Isolation and characterization of diadenosine 5',5"'-P1,P4-tetraphosphate pyrophosphohydrolase from Physarum polycephalum

A new enzyme that hydrolyzes diadenosine 5',5"'-P1,P4-tetraphosphate has been purified by a factor of 250 from the acellular slime mold Physarum polycephalum. Activity was assayed radioisotopically with [3H]Ap4A. Isolation of the enzyme was facilitated by dye-ligand chromatography. The enzyme symmetrically hydrolyzes Ap4A to ADP and exhibits biphasic kinetics for the substrate with values for the apparent Km of 2.6 micro M and 37 micro M. The two values of Vmax differ by a factor of 10. Mg2+, Ca2+, and other divalent cations inhibit the activity with 40-80% inhibition occurring at 0.5 mM. Mg2+, at 0.5 mM, decreases both values of Vmax by 50%, decreases the low Km value by about 30%, and increases the high Km value by about 100%. (Ethylenedinitrilo)tetraacetic acid (EDTA) and [ethylenebis(oxyethylenenitrilo)]tetraacetic acid (EGTA), at 10 mM, inhibit the activity by 50%. ADP, ATP, Ap4, and Gp4 are equipotent inhibitors with 50% inhibition occurring at 30 micro M. AMP is a relatively weak inhibitor. The molecular weight of the enzyme is 26000 on the basis of elution of activity from a calibrated Sephadex G-75 column.     

Particulate diadenosine 5',5"'-P1,P3-triphosphate hydrolases in rat brain: two specific dinucleoside triphosphatases and two phosphodiesterase I-like hydrolases

Rat liver and brain differ in the distribution pattern of the total hydrolytic activity on diadenosine 5',5"'-P1,P3-triphosphate (Ap3A) between the soluble and particulate fractions. The Ap3A-hydrolase activity in both the soluble and particulate liver fractions and in the brain soluble fraction had been previously studied in detail. We report now on the brain particulate fraction which, unlike liver, showed a low unspecific phosphodiesterase I-like (PDEaseI, EC 3.1.4.1) activity relative to the specific dinucleoside triphosphatase (Ap3Aase, EC 3.6.1.29). Two PDEaseI-like forms (PDEaseI-A and PDEaseI-B), with different apparent Mrs and kinetic properties, and two Ap3Aases (Ap3Aase-alpha and Ap3Aase-beta) were solubilized with 0.5% Triton X-100 from the particulate fraction. Ap3Aase-alpha resembled the cytosolic Ap3Aase (Ap3Aase-c), a known situation in liver. Comparative to Ap3Aase-alpha, Ap3Aase-beta showed a slightly higher Km (35 vs. 15 micron) and lower isoelectric point (5.25 vs. 5.45); Ap3Aase-beta was absent from the soluble fraction, and its recovery was unaffected by proteinase inhibitors, strongly arguing for distinct soluble and particulate turnover pathways for dinucleoside polyphosphates.     

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.     

Alteration of hemoglobin function by diadenosine 5',5'-P1,P4-tetraphosphate and other alarmones.

Heat-shocked organisms are known to produce not only "heat shock proteins" but also diadenosine tetraphosphate (Ap4A) and related compounds that may act as "alarmones" that alert the cell to the onset of metabolic stress. We found that Ap4A is synthesized in chicken erythrocytes and that the Ap4A level in the whole blood of heat-stressed birds increases about 10-fold. In searching for alarmone receptors, we found that the diadenosine polyphosphates bind preferentially with high affinity to the deoxy conformation of hemoglobin in a ratio of one/tetramer. The binding affinity of this new class of effectors of hemoglobin function is directly related to the number of phosphates which bridge the nucleotide moieties, with the most dramatic in vitro effect on oxygen affinity being shown by Ap6A. Decreasing effects are brought about by diadenosine penta-, tetra-, tri-, di-, and monophosphates. The association constant for Ap4A binding to deoxygenated human hemoglobin at pH 7.25 is 26 microM-1, close to that for 2,3-diphosphoglycerate. At 100-fold excess over heme, Ap4A increases the P50 of stripped Hb A in 0.05 M HEPES buffer at pH 7.25, 20 degrees C, from 0.85 to 6.03 mm Hg. The binding, which markedly enhances the Bohr effect, involves the beta chain anion-binding site. The kinetics of both ligand binding and dissociation are affected, with a greater quantitative effect on the oxygen dissociation process. Although the low concentration of the diadenosine polyphosphates in red cells precludes a physiologically significant modulation of oxygen delivery, competition with the ATP- and NAD(P)H-binding sites on hemoglobin or regulatory enzymes may prove to be of adaptive significance.     

Immunoaffinity chromatography of diadenosine 5',5'-P1,P4-tetraphosphate phosphorylase from Saccharomyces cerevisiae

Diadenosine 5',5'-P1,P4-tetraphosphate (Ap4A) phosphorylase has been isolated previously using classical protein isolation techniques [A. Guranowski and S. Blanquet (1985) J. Biol. Chem. 260, 3542-3547]. A protein A-Sepharose immunoaffinity column was prepared to simplify the purification procedure. The immunoaffinity column was prepared using specific polyclonal antibodies to Ap4A phosphorylase covalently coupled to protein A-Sepharose with dimethyl pimelimidate by a modification of the procedure of C. Schneider et al. [(1982) J. Biol. Chem. 257, 10,766-10,769]. The specific activity of the immunoaffinity-purified enzyme showed an increase equivalent to the specific activity obtained by chromatography on DEAE-cellulose and hydroxyapatite columns.     

Isolation and identification of diadenosine 5',5'-P1,P4-tetraphosphate binding proteins using magnetic bio-panning

We report the development of a synthetic, biotin-conjugated diadenosine tetraphosphate (Ap(4)A)-'molecular hook' attached to magnetic beads enabling the isolation of Ap(4)A-binding proteins from bacterial cells or mammalian tissue lysates. Characterisation and identification of isolated binding proteins is performed sequentially by mass spectrometry. The observation of positive controls suggests that these newly observed proteins are putative Ap(4)A-binding partners, and we have expectations that others can be found with further technical improvements in our methods.     

Specific magnesium-dependent diadenosine 5'',5''''''-P1,P3-triphosphate pyrophosphohydrolase in Escherichia coli.

A specific Mg2+-dependent bis(5'-adenosyl)-triphosphatase (EC 3.6.1.29) was purified 270-fold from Escherichia coli. The enzyme had a strict requirement for Mg2+. Other divalent cations, such as Mn2+, Ca2+, or Co2+, were not effective. The products of the reaction with bis(5'-adenosyl) triphosphate (Ap3A) as the substrate were ADP and AMP in stoichiometric amounts. The Km for Ap3A was 12 +/- 5 microM. Bis(5'-adenosyl) di-, tetra-, and pentaphosphates, NAD+, ATP, ADP, AMP, glucose 6-phosphate, p-nitrophenylphosphate, bis-p-nitrophenylphospate, and deoxyribosylthymine-5'-(4-nitrophenylphosphate) were not substrates of the reaction. The enzyme had a molecular mass of 36 kilodaltons (as determined both by gel filtration and sodium dodecyl sulfate-polyacrylamide gel electrophoresis), an isoelectric point of 4.84 +/- 0.05, and a pH optimum of 8.2 to 8.5. Zn2+, a known potent inhibitor of rat liver bis(5'-adenosyl)-triphosphatase and bis(5'-guanosyl)-tetraphosphatase (EC 3.6 1.17), was without effect. The enzyme differs from the E. coli diadenosine 5',5'-P1, P4-tetraphosphate pyrophosphohydrolase which, in the presence of Mn2+, also hydrolyzes Ap3A.     

Pig lung 5'-nucleotidase: effect of diadenosine 5',5'-P1, P4-tetraphosphate and its related compounds

1. A 5'-nucleotidase was purified from pig lung to apparent homogeneity. 2. Its kinetic properties were similar to those of the previously reported cytoplasmic 5'-nucleotidase, which preferentially hydrolyses IMP and GMP. 3. It was a tetramer composed of 69 kDa subunit. 4. It was effectively stimulated by diadenosine tetraphosphate and glycerate 2,3-bisphosphate.     

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.     

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.     

Synthesis of diadenosine 5',5'-P1,P4-tetraphosphate by organellar and cytoplasmic phenylalanyl-tRNA synthetases of Euglena gracilis

Purified phenylalanyl-tRNA synthetases present in chloroplasts, mitochondria and cytoplasm of green and bleached Euglena gracilis strains, respectively, are able to synthesize diadenosine 5',5'-P1,P4-tetraphosphate (Ap4A). Ap4A synthesis is strictly dependent on zinc ions. This is the first evidence that chloroplasts should be able to synthesize Ap4A. Synthesis of Ap4A by phenylalanyl-tRNA synthetases of the three compartments of a plant cell or by other enzymes such as Ap4A phosphorylase is discussed.     

Cloning, characterisation and crystallisation of a diadenosine 5',5"'-P(1),P(4)-tetraphosphate pyrophosphohydrolase from Caenorhabditis elegans.

Asymmetrically cleaving diadenosine 5',5"'-P(1),P(4)-tetraphosphate (Ap4A) hydrolase activity has been detected in extracts of adult Caenorhabditis elegans and the corresponding cDNA amplified and expressed in Escherichia coli. As expected, sequence analysis shows the enzyme to be a member of the Nudix hydrolase family. The purified recombinant enzyme behaves as a typical animal Ap4A hydrolase. It hydrolyses Ap4A with a K(m) of 7 microM and k(cat) of 27 s(-1) producing AMP and ATP as products. It is also active towards other adenosine and diadenosine polyphosphates with four or more phosphate groups, but not diadenosine triphosphate, always generating ATP as one of the products. It is inhibited non-competitively by fluoride (K(i)=25 microM) and competitively by adenosine 5'-tetraphosphate with Ap4A as substrate (K(i)=10 nM). Crystals of diffraction quality with the morphology of rectangular plates were readily obtained and preliminary data collected. These crystals diffract to a minimum d-spacing of 2 A and belong to either space group C222 or C222(1). Phylogenetic analysis of known and putative Ap4A hydrolases of the Nudix family suggests that they fall into two groups comprising plant and Proteobacterial enzymes on the one hand and animal and archaeal enzymes on the other. Complete structural determination of the C. elegans Ap4A hydrolase will help determine the basis of this grouping.     

Synthesis of diadenosine 5',5'-P1,P4-tetraphosphate (AP4A) in sea urchin embryos

Sea urchin embryos were labeled with [3H]adenosine at two developmental stages (morula and prism) and the labeled acid-soluble nucleotides were fractionated successively by column chromatography with DEAE-Sephadex and DEAE-cellulose, and by thin-layer chromatography on a PEI-cellulose plate. Significant radioactivity was detected on the PEI-cellulose plate at the region of diadenosine 5',5'-P1,P4-tetraphosphate (AP4A). After treatment of this fraction with phosphodiesterase, the radioactivity was all recovered in the AMP region, while alkaline phosphatase had no effect on the AP4A fraction. The present result suggests that AP4A is actively synthesized in the sea urchin embryos.     

Relationship between cellular diadenosine 5',5'-P1,P4-tetraphosphate level, cell density, cell growth stimulation and toxic stresses

In order to elucidate the postulated role of diadenosine 5',5'-P1,P4-tetraphosphate (Ap4A) in cell growth regulation, the Ap4A cellular content was measured in cells submitted to various treatments affecting the cell growth. Ap4A level was found to increase ten times when cells reached confluence, whereas no significant variation of the ATP pool was observed. Cell growth arrest after serum depletion did not cause any variation in the Ap4A pool. A limited increase in the Ap4A pool was observed when growth of arrested cells was reinitiated but this variation reflected only the increase of cell density. No significant variation in the Ap4A intracellular level was observed after submitting two eukaryotic cell lines to various stresses (cytotoxic drugs, ethanol and heat-shock treatments). These results suggest that, in eukaryotic cells, Ap4A is not involved in cell growth stimulation but rather is associated with cell contact growth inhibition. They also suggest that Ap4A is not an 'alarmone', contrary to what has been proposed for bacteria.     

Diadenosine 5',5"'-P1,P4-tetraphosphate and related adenylylated nucleotides in Salmonella typhimurium

Salmonella typhimurium LT2 rapidly accumulates high levels of a family of five adenylylated nucleotides following exposure to a bacteriostatic quinone, 6-amino-7-chloro-5,8-dioxoquinoline. These compounds have been analyzed using our recently described two-dimensional thin layer chromatographic method. The five dinucleotides, which cannot be detected in exponentially growing cells, have been identified as diadenosine 5',5"'-P1,P4-tetraphosphate (AppppA), ApppGpp (guanosine 3'-diphosphate-5'-adenosine-5'-(P1,P3-triphosphate)), AppppG (adenosine 5'-guanosine-5'-(P1,P4-tetraphosphate)), ApppG (adenosine 5'-guanosine-5'-(P1,P3-triphosphate)), and ApppA (diadenosine 5',5"'-P1,P3-triphosphate). AppppA has been previously detected in vitro as an enzymatic product of aminoacyl-tRNA synthetases and in vivo at submicromolar levels in eucaryotic cells. The induced intracellular concentration of AppppA and the other adenylylated nucleotides in S. typhimurium is approximately 100-fold higher than that found in eucaryotic cells. We propose that these dinucleotides are alarmones, regulatory molecules signaling a particular metabolic stress.     

Effects of diadenosine 5',5'-P1, P4-tetraphosphate and of adenosine 5'-triphosphate on the higher order structure of calf thymus DNA

The effective length and the hard core radius were calculated by scaled particle theory for high molecular weight calf thymus DNA in the presence of varying concentrations of diadenosine 5',5'-P1, P4-tetraphosphate and of adenosine 5'-triphosphate in aqueous millimolar NaCl. DNA became slightly more flexible in the presence of diadenosine 5',5'-P1, P4-tetraphosphate at concentrations of 10(-9)-10(-7) M. DNA was denatured in the presence of 5 X 10(-5) M adenosine triphosphate.     

Phosphonate analogues of diadenosine 5',5'-P1,P4-tetraphosphate as substrates or inhibitors of procaryotic and eucaryotic enzymes degrading dinucleoside tetraphosphates

The substrate specificity of procaryotic and eucaryotic AppppA-degrading enzymes was investigated with phosphonate analogues of diadenosine 5',5'-P1,P4-tetraphosphate (AppppA). App(CH2)ppA (I), App(CHBr)ppA (II), and Appp(CH2)pA (III), but not Ap(CH2)pp(CH2)pA (IV), are substrates for lupin AppppA hydrolase (EC 3.6.1.17) and phosphodiesterase I (EC 3.1.4.1). None of the four analogues is hydrolyzed by bacterial AppppA hydrolase (EC 3.6.1.41), and only analogue III is degraded by yeast AppppA phosphorylase (EC 2.7.7.53). The analogues are competitive inhibitors of all four enzymes. The affinity of analogue IV is 3-40-fold lower than that of analogues I-III for all four enzymes. Introduction of one methylene (as in I and III) [or bromomethylene (as in II)] group into AppppA results in a 3-15-fold increase of its affinity for lupin and Escherichia coli AppppA hydrolases. The same modifications only negligibly (10-30%) affect its affinity for yeast AppppA phosphorylase and decrease its affinity for lupin phosphodiesterase I about 2.5-fold. The data provide further evidence for the heterogeneity among catalytic sites of all four AppppA-degrading enzymes.