Mitochondrial location of rat liver dinucleoside triphosphatase

Rat liver dinucleoside triphosphatase (EC 3.6.1.29) is associated with sucrose-gradient purified mitochondria and can be extracted by freeze and thaw treatment. The proportion of mitochondrial dinucleoside triphosphatase approaches 50% of total liver enzyme. Evidence is also presented that 10% of total liver bis(5'-guanosyl)tetraphosphatase (EC 3.6.1.17) might be equally linked to mitochondria. Those data suggest that diadenosine 5',5'-P1,P3-triphosphate, diadenosine 5',5'-P1,P4-tetraphosphate, or other substrates of those enzymes, might be somehow related to mitochondria or mitochondrial function(s), although the occurrence of dinucleoside polyphosphates has not been reported in that organelle.     

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.     

Synthesis of dinucleoside polyphosphates catalyzed by firefly luciferase.

In the presence of ATP, luciferin (LH2), Mg2+ and pyrophosphatase, the firefly (Photinus pyralis) luciferase synthesizes diadenosine 5',5"'-P1,P4-tetraphosphate (Ap4A) through formation of the E-LH2-AMP complex and transfer of AMP to ATP. The maximum rate of the synthesis is observed at pH 5.7. The Km values for luciferin and ATP are 2-3 microM and 4 mM, respectively. The synthesis is strictly dependent upon luciferin and a divalent metal cation. Mg2+ can be substituted with Zn2+, Co2+ or Mn2+, which are about half as active as Mg2+, as well as with Ni2+, Cd2+ or Ca2+, which, at 5 mM concentration, are 12-20-fold less effective than Mg2+. ATP is the best substrate of the above reaction, but it can be substituted with adenosine 5'-tetraphosphate (p4A), dATP, and GTP, and thus the luciferase synthesizes the corresponding homo-dinucleoside polyphosphates:diadenosine 5',5"'-P1,P5-pentaphosphate (Ap5A), dideoxyadenosine 5',5"'-P1,P4-tetraphosphate (dAp4dA) and diguanosine 5',5"'-P1,P4-tetraphosphate (Gp4G). In standard reaction mixtures containing ATP and a different nucleotide (p4A, dATP, adenosine 5'-[alpha,beta-methylene]-triphosphate, (Ap[CH2]pp), (S')-adenosine-5'-[alpha-thio]triphosphate [Sp)ATP[alpha S]) and GTP], luciferase synthesizes, in addition to Ap4A, the corresponding hetero-dinucleoside polyphosphates, Ap5A, adenosine 5',5"'-P1,P4-tetraphosphodeoxyadenosine (Ap4dA), diadenosine 5',5"'-P1,P4-[alpha,beta-methylene] tetraphosphate (Ap[CH2]pppA), (Sp-diadenosine 5',5"'-P1,P4-[alpha-thio]tetraphosphate [Sp)Ap4A[alpha S]) and adenosine-5',5"'-P1,P4-tetraphosphoguanosine (Ap4G), respectively. Adenine nucleotides, with at least a 3-phosphate chain and with an intact alpha-phosphate, are the preferred substrates for the formation of the enzyme-nucleotidyl complex. Nucleotides best accepting AMP from the E-LH2-AMP complex are those which contain at least a 3-phosphate chain and an intact terminal pyrophosphate moiety. ADP or other NDP are poor adenylate acceptors as very little diadenosine 5',5"'-P1,P3-triphosphate (Ap3A) or adenosine-5',5"'-P1,P3-triphosphonucleosides (Ap3N) are formed. In the presence of NTP (excepting ATP), luciferase is able to split Ap4A, transferring the resulting adenylate to NTP, to form hetero-dinucleoside polyphosphates. In the presence of PPi, luciferase is also able to split Ap4A, yielding ATP. The cleavage of Ap4A in the presence of Pi or ADP takes place at a very low rate. The synthesis of dinucleoside polyphosphates, catalyzed by firefly luciferase, is compared with that catalyzed by aminoacyl-tRNA synthetases and Ap4A phosphorylase.     

Location of dinucleoside triphosphatase in the matrix space of rat liver mitochondria.

The submitochondrial location of dinucleoside triphosphatase (EC 3.6.1.29), previously shown to be in part associated with mitochondria, has been studied in rat liver. The precipitability and latency of activity in organelle suspensions, and the profile of solubilization by digitonin, were like those of the matrix space marker glutamate dehydrogenase, and differed from those of other submitochondrial fractions. This, and the synthesis of diadenosine polyphosphates by mitochondrial aminoacyl-tRNA synthetases, suggest the occurrence of a pathway for the intramitochondrial turnover of diadenosine 5',5'-P1,P3-triphosphate (Ap3A).     

Isothermal titration calorimetry reveals a zinc ion as an atomic switch in the diadenosine polyphosphates

Diadenosine polyphosphates (diadenosine 5',5'-P(1),P(n)-polyphosphate (Ap(n)A)) are 5'-5'-phosphate-bridged dinucleosides that have been proposed to act as signaling molecules in a variety of biological systems. Isothermal titration calorimetry was used to measure the affinities of a variety of metal cations for ATP, diadenosine 5',5'-P(1),P(3)-triphosphate (Ap(3)A), diadenosine 5',5'-P(1),P(4)-tetraphosphate (Ap(4)A), and diadenosine 5',5'-P(1),P(5)-pentaphosphate (Ap(5)A). The binding of Mg(2+), Ca(2+), and Mn(2+) to ATP is shown to take place with the beta,gamma-phosphates (primary site) and be endothermic in character. The binding of Ni(2+), Cd(2+), and Zn(2+) to ATP is found to take place at both the primary site and at a secondary site identified as N-7 of the adenine ring. Binding to this second site is exothermic in character. Generally, the binding of metal cations to diadenosine polyphosphates involves a similar primary site to ATP. No exothermic binding events are identified. Critically, the binding of Zn(2+) to diadenosine polyphosphates proves to be exceptional. This appears to involve a very high affinity association involving the N-7 atoms of both adenine rings in each Ap(n)A, as well as the more usual endothermic association with the phosphate chain. The high affinity association is also endothermic in character. A combination of NMR and CD evidence is provided in support of the calorimetry data demonstrating chemical shift changes and base stacking disruptions entirely consistent with N-7 bridging interactions. N-7 bridging interactions are entirely reversible, as demonstrated by EDTA titration. Considering the effects of Zn(2+) on a wide variety of dinucleoside polyphosphate-metabolizing enzymes, we examine the possibility of Zn(2+) acting as an atomic switch to control the biological function of the diadenosine polyphosphates.     

Hydrolysis of diadenosine polyphosphates by nucleotide pyrophosphatases/phosphodiesterases.

Diadenosine polyphosphates (ApnAs) act as extracellular signaling molecules in a broad variety of tissues. They were shown to be hydrolyzed by surface-located enzymes in an asymmetric manner, generating AMP and Apn-1 from ApnA. The molecular identity of the enzymes responsible remains unclear. We analyzed the potential of NPP1, NPP2, and NPP3, the three members of the ecto-nucleotide pyrophosphatase/phosphodiesterase family, to hydrolyze the diadenosine polyphosphates diadenosine 5',5"'-P1,P3-triphosphate (Ap3A), diadenosine 5',5"'-P1,P4-tetraphosphate (Ap4A), and diadenosine 5',5"'-P1,P5-pentaphosphate, (Ap5A), and the diguanosine polyphosphate, diguanosine 5',5"'-P1,P4-tetraphosphate (Gp4G). Each of the three enzymes hydrolyzed Ap3A, Ap4A, and Ap5A at comparable rates. Gp4G was hydrolyzed by NPP1 and NPP2 at rates similar to Ap4A, but only at half this rate by NPP3. Hydrolysis was asymmetric, involving the alpha,beta-pyrophosphate bond. ApnA hydrolysis had a very alkaline pH optimum and was inhibited by EDTA. Michaelis constant (Km) values for Ap3A were 5.1 micro m, 8.0 micro m, and 49.5 micro m for NPP1, NPP2, and NPP3, respectively. Our results suggest that NPP1, NPP2, and NPP3 are major enzyme candidates for the hydrolysis of extracellular diadenosine polyphosphates in vertebrate tissues.     

Selective degradation of 2'-adenylated diadenosine tri- and tetraphosphates, Ap(3)A and Ap(4)A, by two specific human dinucleoside polyphosphate hydrolases

It is known that the interferon-inducible 2',5'-oligoadenylate synthetase can catalyze the 2'-adenylation of various diadenosine polyphosphates. However, catabolism of those 2'-adenylated compounds has not been investigated so far. This study shows that the mono- and bis-adenylated (or mono- and bis-deoxyadenylated) diadenosine triphosphates are not substrates of the human Fhit (fragile histidine triad) protein, which acts as a typical dinucleoside triphosphate hydrolase (EC 3.6.1.29). In contrast, the diadenosine tetraphosphate counterparts are substrates for the human (asymmetrical) Ap(4)A hydrolase (EC 3.6.1.17). The relative rates of the hydrolysis of 0.15 mM AppppA, (2'-pdA)AppppA, and (2'-pdA)AppppA(2"'-pdA) catalyzed by the latter enzyme were determined as 100:232:38, respectively. The asymmetrical substrate was hydrolyzed to ATP + (2'-pdA)AMP (80%) and to (2'-pdA)ATP + AMP (20%). The human Fhit protein, for which Ap(4)A is a poor substrate, did not degrade the 2'-adenylated diadenosine tetraphosphates either. The preference of the interferon-inducible 2'-5' oligoadenylate synthetase to use Ap(3)A over Ap(4)A as a primer for 2'-adenylation and the difference in the recognition of the 2'-adenylated diadenosine triphosphates versus the 2'-adenylated diadenosine tetraphosphates by the dinucleoside polyphosphate hydrolases described here provide a mechanism by which the ratio of the 2'-adenylated forms of the signalling molecules, Ap(3)A and Ap(4)A, could be regulated in vivo.     

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.     

The Saccharomyces cerevisiae YOR163w gene encodes a diadenosine 5', 5"'-P1,P6-hexaphosphate (Ap6A) hydrolase member of the MutT motif (Nudix hydrolase) family

The YOR163w open reading frame on chromosome XV of the Saccharomyces cerevisiae genome encodes a member of the MutT motif (nudix hydrolase) family of enzymes of Mr 21,443. By cloning and expressing this gene in Escherichia coli and S. cerevisiae, we have shown the product to be a (di)adenosine polyphosphate hydrolase with a previously undescribed substrate specificity. Diadenosine 5',5"'-P1, P6-hexaphosphate is the preferred substrate, and hydrolysis in H218O shows that ADP and adenosine 5'-tetraphosphate are produced by attack at Pbeta and AMP and adenosine 5'-pentaphosphate are produced by attack at Palpha with a Km of 56 microM and kcat of 0.4 s-1. Diadenosine 5',5"'-P1,P5-pentaphosphate, adenosine 5'-pentaphosphate, and adenosine 5'-tetraphosphate are also substrates, but not diadenosine 5',5"'-P1,P4-tetraphosphate or other dinucleotides, mononucleotides, nucleotide sugars, or nucleotide alcohols. The enzyme, which was shown to be expressed in log phase yeast cells by immunoblotting, displays optimal activity at pH 6.9, 50 degrees C, and 4-10 mM Mg2+ (or 200 microM Mn2+). It has an absolute requirement for a reducing agent, such as dithiothreitol (1 mM), and is inhibited by Ca2+ with an IC50 of 3.3 mM and F- (noncompetitively) with a Ki of 80 microM. Its function may be to eliminate potentially toxic dinucleoside polyphosphates during sporulation.     

Crystal structure of human cytosolic 5'-nucleotidase II: insights into allosteric regulation and substrate recognition

Cytosolic 5'-nucleotidase II catalyzes the dephosphorylation of 6-hydroxypurine nucleoside 5'-monophosphates and regulates the IMP and GMP pools within the cell. It possesses phosphotransferase activity and thereby also catalyzes the reverse reaction. Both reactions are allosterically activated by adenine-based nucleotides and 2,3-bisphosphoglycerate. We have solved structures of cytosolic 5'-nucleotidase II as native protein (2.2 Angstrom) and in complex with adenosine (1.5 Angstrom) and beryllium trifluoride (2.15 Angstrom) The tetrameric enzyme is structurally similar to enzymes of the haloacid dehalogenase (HAD) superfamily, including mitochondrial 5'(3')-deoxyribonucleotidase and cytosolic 5'-nucleotidase III but possesses additional regulatory regions that contain two allosteric effector sites. At effector site 1 located near a subunit interface we modeled diadenosine tetraphosphate with one adenosine moiety in each subunit. This efficiently glues the tetramer subunits together in pairs. The model shows why diadenosine tetraphosphate but not diadenosine triphosphate activates the enzyme and supports a role for cN-II during apoptosis when the level of diadenosine tetraphosphate increases. We have also modeled 2,3-bisphosphoglycerate in effector site 1 using one phosphate site from each subunit. By comparing the structure of cytosolic 5'-nucleotidase II with that of mitochondrial 5'(3')-deoxyribonucleotidase in complex with dGMP, we identified residues involved in substrate recognition.     

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.     

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.     

4-Coumarate:coenzyme A ligase has the catalytic capacity to synthesize and reuse various (di)adenosine polyphosphates

4-Coumarate:coenzyme A ligase (4CL) is known to activate cinnamic acid derivatives to their corresponding coenzyme A esters. As a new type of 4CL-catalyzed reaction, we observed the synthesis of various mono- and diadenosine polyphosphates. Both the native 4CL2 isoform from Arabidopsis (At4CL2 wild type) and the At4CL2 gain of function mutant M293P/K320L, which exhibits the capacity to use a broader range of phenolic substrates, catalyzed the synthesis of adenosine 5'-tetraphosphate (p(4)A) and adenosine 5'-pentaphosphate when incubated with MgATP(-2) and tripolyphosphate or tetrapolyphosphate (P(4)), respectively. Diadenosine 5',5',-P(1),P(4)-tetraphosphate represented the main product when the enzymes were supplied with only MgATP(2-). The At4CL2 mutant M293P/K320L was studied in more detail and was also found to catalyze the synthesis of additional dinucleoside polyphosphates such as diadenosine 5',5'-P(1),P(5)-pentaphosphate and dAp(4)dA from the appropriate substrates, p(4)A and dATP, respectively. Formation of Ap(3)A from ATP and ADP was not observed with either At4CL2 variant. In all cases analyzed, (di)adenosine polyphosphate synthesis was either strictly dependent on or strongly stimulated by the presence of a cognate cinnamic acid derivative. The At4CL2 mutant enzyme K540L carrying a point mutation in the catalytic center that is critical for adenylate intermediate formation was inactive in both p(4)A and diadenosine 5',5',-P(1),P(4)-tetraphosphate synthesis. These results indicate that the cinnamoyl-adenylate intermediate synthesized by At4CL2 not only functions as an intermediate in coenzyme A ester formation but can also act as a cocatalytic AMP-donor in (di)adenosine polyphosphate synthesis.     

Identification of dinucleoside polyphosphates in adrenal glands

Dinucleoside polyphosphates have been characterised as extracellular mediators controlling numerous physiological functions like vascular tone or cell proliferation. Here we describe the isolation and identification of dinucleoside polyphosphates Ap(n)A (with n=2-3), Ap(n)G (with n=2-6) as well as Gp(n)G (with n=2-6) from adrenal glands. These dinucleoside polyphosphates are localised in granules of the adrenal glands. The dinucleoside polyphosphates diadenosine diphosphate (Ap(2)A), diadenosine triphosphate (Ap(3)A), adenosine guanosine polyphosphates (Ap(n)G) and diguanosine polyphosphates (Gp(n)G), both with phosphate group (p) numbers (n) ranging from 2 to 6, were identified by fractionating them to homogeneity by preparative size-exclusion- and affinity-chromatography as well as analytical anion-exchange and reversed-phase-chromatography from deproteinised adrenal glands and by analysis of the homogeneous dinucleoside polyphosphates containing fractions with post-source-decay (PSD) matrix-assisted laser desorption/ionisation mass spectrometry (MALDI-MS). The identity of the dinucleoside polyphosphates was confirmed by retention time comparison with authentic dinucleoside polyphosphates. Enzymatic analysis demonstrated an interconnection of the phosphate groups with the adenosines in the 5(')-positions of the riboses in all dinucleoside polyphosphates purified from adrenal glands. In conclusion, the identification of these dinucleoside polyphosphates in adrenal gland granules emphasises that these dinucleoside polyphosphates can be released from the adrenal glands upon stimulation into the circulation.     

Analogs of diadenosine tetraphosphate (Ap4A)

This review summarizes our knowledge of analogs and derivatives of diadenosine 5',5"'-P1,P4-tetraphosphate (Ap4A), the most extensively studied member of the dinucleoside 5',5"'-P1,Pn-polyphosphate (NpnN) family. After a short discussion of enzymes that may be responsible for the accumulation and degradation of Np4)N's in the cell, this review focuses on chemically and/or enzymatically produced analogs and their practical applications. Particular attention is paid to compounds that have aided the study of enzymes involved in the metabolism of Ap4A (Np4N'). Certain Ap4A analogs were alternative substrates of Ap4A-degrading enzymes and/or acted as enzyme inhibitors, some other helped to establish enzyme mechanisms, increased the sensitivity of certain enzyme assays or produced stable enzyme:ligand complexes for structural analysis.     

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.     

Inhibition of adenylate kinase by P1-(lin-benzo-5'-adenosyl)-P4-(5'-adenosyl) tetraphosphate and P1-(lin-benzo-5'-adenosyl)-P5-(5'-adenosyl pentaphosphate.

P1-(lin-Benzo-5'-adenosyl)-P5-(5'-adenosyl) penraphosphate and P1-(lin-benzo-5'-adenosyl)-P4-(5'-adenosyl) tetraphosphate have been synthesized from lin-benzoadenosine 5'-monophosphoromorpholidate and adenosine 5'-tetraphosphate and adenosine 5'-triphosphate. These mixed dinucleoside polyphosphates are potent inhibitors of porcine muscle adenylate kinase, with association constants of 2 x 10(5) M-1 for the pentaphosphate and 2 x 10(6) M-1 for the tetraphosphate, respectively, as determined by kinetics and fluorescence experiments. The increase in fluorescence intensities and fluorescence lifetimes of both inhibitors upon binding to adenylate kinase results from a breaking of the intramolecular stacking interaction observed when these ligands are free in solution and implicates their binding to the enzyme in an "open" or "extended" form. These results and the dimensional requirements of these inhibitors are discussed in relation to our current knowledge of the active site of adenylate kinase and to the known inhibitors of adenylate kinase, P1,P5-bis(5'-adenosyl) pentaphosphate and P1,P4-bis-(5'-adenosyl) tetraphosphate.     

ADP-ribosylation of diadenosine 5',5"-P1,P4-tetraphosphate by poly(ADP-ribose) polymerase in vitro

When the effect of diadenosine 5',5"'-P1,P4-tetraphosphate on a purified poly(ADP-ribose) polymerase reaction was examined, the compound strongly inhibited ADP-ribosylation reaction of histone, while the compound was much less inhibitory of the Mg2+-dependent automodification of this enzyme. In an attempt to study the mechanism of the inhibition, we analyzed the total reaction products, which were synthesized from NAD+ in the presence of diadenosine 5',5"'-P1,P4-tetraphosphate in a reaction mixture for ADP-ribosylation of histone, and found that a new, low molecular product was predominantly synthesized instead of ADP-ribosylated histone in the reaction. Approximately 90% of added NAD+ was converted into this low molecular product under an appropriate reaction condition. Further analysis revealed that the product was mono- and oligo(ADP-ribosyl)ated diadenosine nucleotide and that the bound oligo(ADP-ribose) is elongating at one end of the product during the reaction. Thus, the present study clearly demonstrated that diadenosine 5',5"'-P1,P4-tetraphosphate functions as an acceptor for ADP-ribose in a poly(ADP-ribose) polymerase reaction in vitro. The finding that histone H1 is required in the reaction mixture for the synthesis of this new product suggests that histone H1 and the diadenosine compound interact during this modification reaction.     

Purification to homogeneity of rat liver dinucleoside tetraphosphatase by affinity elution with adenosine 5'-tetraphosphate

Starting from a partially purified dinucleoside tetraphosphatase (Np4Nase; EC 3.6.1.17), we developed an affinity elution purification protocol involving the strong competitive inhibitor adenosine 5'-tetraphosphate. Np4Nase bound to Cibacron Blue F3G-A-Sepharose 4B or to Reactive Blue 2-Sepharose CL-6B was specifically eluted with 10 microM adenosine 5'-tetraphosphate and 5 mM MgCl2, but not by either of them separately. The final Np4Nase preparation was homogeneous by sodium dodecyl sulfate polyacrylamide gel electrophoresis followed by Coomassie blue or silver staining. The protein band showed an apparent 18 kDa molecular mass. The specific activity of the homogeneous Np4Nase was about 150 units/mg, meaning a 45,000-fold increase and a 10% recovery with respect to the crude extract. After preparative polyacrylamide gel electrophoresis, protein visualization with KCl, fragmentation of the gel lane, and extraction, all the renatured Np4Nase activity was found associated to the 18 kDa band. The renatured enzyme showed the same Km value for diadenosine 5',5"'-P1,P4-tetraphosphate as the partially purified or the native homogeneous Np4Nase.     

Dinucleoside polyphosphate NAD analogs as potential NMN adenylyltransferase inhibitors. Synthesis and biological evaluation

Two dinucleoside polyphosphate NAD analogs, P1-(adenosine-5')-P3-(nicotinamide riboside-5')triphosphate (Np3A, 1) and P1-(adenosine-5')-P4-(nicotinamide riboside-5')tetraphosphate (Np4A, 2), were synthesized and tested as inhibitors of both microbial and human recombinant NMN adenylyltransferase. Compounds 1 and 2 proved to be selective inhibitors of microbial enzymes.