Poly(A) polymerase from Escherichia coli adenylylates the 3'-hydroxyl residue of nucleosides, nucleoside 5'-phosphates and nucleoside(5')oligophospho(5')nucleosides (NpnN).

The capacity of Escherichia coli poly(A) polymerase to adenylylate the 3'-OH residue of a variety of nucleosides, nucleoside 5'-phosphates and dinucleotides of the type nucleoside(5')oligophospho(5')nucleoside is described here for the first time. Using micromolar concentrations of [alpha-32P]ATP, the following nucleosides/nucleotides were found to be substrates of the reaction: guanosine, AMP, CMP, GMP, IMP, GDP, CTP, dGTP, GTP, XTP, adenosine(5')diphospho(5')adenosine (Ap2A), adenosine (5')triphospho(5')adenosine (Ap3A), adenosine(5')tetraphospho(5')adenosine (Ap4A), adenosine(5')pentaphospho(5')adenosine (Ap5A), guanosine(5')diphospho(5') guanosine (Gp2G), guanosine(5')triphospho(5')guanosine (Gp3G), guanosine(5')tetraphospho(5')guanosine (Gp4G), and guanosine(5')pentaphospho(5')guanosine (Gp5G). The synthesized products were analysed by TLC or HPLC and characterized by their UV spectra, and by treatment with alkaline phosphatase and snake venom phosphodiesterase. The presence of 1 mM GMP inhibited competitively the polyadenylylation of tRNA. We hypothesize that the type of methods used to measure polyadenylation of RNA is the reason why this novel property of E. coli poly(A) polymerase has not been observed previously.     

Specific phosphorylase from Euglena gracilis splits diadenosine 5',5''-P(1),P(4)-tetraphosphate (Ap(4)A) and diadenosine 5',5''-P(1),P(3)-triphosphate (Ap(3)A)

1. Phosphorolytic cleavage of Ap(4),A was demonstrated in cell-free extracts from two protozoan organisms, Euglena gracilis and Acanthamoeba castellanii. 2. A specific dinucleoside oligophosphate (DNOP) alpha, beta-phosphorylase which degrades substrates with formation of corresponding nucleoside 5'-diphosphate (NDP) as one of the reaction products was purified 625-fold from Euglena gracilis cells. 3. In addition to Ap(4)A, the phosphorylase degrades AP(3)A, Ap(5)A, Gp(4)G and one of phosphonate analogs, ApppCH(2)pA. The K(m) values for Ap(4), A and Ap(3) A are 27 and 25 micron, and relative velocities 100 and 14, respectively. The K(m) for phosphate is 0.5 mM. 4. Some anions (arsenate, chromate, molybdate and vanadate) can substitute for phosphate in the catalyzed reactions and in their presence the DNOPs yield corresponding nucleoside 5'-monophosphate as one of the reactions' product. The enzyme supports also an anion-dependent dephosphorylation of NDPs. 5. Molecular weight of the native Euglena phosphorylase is 30,000. Optimum pH for its activity is at 8.0 Divalent metal cations are essential for the phosphorolysis of DNOPs but are not for the NDP dephosphorylation mentioned.     

Catabolism of bis(5'-nucleosidyl) tetraphosphates in Saccharomyces cerevisiae.

Bis(5'-adenosyl) tetraphosphate (Ap4A) phosphorylase II (P. Plateau, M. Fromant, J. M. Schmitter, J. M. Buhler, and S. Blanquet, J. Bacteriol. 171:6437-6445, 1989) was obtained in a homogeneous form through a 40,000-fold purification, starting from a Saccharomyces cerevisiae strain devoid of Ap4A phosphorylase I activity. The former enzyme behaves as a 36.8K monomer. As with Ap4A phosphorylase I, the addition of divalent cations is required for the expression of activity. Mn2+, Mg2+, and Ca2+ sustain phosphorolysis by the two enzymes, whereas Co2+ and Cd2+ stimulate only phosphorylase II activity. All bis(5'-nucleosidyl) tetraphosphates assayed (Ap4A, Ap4C, Ap4G, Ap4U, Gp4G, and Gp4U) are substrates of the two enzymes. However, Ap4A phosphorylase II shows a marked preference for A-containing substrates. The two enzymes catalyze adenosine 5'-phosphosulfate phosphorolysis or an exchange reaction between Pi and the beta-phosphate of any nucleoside diphosphate. They can also produce Ap4A at the expense of ATP and ADP. The gene (APA2) encoding Ap4A phosphorylase II was isolated and sequenced. The deduced amino acid sequence shares 60% identity with that of Ap4A phosphorylase I. Disruption of APA2 and/or APA1 shows that none of these genes is essential for the viability of Saccharomyces cerevisiae. The concentrations of all bis(5'-nucleosidyl) tetraphosphates are increased in an apa1 apa2 double mutant, as compared with the parental wild-type strain. The factor of increase is 5 to 50 times, depending on the nucleotide. This observation supports the conclusion that, in vivo, Ap4A phosphorylase II, like Ap4A phosphorylase I, participates in the catabolism rather than the synthesis of the bis(5'-nucleosidyl) tetraphosphates.     

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)     

Chromate, molybdate, tungstate and vanadate behave as substrates of yeast diadenosine 5',5'-p1,p4-tetraphosphate alpha, beta-phosphorylase

Diadenosine 5',5'-p1,p4-tetraphosphate (Ap4A) alpha, beta-phosphorylase from yeast Saccharomyces cerevisiae catalyzes two reactions: Ap4A cleavage and nucleoside diphosphate--phosphate (NDP-Pi) exchange. In both reactions phosphate can be substituted by arsenate, chromate, molybdate, tungstate or vanadate. In the presence of each anion, nucleoside 5'-monophosphate (NMP) always accumulates as a product of the reaction. This indicates that an unstable NMP anion is formed as an intermediate.     

Isolation and characterization of a dinucleoside triphosphatase from Saccharomyces cerevisiae.

An enzyme able to cleave dinucleoside triphosphates has been purified 3,750-fold from Saccharomyces cerevisiae. Contrary to the enzymes previously shown to catabolize Ap4A in yeast, this enzyme is a hydrolase rather than a phosphorylase. The dinucleoside triphosphatase molecular ratio estimated by gel filtration is 55,000. Dinucleoside triphosphatase activity is strongly stimulated by the presence of divalent cations. Mn2+ displays the strongest stimulating effect, followed by Mg2+, Co2+, Cd2+, and Ca2+. The Km value for Ap3A is 5.4 microM (50 mM Tris-HCl [pH 7.8], 5 mM MgCl2, and 0.1 mM EDTA; 37 degrees C). Dinucleoside polyphosphates are substrates of this enzyme, provided that they contain more than two phosphates and that at least one of the two bases is a purine (Ap3A, Ap3G, Ap3C, Gp3G, Gp3C, m7Gp3A, m7Gp3G, Ap4A, Ap4G, Ap4C, Ap4U, Gp4G, and Ap5A are substrates; AMP, ADP, ATP, Ap2A, and Cp4U are not). Among the products, a nucleoside monophosphate is always formed. The specificity of cleavage of methylated dinucleoside triphosphates and the molecular weight of dinucleoside triphosphatase indicate that this enzyme is different from the mRNA decapping enzyme previously characterized (A. Stevens, Mol. Cell. Biol. 8:2005-2010, 1988).     

Thermostable Pyrococcus furiosus DNA ligase catalyzes the synthesis of (di)nucleoside polyphosphates

DNA ligase from the hyperthermophilic marine archaeon Pyrococcus furiosus (Pfu DNA ligase) synthesizes adenosine 5'-tetraphosphate (p4A) and dinucleoside polyphosphates by displacement of the adenosine 5'-monophosphate (AMP) from the Pfu DNA ligase-AMP (E-AMP) complex with tripolyphosphate (P3), nucleoside triphosphates (NTP), or nucleoside diphosphates (NDP). The experiments were performed in the presence of 1-2 microM [alpha-32P]ATP and millimolar concentrations of NTP or NDP. Relative rates of synthesis (%) of the following adenosine(5')tetraphospho(5')nucleosides (Ap4N) were observed: Ap4guanosine (Ap4G) (from GTP, 100); Ap4deoxythymidine (Ap4dT) (from dTTP, 95); Ap4xanthosine (Ap4X) (from XTP, 94); Ap4deoxycytidine (Ap4dC) (from dCTP, 64); Ap4cytidine (Ap4C) (from CTP, 60); Ap4deoxyguanosine (Ap4dG) (from dGTP, 58); Ap4uridine (Ap4U) (from UTP, <3). The relative rate of synthesis (%) of adenosine(5')triphospho(5')nucleosides (Ap3N) were: Ap3guanosine (Ap3G) (from GDP, 100); Ap3xanthosine (Ap3X) (from XDP, 110); Ap3cytidine (Ap3C) (from CDP, 42); Ap3adenosine (Ap3A) (from ADP, <1). In general, the rate of synthesis of Ap4N was double that of the corresponding Ap3N. The enzyme presented optimum activity at a pH value of 7.2-7.5, in the presence of 4 mM Mg2+, and at 70 degrees C. The apparent Km values for ATP and GTP in the synthesis of Ap4G were about 0.001 and 0.4mM, respectively, lower values than those described for other DNA or RNA ligases. Pfu DNA ligase is used in the ligase chain reaction (LCR) and some of the reactions here reported [in particular the synthesis of Ap4adenosine (Ap4A)] could take place during the course of that reaction.     

Catabolism of diadenosine 5',5"'-P1,P4-tetraphosphate in procaryotes. Purification and properties of diadenosine 5',5"'-P1,P4-tetraphosphate (symmetrical) pyrophosphohydrolase from Escherichia coli K12

Enzymatic activity which hydrolyzes diadenosine 5',5"'-P1,P4-tetraphosphate (Ap4A) yielding ADP has been identified in extracts of eubacteria, Escherichia coli and Acidaminococcus fermentans, and of a highly thermophilic archaebacterium, Pyrodictum occultum. Specific Ap4A (symmetric) pyrophosphohydrolase from Escherichia coli K12 has been purified almost 400-fold. The preparation was free of phosphatase, ATPase, phosphodiesterase, AMP-nucleosidase, and adenylate kinase. The Ap4A pyrophosphohydrolase molecular weight estimated by gel filtration is 27,000 +/- 1,000. Activity maximum is at pH 8.3. The Km value computed for Ap4A is 25 +/- 3 microM. The sulfhydryl group(s) is essential for enzyme activity. Metal chelators, EDTA, and o-phenanthroline, inhibit Ap4A hydrolysis; I0.5 values are 3 and 50 microM, respectively. Co2+ is a strong stimulator with an almost 100-fold increase in rate of Ap4A hydrolysis and a plateau in the range of 100-500 microM Co2+, when compared with the nonstimulated hydrolysis. Other transition metal ions, Mn2+, Cd2+, and Ni2+, stimulate by factors of 8, 3.5, and 3.5, respectively, with optimal concentrations in the range 200-500, 2-5, and 4-8 microM, respectively. Zn2+, Cu2+, and Fe2+, up to 30 microM, are without effect and they inhibit at higher concentrations. Mg2+ or Ca2+, in the absence of other divalent metal ions, are weak stimulators (1.5-fold stimulation occurs at 1-2 mM concentration), but act synergistically with Co2+ at its suboptimal concentrations. Stimulation in the presence of 10 microM Co2+ and either 1 mM MgCl2 or CaCl2 increases up to 75-fold. The same degree of synergy is found at 10 microM Co2+ and either 2-5 mM spermidine or 0.5-1.5 mM spermine. Besides Ap4A, bacterial Ap4A pyrophosphohydrolase hydrolyzes effectively Ap5A and Gp4G, and, to some extent, p4A, Ap6A, and Ap3A yielding in each case corresponding nucleoside diphosphate as one of the products.     

Characterization of the bis(5''-nucleosidyl) tetraphosphate pyrophosphohydrolase from encysted embryos of the brine shrimp Artemia.

The P1P4-bis(5'-nucleosidyl) tetraphosphate asymmetrical-pyrophosphohydrolase from encysted embryos of the brine shrimp Artemia has been purified over 11,000-fold to homogeneity. Anion-exchange chromatography resolves two major species with very similar properties. The enzyme is a single polypeptide of Mr 17,600 and is maximally active at pH 8.4 and 2 mM-Mg2+. It is inhibited by Ca2+ (IC50 = 0.9 mM with 2 mM-Mg2+) but not by Zn2+ ions. It preferentially hydrolyses P1P4-bis(5'-nucleosidyl) tetraphosphates, e.g. P1P4-bis(5'-adenosyl) tetraphosphate (Ap4A) (kcat. = 12.7 s-1; Km = 33 microM) and P1P4-bis(5'-guanosyl) tetraphosphate (Gp4G) (kcat. = 6.2 s-1; Km = 5 microM). With adenosine 5'-P1-tetraphospho-P4-5"'-guanosine (Ap4G) as substrate, there is a 4.5-fold preference for AMP and GTP as products and biphasic reaction kinetics are observed giving Km values of 4.7 microM and 34 microM, and corresponding rate constants of 6.5 s-1 and 11.9 s-1. The net rate constant for Ap4G hydrolysis is 7.6 s-1. The enzyme will also hydrolyse nucleotides with more than four phosphate groups, e.g. Ap5G, Ap6A and Gp5G are hydrolysed at 25%, 18% and 10% of the rate of Ap4A respectively. An NTP is always one of the products. Ap2A and Gp2G are not hydrolysed, while Ap3A and Gp3G are very poor substrates. When the enzyme is partially purified from embryos and larvae at different stages of development by sedimentation through a sucrose density gradient, its activity increases 3-fold during the first 12 h of pre-emergence development. This is followed by a slow decline during subsequent larval development. The similarity of this enzyme to other asymmetrical-pyrophosphohydrolases suggests that it did not evolve specifically to degrade the large yolk platelet store of Gp4G which is found in Artemia embryos, but that it probably serves the same general function in bis(5'-nucleosidyl) oligophosphate metabolism as in other cells.     

Dinucleoside polyphosphates stimulate the primer independent synthesis of poly(A) catalyzed by yeast poly(A) polymerase.

Novel properties of the primer independent synthesis of poly(A), catalyzed by the yeast poly(A) polymerase are presented. The commercial enzyme from yeast, in contrast to the enzyme from Escherichia coli, is unable to adenylate the 3'-OH end of nucleosides, nucleotides or dinucleoside polyphosphates (NpnN). In the presence of 0.05 mm ATP, dinucleotides (at 0.01 mm) activated the enzyme velocity in the following decreasing order: Gp4G, 100; Gp3G, 82; Ap6A, 61; Gp2G, 52; Ap4A, 51; Ap2A, 41; Gp5G, 36; Ap5A, 27; Ap3A, 20, where 100 represents a 10-fold activation in relation to a control without effector. The velocity of the enzyme towards its substrate ATP displayed sigmoidal kinetics with a Hill coefficient (nH) of 1.6 and a Km(S0.5) value of 0.308 +/- 0.120 mm. Dinucleoside polyphosphates did not affect the maximum velocity (Vmax) of the reaction, but did alter its nH and Km(S0.5) values. In the presence of 0.01 mm Gp4G or Ap4A the nH and Km(S0.5) values were (1.0 and 0.063 +/- 0.012 mm) and (0.8 and 0.170 +/- 0.025 mm), respectively. With these kinetic properties, a dinucleoside polyphosphate concentration as low as 1 micro m may have a noticeable activating effect on the synthesis of poly(A) by the enzyme. These findings together with previous publications from this laboratory point to a potential relationship between dinucleoside polyphosphates and enzymes catalyzing the synthesis and/or modification of DNA or RNA.     

Characterisation of a bis(5'-nucleosidyl) triphosphate pyrophosphohydrolase from encysted embryos of the brine shrimp Artemia.

1. A P1,P3-bis(5'-nucleosidyl)triphosphate pyrophosphohydrolase (Np3 Nase) has been partially purified from Artemia embryos. 2. The Np3 Nase has a native Mr of 115,000 and preferentially hydrolyses substrates of the form Np3 N. Relative rates of hydrolysis are Ap3A (Vrel = 1.0), Gp3G (Vrel = 0.71), Ap4A (Vrel = 0.08), Ap5A (Vrel = 0.09), Gp4G (Vrel = 0.3) and Gp5G (Vrel = 0.33). An NMP is always one of the products. 3. The Km values for Ap3A and Gp3G are 15 and 10 microM respectively. 4. Mg2+, Mn2+ and Ca2+ ions all stimulate the activity, while Zn2+, Co2+ and Ni2+ ions are inhibitory. 5. The activity of the Np3 Nase remains constant during pre-emergence development of encysted embryos but decreases slightly after hatching.     

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.     

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.     

Adenosine(5')tetraphospho(5')adenosine-binding protein of calf thymus

An adenosine(5')tetraphospho(5')adenosine (Ap4A) binding protein has been purified from calf thymus. The protein is comprised of a single polypeptide of Mr 54000 and is capable of high-affinity (Kd = 13 microM) binding of Ap4A with great substrate specificity. The Ap4A binding protein has been isolated in two forms: a 'free', or non-polymerase-bound, form which predominates, and a similar form which copurifies with DNA polymerase alpha, but which can be resolved from it. The free form of Ap4A binding protein contains associated adenosine(5')tetraphospho(5')adenosine phosphohydrolase (Ap4Aase) activity, while the form resolved from DNA polymerase alpha contains no such activity. The Ap4Aase activity, which catalyzes the phosphohydrolysis of Ap4A to ATP and AMP, is strongly inhibited by low levels (50-100 microM) of Zn2+ without any effect on the Ap4A binding protein activity. This difference in associated Ap4Aase activity between free and polymerase-bound forms of the protein, plus the copurification mentioned above, indicate a specific association between Ap4A binding protein and DNA polymerase alpha.     

Identification of a unique membrane receptor for adenosine 5',5"'-P1,P4-tetraphosphate

Adenosine 5',5"'-P1,P4-tetraphosphate (Ap4A) has been implicated as a modulator of cell stress. We have performed binding studies which indicate that membranes from all tissues tested bind tritium-labeled Ap4A. The characteristics of Ap4A binding were determined on brain membrane homogenates after development of an optimized in vitro filter-binding assay. Ap4A binding is specific for adenylated dinucleotides and for the length of the phosphate bridge. A Kd of 0.71 microM for Ap4A was determined.     

Existence of high and low affinity dinucleotides pentaphosphate-induced calcium responses in individual synaptic terminals and lack of correlation with the distribution of P2X1-7 subunits

Individual analysis of synaptic terminals calcium responses, induced by dinucleotides pentaphosphate, Ap(5)A or Gp(5)G, demonstrates the presence of two main groups considering the concentration required for stimulation. The first group corresponds to those responding to Ap(5)A or Gp(5)G at nanomolar concentration, representing 16% and 12%, respectively, and the second one responds to micromolar concentration and represents, respectively, 17% and 14%, of the total functional synaptosomal population in rat midbrain. Dose-response curves in single terminals showed an Ap(5)A EC(50) values of 0.9+/-0.2 nM and 11.8+/-0.9 microM, being the maximal intrasynaptosomal calcium increase of 200+/-0.3 and 125+/-0.2 nM for the high and low affinity responding terminals, respectively. Combination of microfluorimetric and immunocytochemical studies showed lack of correlation between dinucleotides pentaphosphate responses and P2X receptor subunits expression, in spite of the abundance of P2X(2), P2X(3) and P2X(7) at the presynaptic level in rat midbrain synaptosomes. Pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid (PPADS), a P2X receptors antagonist, showed no effect on low affinity dinucleotides receptors population, and partial inhibition on the high affinity one. On the other hand, diinosine pentaphosphate (Ip(5)I) completely abolished the low affinity dinucleotides responses, and 60% inhibition of the high affinity ones.     

Identification and partial characterization of an adenosine(5'')tetraphospho(5'')adenosine hydrolase on intact bovine aortic endothelial cells.

The biologically active dinucleotides adenosine(5')tetraphospho(5')adenosine (Ap4A) and adenosine(5')-triphospho(5')adenosine (Ap3A), which are both releasable into the circulation from storage pools in thrombocytes, are catabolized by intact bovine aortic endothelial cells. 1. Compared with extracellular ATP and ADP, which are very rapidly hydrolysed, the degradation of Ap4A and Ap3A by endothelial ectohydrolases is relatively slow, resulting in a much longer half-life on the endothelial surface of the blood vessel. The products of hydrolysis are further degraded and finally taken up as adenosine. 2. Ap4A hydrolase has high affinity for its substrate (Km 10 microM). 3. ATP as well as AMP transiently accumulates in the extracellular fluid, suggesting an asymmetric split of Ap4A by the ectoenzyme. 4. Mg2+ or Mn2+ at millimolar concentration are needed for maximal activity; Zn2+ and Ca2+ are inhibitory. 5. The hydrolysis of Ap4A is retarded by other nucleotides, such as ATP and Ap3A, which are released from platelets simultaneously with Ap4A.     

Presence and coding properties of 2'-O-methyl-5-carbamoylmethyluridine (ncm5Um) in the wobble position of the anticodon of tRNA(Leu) (U*AA) from brewer's yeast.

The unknown modified nucleoside U* has been isolated by enzymatic and HPLC protocols from tRNA(Leu) (U*AA) recently discovered in brewer's yeast. The pure U* nucleoside has been characterized by electron impact mass spectroscopy, and comparison of its chromatographic and UV-absorption properties with those of appropriate synthetic compounds. The structure of U* was established as 2'-O-methyl-5-carbamoylmethyluridine (ncm5Um). The yeast tRNA(Leu) (U*AA) is the only tRNA so far sequenced which has been shown to contain ncm5Um. The location of such a modified uridine at the first position of the anticodon restricts the decoding property to A of the leucine UUA codon.     

Characterization of P1,P4-diadenosine 5'-tetraphosphate binding on bovine aortic endothelial cells

In recent years it has become increasingly clear that alpha, omega-dinucleotides act as extracellular modulators of various biological processes. P1,P4-diadenosine 5'-tetraphosphate (Ap4A) is the best characterized alpha,omega-dinucleotides and acts as an extracellular signal molecule by inducing the release of nitric oxide (NO) from bovine aortic endothelial cells (BAEC) (R. H. Hilderman, and E. F. Christensen (1998) FEBS Lett. 407, 320-324). However, the characteristics of Ap4A binding to endothelial cells have not been determined. In this report we demonstrate that Ap4A binds to a heterogeneous population of receptors on BAEC. Competition ligand-binding studies using various adenosine dinucleotides, guanosine dinucleotides, adenosine/guanosine dinucleotides, and synthetic P2 purinoceptor agonists and antagonists demonstrate that Ap4A binds to a receptor on BAEC that has a high affinity for some of the adenosine dinucleotides. The apparent IC50 values for Ap4A, Ap2A, and Ap3A are between 12 and 15 microM, while the apparent IC50 values for Ap5A and Ap6A are greater than 500 microM. Evidence is also presented which suggests that this receptor can be classified as a putative P4 purinoceptor. Competition studies also demonstrate that Ap4A binds at a lower affinity to a second class of binding sites.     

Inhibition of thymidine kinase by P1-(adenosine-5')-P5-(thymidine-5')-pentaphosphate

Potential bisubstrate analogs, with adenosine and thymidine joined at their 5' positions by polyphosphoryl linkages of varying lengths (ApndT, where n = the number of phosphoryl groups), were examined as inhibitors of cytosolic thymidine kinase from blast cells of patients with acute myelocytic leukemia. Ki values were 1.2 microM for Ap3dT, 0.31 microM for Ap4dT, 0.12 microM for Ap5dT, and 0.19 microM for Ap6dT. The best inhibitor of the cytosolic enzyme, Ap5dT, was somewhat less effective as an inhibitor of the mitochondrial enzyme (Ki = 0.50 microM). In addition to their inhibitory modes of binding by the cytosolic enzyme, these compounds were bound at considerably lower concentrations (Kd = 0.029 microM for Ap4dT, 0.0025 microM for Ap5dT, and 0.0027 microM for Ap4dT), in such a way as to protect the cytosolic enzyme from thermal inactivation at 37 degrees C in the absence of substrates.