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P Plateau

Publications and source records attributed to P Plateau.

50 records · Page 3Linked to original sources
PubMed1987

Non-adenylylated bis(5'-nucleosidyl) tetraphosphates occur in Saccharomyces cerevisiae and in Escherichia coli and accumulate upon temperature shift or exposure to cadmium.

A new set of bis(5'-nucleosidyl) tetraphosphates, the Bp4B' nucleotides (B and B' = C, G, or U not equal to A), are demonstrated in living cells. In exponentially growing Saccharomyces cerevisiae, cellular concentrations of Cp4U, Up4U, Gp4G, Cp4C, Gp4U, and Gp4C are 210, 200, 60, 50, 40, and 30 nM, respectively. It is likely that these nucleotides originate from the action of diadenosine-5',5"'-P1,P4-tetraphosphate alpha,beta-phosphorylase, an enzyme recently found in yeast. Upon temperature shift or exposure to cadmium, the Bp4B' nucleotides strongly accumulate in the yeast cells. In Escherichia coli, the same nucleotides occur, and similar effects of temperature shift or of cadmium are observed. However, in the bacterium, the origin of these nucleotides is not known. To quantitate these nucleotides in cellular extracts, specific procedures were developed. In the first step, after purification of the mixture of Np4N' (N and N' = A, C, G, or U) nucleotides, the Ap4N nucleotides are specifically removed by incubation with lysyl-tRNA synthetase. In the second step, the Bp4B' species are resolved with the help of anion-exchange high performance liquid chromatography. In the third step, the concentration of each Bp4B' is measured using three coupled enzymatic reactions to produce ATP and bioluminescence. With this strategy, 0.01 pmol of any Bp4B' nucleotide can be reliably detected.

Adenine Nucleotides↗
PubMed1987

Yeast diadenosine 5',5'''-P1,P4-tetraphosphate alpha,beta-phosphorylase behaves as a dinucleoside tetraphosphate synthetase.

The diadenosine 5',5'''-P1,P4-tetraphosphate alpha,beta-phosphorylase (Ap4A phosphorylase), recently observed in yeast [Guaranowski, A., & Blanquet, S. (1985) J. Biol. Chem. 260, 3542-3547], is shown to be capable of catalyzing the synthesis of Ap4A from ATP + ADP, i.e., the reverse reaction of the phosphorolysis of Ap4A. The synthesis of Ap4A markedly depends on the presence of a divalent cation (Ca2+, Mn2+, or Mg2+). In vitro, the equilibrium constant K = ([Ap4A][Pi])/[(ATP][ADP]) is very sensitive to pH. Ap4A synthesis is favored at low pH, in agreement with the consumption of one to two protons when ATP + ADP are converted into Ap4A and phosphate. Optimal activity is found at pH 5.9. At pH 7.0 and in the presence of Ca2+, the Vm for Ap4A synthesis is 7.4 s-1 (37 degrees C). Ap4A phosphorylase is, therefore, a valuable candidate for the production of Ap4A in vivo. Ap4A phosphorylase is also capable of producing various Np4N' molecules from NTP and N'DP. The NTP site is specific for purine ribonucleotides (N = A, G), whereas the N'DP site has a broader specificity (N' = A, C, G, U, dA). This finding suggests that the Gp4N' nucleotides, as well as the Ap4N' ones, could occur in yeast cells.

Acid Anhydride Hydrolases↗
PubMed1987

Intracellular 5',5'-dinucleoside polyphosphate levels remain constant during the Escherichia coli cell cycle.

All AppppN and ApppN nucleotides (N = A, C, G, or U) occur in Escherichia coli. Measured cellular concentrations were 2.42 microM AppppA, 0.61 microM AppppC, 0.95 microM AppppG, 1.17 microM AppppU, 0.47 microM ApppA, 0.14 microM ApppC, 0.20 microM ApppG, and 0.12 microM ApppU. These concentrations remained constant during the cell cycle in synchronized exponentially growing cells.

Cell Cycle↗
PubMed1987

Heat shock and hydrogen peroxide responses of Escherichia coli are not changed by dinucleoside tetraphosphate hydrolase overproduction.

In Escherichia coli strains overproducing dinucleoside tetraphosphate hydrolase, the accumulation of dinucleoside tetraphosphates (AppppN, with N = A, C, G, or U) during heat shock or H2O2 treatment was reduced about 10-fold as compared with a control strain. This accumulation neither modified the pattern of the proteins induced by a temperature shift or H2O2 nor reduced the protection against oxidative damage induced by moderate H2O2 levels.

Acid Anhydride Hydrolases↗
PubMed1985

Variation of Ap4A and other dinucleoside polyphosphates in stressed Drosophila cells.

The unusual bis(5'-nucleosidyl)oligophosphates: Ap4A, Ap4G, Ap3A, and Ap3G, have been measured in cultures of Drosophila cells. Exponentially growing cells contain concentrations of 0.25, 0.31, 0.87, and 2.25 microM, respectively. These nucleotides have been followed after stressing the cells either by CdCl2 addition or by heat-shock treatment. Their concentrations are not affected by exposure to 500 microM CdCl2 during 6 h. Beyond this threshold of cadmium concentration, the nucleotides increase. With 5 mM CdCl2, an enhancement by 2 orders of magnitude of all the dinucleoside tri- and tetra-phosphates is observed. Upon heat-shock from 19 to 37 degrees C, Ap4A, Ap3A, and Ap3G increase up to 2.2, 3, and 3.3 times their initial levels, respectively. The increase is achieved within 1 h.

Adenine Nucleotides↗
PubMed1985

Catabolism of bis(5'-nucleosidyl) oligophosphates in Escherichia coli: metal requirements and substrate specificity of homogeneous diadenosine-5',5'''-P1,P4-tetraphosphate pyrophosphohydrolase.

Diadenosine-5',5'''-P1,P4-tetraphosphate pyrophosphohydrolase (diadenosinetetraphosphatase) from Escherichia coli strain EM20031 has been purified 5000-fold from 4 kg of wet cells. It produces 2.4 mg of homogeneous enzyme with a yield of 3.1%. The enzyme activity in the reaction of ADP production from Ap4A is 250 s-1 [37 degrees C, 50 mM tris(hydroxymethyl)aminomethane, pH 7.8, 50 microM Ap4A, 0.5 microM ethylenediaminetetraacetic acid (EDTA), and 50 microM CoCl2]. The enzyme is a single polypeptide chain of Mr 33K, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis and high-performance gel permeation chromatography. Dinucleoside polyphosphates are substrates provided they contain more than two phosphates (Ap4A, Ap4G, Ap4C, Gp4G, Ap3A, Ap3G, Ap3C, Gp3G, Gp3C, Ap5A, Ap6A, and dAp4dA are substrates; Ap2A, NAD, and NADP are not). Among the products, a nucleoside diphosphate is always formed. ATP, GTP, CTP, UTP, dATP, dGTP, dCTP, and dTTP are not substrates; Ap4 is. Addition of Co2+ (50 microM) to the reaction buffer containing 0.5 microM EDTA strongly stimulates Ap4A hydrolysis (stimulation 2500-fold). With 50 microM MnCl2, the stimulation is 900-fold. Ca2+, Fe2+, and Mg2+ have no effect. The Km for Ap4A is 22 microM with Co2+ and 12 microM with Mn2+. The added metals have similar effects on the hydrolysis of Ap3A into ADP + AMP. However, in the latter case, the stimulation by Co2+ is small, and the maximum stimulation brought by Mn2+ is 9 times that brought by Co2+. Exposure of the enzyme to Zn2+ (5 microM), prior to the assay or within the reaction mixture containing Co2+, causes a marked inhibition of Ap4A hydrolysis.(ABSTRACT TRUNCATED AT 250 WORDS)

Acid Anhydride Hydrolases↗
PubMed1985

Internal motions of transfer RNA: a study of exchanging protons by magnetic resonance.

Proton exchange is a probe of macromolecular structure and kinetics. Its value is enhanced when the exchanging protons can be identified by nmr. After dilution of tRNA-H2O samples in D2O, slowly exchanging imino protons are observed, with exchange times ranging from minutes to days. In many cases they originate from the dihydro-uracil region. Most slow exchangers are sensitive to buffer catalysis. Extrapolation to infinite buffer concentration yields the life-time of the closed form, in a two-state model of each base-pair. As predicted by the model, the lifetime obtained by extrapolation is independent of the buffer. Typical lifetimes are 14 minutes for CG11 of yeast tRNAPhe at 17 degrees C, or 5 minutes for U8-A14 of yeast tRNA(Asp) at 20 degrees C, without magnesium. For most slow exchangers, magnesium increases the lifetime of the closed form, but moderately, by factors never more than five. The exchange rates of other, fast-exchanging, imino protons, as determined by line-broadening, are found to depend on buffer concentration. Base-pair lifetimes are determined as above. For instance UA6 of yeast tRNA(Phe) has a lifetime of 14 ms at 17 degrees C. Base-pairs 4 and 6 have shorter lifetimes than the rest of the acceptor stem. Imidazole is a good catalyst for proton exchange of both the long-and the short-lived base-pairs, whereas phosphate is not. Tris is efficient except for cases where, possibly, access is impeded by its size; magnesium reduces the efficiency of catalysis by tris buffer. From the variation of exchange time vs buffer concentration, one determines the buffer concentration for which the exchange rate from the open state is equal to the closing rate. Remarquably, this concentration takes comparable values for most base-pairs, whether short-lived or long-lived. Buffer effects have also been observed in poly(rA).poly(rU), for which we derive a lifetime of 2.5 ms at 27 degrees C, and in other polynucleotides. Some of the exchange times identified in the literature as base-pair lifetimes may instead reflect incomplete catalysis.

Base Composition↗
PubMed1985

Molecular cloning of the Escherichia coli gene for diadenosine 5',5'''-P1,P4-tetraphosphate pyrophosphohydrolase.

A clone overproducing diadenosine tetraphosphatase (diadenosine 5', 5'''-P1, P4-tetraphosphate pyrophosphohydrolase) activity was isolated from an Escherichia coli cosmid library. Localization of the DNA region responsible for stimulation of this activity was achieved by deletion mapping and subcloning in various vectors. Maxicell experiments and immunological assays demonstrated that a 3.5-kilobase-pair DNA fragment carried the structural gene apaH encoding the E. coli diadenosine tetraphosphatase. The DNA coding strand was determined by cloning this fragment in both orientations in pUC plasmids. It was also shown that the overproduction of diadenosine tetraphosphatase decreased the dinucleoside tetraphosphate concentration in E. coli by a factor of 10.

Acid Anhydride Hydrolases↗
PubMed1983

The role of zinc in 5',5'-diadenosine tetraphosphate production by aminoacyl-transfer RNA synthetases.

Aminoacyl-tRNA synthetases are capable of converting 5'-ATP into 5',5'-diadenosine tetraphosphate. The reaction reflects the reversal of enzyme-bound aminoacyl-adenylate by ATP instead of PPi. In the case of a few prokaryotic as well as eukaryotic aminoacyl-tRNA synthetases, the initial rate of diadenosine tetraphosphate synthesis can be greatly enhanced upon adding small amounts of zinc. This observation enables us to establish a relationship between diadenosine tetraphosphate, a nucleotide possibly involved in controlling cell proliferation, and a metallic cofactor, which is believed to play a role in tumour growth.

Adenine Nucleotides↗
PubMed1983

NMR study of slowly exchanging imino protons in yeast tRNAasp.

We have monitored the exchange of imino and amino protons by NMR after quick transfer of yeast tRNAAsp in 2H2O solvent. When the concentration of exchange-catalyzing buffer is not too high, one imino proton exchanges considerably more slowly than any other (e.g., 100 hr versus 4 hr for the second-slowest imino proton at 18 degrees C in 15 mM Mg). This provides excellent conditions for identification, by the nuclear Overhauser effect, of the slowest exchanging proton, which we show to be the imino proton of the U-8 . A-14 reverse Hoogsteen tertiary-structure base pair; other slowly exchanging protons are identified as imino protons from A . U-11 and G . psi-13. In preliminary experiments, we find that the exchange of these protons is catalyzed by cacodylate or Tris buffer. The lifetimes of two other imino protons, ca. 10 min at 28 degrees C, are buffer independent. Slowly exchanging amino protons have also been observed. Correlation with the exchange of the uracil-8 imino proton suggests that they may be from adenine-14.

Base Sequence↗
PubMed1982

Zinc-dependent synthesis of 5',5'-diadenosine tetraphosphate by sheep liver lysyl- and phenylalanyl-tRNA synthetases.

Zinc greatly stimulates the initial rate of in vitro synthesis of 5',5'-diadenosine tetraphosphate by sheep liver lysyl- and phenylalanyl-tRNMA synthetases. In the case of each enzyme, maximum stimulation of 50- to 100-fold is reached upon addition of 50 microM ZnC2 to a reaction mixture (37 degrees c, pH 7.8) containing 50 microM dithioerythritol, 150 mM KCl, 5 mM ATP, 10 mM MgCl2, 0.1 mM cognate L-aminoacid, catalytic amounts of the aminoacyl-tRNA synthetase, and unlimiting pyrophosphatase activity. This observation made with aminoacyl-tRNA synthetases of mammalian origin supports the proposal that changes in cellular free zinc ion concentration could contribute to 5',5'-diadenosine tetraphosphate variations in animal cells as a function of growth activity.

Adenine Nucleotides↗
PubMed1981

Zinc(II)-dependent synthesis of diadenosine 5', 5"' -P(1) ,P(4) -tetraphosphate by Escherichia coli and yeast phenylalanyl transfer ribonucleic acid synthetases.

A new activity of Escherichia coli and yeast phenylalanyl-tRNA synthetases, the conversion adenosine 5' -triphosphate into diadenosine 5' ,5"' -P(1) ,P(4) -tetraphosphate, is reported. This activity is followed by (31)P NMR and chromatography on poly(ethylenimine)-cellulose. It is revealed by the addition of ZnCl2 to a reaction mixture containing the enzyme, ATP-Mg(2+), L-phenylalanine, and pyrophosphatase It reflects the reaction enzyme-bound phenylalanyl adenylate with ATP instead of PPi and strongly depends on the hydrolysis of pyrophosphate in the assay medium. The zinc dependence of this reaction parallels that of the inhibition of tRNA(phe) aminoacylation which is described in the accompanying paper [Mayaux, J. F., & Blanquet, S. (1981) Biochemistry (preceding paper in this issue)]. In the presence of an unlimiting pyrophosphatase activity, diadenosine tetraphosphate synthesis by E. coli and yeast phenylalanyl-tRNA synthetases occurs at maximal rates of 0.5 and 2 s-1, respectively (37 degrees C, pH 7.8, 150 mM KC1, 5 mM ATP, 10 mM MgCl2, 2 mM L-phenylalanine, and 80 muM ZnCl2). Under identical experimental conditions, E coli isoleucyl-, methionyl-, and tyrosyl-tRNA synthetases produce small amounts of diadenosine tetraphosphate at rates 2 or 3 orders of magnitude lower than that achieved by phenylalanyl-tRNA synthetase. In the case of E. coli phenylalanyl-tRNA synthetase, it is shown that the diadenosine tetraphosphate synthetase activity is accompanied by a diadenosinetetraphosphatase activity. This activity, actually supported by phenylalanyl-tRNA synthetase, is responsible for the appearance of ADP in the assay medium. It requires also the presence of both ZnCl2 and L-phenylalanine. The formation of ADP from diadenosine tetraphosphate and its reaction with enzyme-bound aminoacyl adenylate account for the appearance in the reaction mixture of diadenosine 5' ,5"' -P(1) ,P(3)-triphosphate, after that of diadenosine tetraphosphate. The significance of these findings in the context of the role of diadenosine tetraphosphate in controlling cellular growth is discussed.

Adenine Nucleotides↗