreasing temperature.

Adenosine Diphosphate↗
PubMed2006

Effects of nucleotides and nucleotide analogue inhibitors of HIV-1 reverse transcriptase in a ratchet model of polymerase translocation.

A single cycle of nucleotide incorporation by the reverse transcriptase of the human immunodeficiency virus type 1 (HIV-1 RT) involves the initial binding of an incoming nucleotide, a conformational change that traps the substrate, the formation of a new phosphodiester bond, the release of pyrophosphate (PPi), and ultimately polymerase translocation, which clears the nucleotide binding site. This article reviews different mechanistic models for polymerase translocation with emphasis placed on HIV-1 RT. Structure-function analyses of stalled complexes of HIV-1 RT provide strong evidence to suggest that the enzyme can oscillate between pre- and post-translocational states. Nucleotide hydrolysis is not required for the movement of the polymerase in a stalled configuration; thermal energy is sufficient to allow random bidirectional sliding. The next complementary nucleotide, following the incorporated chain-terminator, acts like a pawl of a ratchet that traps the enzyme in the post-translocation state and prevents the reverse movement. Quantitative footprinting experiments have shown that the concentration of the templated nucleotide required to shift the translocational equilibrium forward depends crucially on the structure of the 3'end of the primer. Changes in the relative population of pre- and post-translocation complexes can influence rates of excision of incorporated NRTIs, which, in turn, affects drug susceptibility. The concept of a ratchet model of HIV-1 RT translocation and its implications for drug action and resistance, and the discovery and development of novel antiviral compounds is discussed.

Catalysis↗
PubMed1986

Growth of Lactobacillus bulgaricus in milk. 2. Characteristics of purine nucleotides, pyrimidine nucleotides, and nucleic acid synthesis.

Lactobacillus bulgaricus incorporated exogenous guanine only into guanine nucleotides and not into adenine nucleotides. Lactobacillus bulgaricus lacks the ability to interconvert adenine nucleotides and guanine nucleotides. Biosynthesis of pyrimidine nucleotides from orotic acid was depressed by addition of guanosine monophosphate or adenosine monophosphate. Some step in pyrimidine biosynthesis from orotic acid may be negatively regulated by the intracellular amount of purine nucleotides. Lactobacillus bulgaricus could not grow in milk from which orotate was removed. This indicates that the activity of de novo pyrimidine synthesis is very low in this organism. When the intracellular amount of purine or pyrimidine base was limited, ribonucleic acid synthesis was markedly depressed, resulting in cell elongation, whereas deoxyribonucleic acid synthesis was not so much affected.

Adenine↗
PubMed2004

A type of nucleotide motif that distinguishes tobamovirus species more efficiently than nucleotide signatures.

The complete genomic sequences of forty-eight tobamoviruses were classified and found to form at least twelve species clusters. Individual species were not conveniently defined by 'nucleotide signatures' (i.e. strings of one or more nucleotides unique to a taxon) as these were scattered sparsely throughout the genomes and were mostly single nucleotides. By contrast all the species were concisely and uniquely distinguished by short nucleotide motifs consisting of conserved genus-specific sites intercalated with variable sites that provided species-specific combinations of nucleotides (nucleotide combination motifs; NC-motifs). We describe the procedure for finding NC-motifs in a convenient and phylogenetically conserved region of the tobamovirus RNA polymerase gene, the '4404-50 motif'. NC-motifs have been found in other sets of homologous sequences, and are convenient for use in published taxonomic descriptions.

Conserved Sequence↗
PubMed2005

Binding of six nucleotide cofactors to the hexameric helicase RepA protein of plasmid RSF1010. 2. Base specificity, nucleotide structure, magnesium, and salt effect on the cooperative binding of the cofactors.

Interactions of the RepA hexameric helicase with nucleotide cofactors have been examined using nucleotide analogues, TNP-ADP and TNP-ATP, and unmodified nucleotides. Thermodynamic parameters for the interactions of modified and unmodified nucleotides have been obtained using quantitative fluorescence titration and competition titration methods. The intrinsic binding constant of ATP is by a factor of approximately 10 and approximately 1000 higher than the value observed for ADP and PO(4)(-). The data suggest that helicase acquires free-energy transducing capabilities when associated with the ssDNA, thus, forming a "holoenzyme". ATP binding is characterized by significantly stronger negative cooperativity than ADP. The cooperative interactions are predominantly induced through the specific interactions of the gamma phosphate and the ribose with the protein. The salt effect on cofactor binding indicates a very different nature of the intrinsic and cooperative interactions. Surprisingly, binding of Mg(2+), to both the cofactor and helicase, predominantly controls the ADP-RepA interactions. Mg(2+) cations seem to play a role in affecting the distribution of high and low ssDNA-affinity states, through the strong effect on the diphosphate versus triphosphate binding. The data indicate that Mg(2+) has a dual function in nucleotide-helicase interactions. At low [Mg(2+)], NTP binds stronger than NDP and the enzyme is predominantly in the high ssDNA-affinity state. At higher [Mg(2+)], NTP binds weaker than NDP and the helicase subunits can exist in alternating low- and high-affinity states that facilitate the efficient dsDNA unwinding. The RepA helicase shows a preference toward purine nucleotides. The cooperative interactions are independent of the type of the base.

Adenosine Diphosphate↗
PubMed2005

Binding of six nucleotide cofactors to the hexameric helicase RepA protein of plasmid RSF1010. 1. Direct evidence of cooperative interactions between the nucleotide-binding sites of a hexameric helicase.

The interactions of nucleotides with RepA hexameric helicase from plasmid RSF1010 have been examined using nucleotide analogues, TNP-ADP, TNP-ATP, and MANT-ADP. The binding of the analogues is accompanied by strong quenching of the protein fluorescence. A quantitative fluorescence titration method has been applied to analyze the interactions, independent of any assumptions of proportionality between the fluorescence quenching and the average degree of binding. The fluorescence quenching as a function of the average degree of binding is expressed by an empirical function that enables analysis of the data, without the necessity of determining quenching parameters for different complexes. At saturation, the RepA hexamer binds six nucleotide molecules, indicating that each subunit of the hexamer can engage in interactions with the cofactor. The nucleotide macroscopic affinity decreases with the increasing degree of binding, indicating heterogeneity among the binding sites. A statistical thermodynamic hexagon model provides an excellent description of the binding process and requires only two interaction parameters, the intrinsic binding constant, K, and cooperativity parameter, sigma. The heterogeneity in affinity reflects negative cooperative interactions between the binding sites. Analyses of the data provide clear evidence that the alternative model of two independent classes of binding sites does not describe the nucleotide binding. Such a model cannot account for both, the binding isotherms and the dependence of the fluorescence quenching upon the degree of binding. Thus, cooperative interactions between the nucleotide-binding sites are an intrinsic property of the RepA helicase. The presence of the cooperative interactions indicates significant communication among the subunits of the helicase.

Adenosine Diphosphate↗