Nucleoside 5'-Phosphoramidates Control the Phenylpropanoid Pathway in Vitis vinifera Suspension-Cultured Cells.

It is known that cells contain various uncommon nucleotides such as dinucleoside polyphosphates (NpnN's) and adenosine 5'-phosphoramidate (NH2-pA) belonging to nucleoside 5'-phosphoramidates (NH2-pNs). Their cellular levels are enzymatically controlled. Some of them are accumulated in cells under stress, and therefore, they could act as signal molecules. Our previous research carried out in Arabidopsis thaliana and grape (Vitis vinifera) showed that NpnN's induced the expression of genes in the phenylpropanoid pathway and favored the accumulation of their products, which protect plants against stress. Moreover, we found that NH2-pA could play a signaling role in Arabidopsis seedlings. Data presented in this paper show that exogenously applied purine (NH2-pA, NH2-pG) and pyrimidine (NH2-pU, NH2-pC) nucleoside 5'-phosphoramidates can modify the expression of genes that control the biosynthesis of both stilbenes and lignin in Vitis vinifera cv. Monastrell suspension-cultured cells. We investigated the expression of genes encoding for phenylalanine ammonia-lyase (PAL1), cinnamate-4-hydroxylase (C4H1), 4-coumarate:coenzyme A ligase (4CL1), chalcone synthase (CHS1), stilbene synthase (STS1), cinnamoyl-coenzyme A:NADP oxidoreductase (CCR2), and cinnamyl alcohol dehydrogenase (CAD1). Each of the tested NH2-pNs also induced the expression of the trans-resveratrol cell membrane transporter VvABCG44 gene and caused the accumulation of trans-resveratrol and trans-piceid in grape cells as well as in the culture medium. NH2-pC, however, evoked the most effective induction of phenylpropanoid pathway genes such as PAL1, C4H1, 4CL1, and STS1. Moreover, this nucleotide also induced at short times the accumulation of N-benzoylputrescine (BenPut), one of the phenylamides that are derivatives of phenylpropanoid and polyamines. The investigated nucleotides did not change either the lignin content or the cell dry weight, nor did they affect the cell viability throughout the experiment. The results suggest that nucleoside 5'-phosphoramidates could be considered as new signaling molecules.

Novel reactivity of Fhit proteins: catalysts for fluorolysis of nucleoside 5'-phosphoramidates and nucleoside 5'-phosphosulfates to generate nucleoside 5'-phosphorofluoridates.

Fragile histidine triad (HIT) proteins (Fhits) occur in all eukaryotes but their function is largely unknown. Human Fhit is presumed to function as a tumour suppressor. Previously, we demonstrated that Fhits catalyse hydrolysis of not only dinucleoside triphosphates but also natural adenosine 5'-phosphoramidate (NH2-pA) and adenosine 5'-phosphosulfate (SO4-pA) as well as synthetic adenosine 5'-phosphorofluoridate (F-pA). In the present study, we describe an Fhit-catalysed displacement of the amino group of nucleoside 5'-phosphoramidates (NH2-pNs) or the sulfate moiety of nucleoside 5'-phosphosulfates (SO4-pNs) by fluoride anion. This results in transient accumulation of the corresponding nucleoside 5'-phosphorofluoridates (F-pNs). Substrate specificity and kinetic characterization of the fluorolytic reactions catalysed by the human Fhit and other examples of involvement of fluoride in the biochemistry of nucleotides are described. Among other HIT proteins, human histidine triad nucleotide-binding protein (Hint1) catalysed fluorolysis of NH2-pA 20 times and human Hint2 40 times more slowly than human Fhit.

Hydrogen-sulfide-mediated vasodilatory effect of nucleoside 5'-monophosphorothioates in perivascular adipose tissue.

Hydrogen sulfide (H2S) is synthesized in perivascular adipose tissue (PVAT) and induces vasorelaxation. We examined whether the sulfur-containing AMP and GMP analogs AMPS and GMPS can serve as the H2S donors in PVAT. H2S production by isolated rat periaortic adipose tissue (PAT) was measured with a polarographic sensor. In addition, phenylephrine-induced contractility of aortic rings with (+) or without (-) PAT was examined. Isolated PAT produced H2S from AMPS or GMPS in the presence of the P2X7 receptor agonist BzATP. Phenylephrine-induced contractility of PAT(+) rings was lower than of PAT(-) rings. AMPS or GMPS had no effect on the contractility of PAT(-) rings, but used together with BzATP reduced the contractility of PAT(+) rings when endogenous H2S production was inhibited with propargylglycine. A high-fat diet reduced endogenous H2S production by PAT. Interestingly, AMPS and GMPS were converted to H2S by PAT of obese rats, and reduced contractility of PAT(+) aortic rings isolated from these animals even in the absence of BzATP. We conclude that (i) AMPS and GMPS can be hydrolyzed to H2S by PAT when P2X7 receptors are activated, (ii) a high-fat diet impairs endogenous H2S production by PAT, (iii) AMPS and GMPS restore the anticontractile effects of PAT in obese animals without P2X7 stimulation.

Nucleoside monophosphorothioates as the new hydrogen sulfide precursors with unique properties.

Hydrogen sulfide (H2S) is the gasotransmitter enzymatically synthesized in mammalian tissues from l-cysteine. H2S donors are considered as the potential drugs for the treatment of cardiovascular, neurological and inflammatory diseases. Recently, it has been demonstrated that synthetic nucleotide analogs, adenosine- and guanosine 5'-monophosphorothioates (AMPS and GMPS) can be converted to H2S and AMP or GMP, respectively, by purified histidine triad nucleotide-binding (Hint) proteins. We examined if AMPS and GMPS can be used as the H2S donors in intact biological systems. H2S production by isolated rat kidney glomeruli was measured by the specific polarographic sensor. H2S production was detected when glomeruli were incubated with AMPS or GMPS and ionotropic purinergic P2X7 receptor/channel agonist, BzATP. More H2S was generated from GMPS than from equimolar amount of AMPS. Nucleoside phosphorothioates together with BzATP relaxed angiotensin II-preconstricted glomeruli. In addition, infusion of AMPS or GMPS together with BzATP into the renal artery increased filtration fraction and glomerular filtration rate but had no effect on renal vascular resistance or renal blood flow. AMPS but not GMPS was converted to adenosine by isolated glomeruli, however, adenosine was not involved in AMPS-induced H2S synthesis because neither adenosine nor specific adenosine receptor agonists had any effect on H2S production. AMPS, but not GMPS, increased phosphorylation level of AMP-stimulated protein kinase (AMPK), but AMPK inhibitor, compound C, had no effect on AMPS-induced H2S production. In conclusion, nucleoside phosphorothioates are converted to H2S which relaxes isolated kidney glomeruli in vitro and increases glomerular filtration rate in vivo. AMPS and GMPS can be used as the H2S donors in experimental studies and possibly also as the H2S-releasing drugs.

Identification of the HIT-45 protein from Trypanosoma brucei as an FHIT protein/dinucleoside triphosphatase: substrate specificity studies on the recombinant and endogenous proteins.

A new member of the FHIT protein family, designated HIT-45, has been identified in the African trypanosome Trypanosoma brucei. Recombinant HIT-45 proteins were purified from trypanosomal and bacterial protein expression systems and analyzed for substrate specificity using various dinucleoside polyphosphates, including those that contain the 5'-mRNA cap, i.e., m(7)GMP. This enzyme exhibited typical dinucleoside triphosphatase activity (EC 3.6.1.29), having its highest specificity for diadenosine triphosphate (ApppA). However, the trypanosome enzyme contains a unique amino-terminal extension, and hydrolysis of cap dinucleotides with monomethylated guanosine or dimethylated guanosine always yielded m(7)GMP (or m(2,7)GMP) as one of the reaction products. Interestingly, m(7)Gpppm(3)(N6, N6, 2'O)A was preferred among the methylated substrates. This hypermethylated dinucleotide is unique to trypanosomes and may be an intermediate in the decay of cap 4, i.e., m(7)Gpppm(3)(N6, N6, 2'O)Apm(2'O)Apm(2'O)Cpm(2)(N3, 2'O)U, that occurs in these organisms.

Adenosine 5'-tetraphosphate is a highly potent purinergic endothelium-derived vasoconstrictor.

Besides serving as a mechanical barrier, the endothelium has important regulatory functions. The discovery of nitric oxide revolutionized our understanding of vasoregulation. In contrast, the identity of endothelium-derived vasoconstrictive factors still remains uncertain. The supernatant from mechanically stimulated human microvascular endothelial cells elicited a potent vasoconstrictive response in the isolated perfused rat kidney. Whereas a nonselective purinoceptor blocker blocked this vasoactivity most potently, the inhibition of the endothelin receptor by BQ123 weakly affected that vasoconstrictive response. As a compound responsible for that vasoconstrictive effect, we have isolated from HMECs and identified the mononucleotide adenosine 5'-tetraphosphate (AP4). This nucleotide proved to be the most potent vasoactive purinergic mediator identified to date, exerting the vasoconstriction predominantly through activation of the P2X1 receptor. The intraarterial application of AP4 in a Wistar-Kyoto rat induced a strong increase of the mean arterial pressure. The plasma concentration of AP4 is in the nanomolar range, which, in vivo, induces a significant change in the mean arterial pressure. To our knowledge, AP4, which exerts vasoactive effects, is the most potent endogenous mononucleotide identified to date in mammals. The effects of AP4, the plasma concentration of AP4, and its release suggest that this compound functions as an important vasoregulator.

Purine and pyrimidine dinucleoside polyphosphates differentially affect the phenylpropanoid pathway in Vitis vinifera L. cv. Monastrell suspension cultured cells.

It is known that the concentration of dinucleoside polyphosphates (NpnN's) in cells increases under stress and that adverse environmental factors induce biosynthesis of phenylpropanoids, which protect the plant against stress. Previously, we showed that purine NpnN's such as Ap3A and Ap4A induce both the activity of enzymes of the phenylpropanoid pathway and the expression of relevant genes in Arabidopsis seedlings. Moreover, we showed that Ap3A induced stilbene biosynthesis in Vitis vinifera cv. Monastrell suspension cultured cells. Data presented in this paper show that pyrimidine-containing NpnN's also modify the biosynthesis of stilbenes, affecting the transcript level of genes encoding key enzymes of the phenylpropanoid pathway and of these, Up4U caused the most effective accumulation of trans-resveratrol in the culture media. Similar effect was caused by Ap3A and Gp3G. Other pyrimidine NpnN's, such as Cp3C, Cp4C, and Ap4C, strongly inhibited the biosynthesis of stilbenes, but markedly (6- to 8-fold) induced the expression of the cinnamoyl-CoA reductase gene that controls lignin biosynthesis. Purine counterparts also clearly induced biosynthesis of trans-resveratrol and trans-piceid, but only slightly induced the expression of genes involved in lignin biosynthesis. In cells, Up3U caused a greater accumulation of trans-resveratrol and trans-piceid than did Up4U. Each of the NpnN's studied induced expression of the gene encoding the resveratrol transporter VvABCG44, which operates within the Vitis vinifera cell membrane. AMP, GMP, UMP, and CMP, potential products of NpnN degradation, did not affect the accumulation of stilbenes. The results obtained strongly support that NpnN's play a role as signaling molecules in plants.

Fhit proteins can also recognize substrates other than dinucleoside polyphosphates.

We show here that Fhit proteins, in addition to their function as dinucleoside triphosphate hydrolases, act similarly to adenylylsulfatases and nucleoside phosphoramidases, liberating nucleoside 5'-monophosphates from such natural metabolites as adenosine 5'-phosphosulfate and adenosine 5'-phosphoramidate. Moreover, Fhits recognize synthetic nucleotides, such as adenosine 5'-O-phosphorofluoridate and adenosine 5'-O-(gamma-fluorotriphosphate), and release AMP from them. With respect to the former, Fhits behave like a phosphodiesterase I concomitant with cleavage of the P-F bond. Some kinetic parameters and implications of the novel reactions catalyzed by the human and plant (Arabidopsis thaliana) Fhit proteins are presented.

Substrate specificity and products of side-reactions catalyzed by jasmonate:amino acid synthetase (JAR1).

Jasmonate:amino acid synthetase (JAR1) is involved in the function of jasmonic acid (JA) as a plant hormone. It catalyzes the synthesis of several JA-amido conjugates, the most important of which appears to be JA-Ile. Structurally, JAR1 is a member of the firefly luciferase superfamily that comprises enzymes that adenylate various organic acids. This study analyzed the substrate specificity of recombinant JAR1 and determined whether it catalyzes the synthesis of mono- and dinucleoside polyphosphates, which are side-reaction products of many enzymes forming acyl approximately adenylates. Among different oxylipins tested as mixed stereoisomers for substrate activity with JAR1, the highest rate of conversion to Ile-conjugates was observed for (+/-)-JA and 9,10-dihydro-JA, while the rate of conjugation with 12-hydroxy-JA and OPC-4 (3-oxo-2-(2Z-pentenyl)cyclopentane-1-butyric acid) was only about 1-2% that for (+/-)-JA. Of the two stereoisomers of JA, (-)-JA and (+)-JA, rate of synthesis of the former was about 100-fold faster than for (+)-JA. Finally, we have demonstrated that (1) in the presence of ATP, Mg(2+), (-)-JA and tripolyphosphate the ligase produces adenosine 5'-tetraphosphate (p(4)A); (2) addition of isoleucine to that mixture halts the p(4)A synthesis; (3) the enzyme produces neither diadenosine triphosphate (Ap(3)A) nor diadenosine tetraphosphate (Ap(4)A) and (4) Ap(4)A cannot substitute ATP as a source of adenylate in the complete reaction that yields JA-Ile.

Methylene analogues of adenosine 5'-tetraphosphate. Their chemical synthesis and recognition by human and plant mononucleoside tetraphosphatases and dinucleoside tetraphosphatases.

Adenosine 5'-polyphosphates have been identified in vitro, as products of certain enzymatic reactions, and in vivo. Although the biological role of these compounds is not known, there exist highly specific hydrolases that degrade nucleoside 5'-polyphosphates into the corresponding nucleoside 5'-triphosphates. One approach to understanding the mechanism and function of these enzymes is through the use of specifically designed phosphonate analogues. We synthesized novel nucleotides: alpha,beta-methylene-adenosine 5'-tetraphosphate (pppCH2pA), beta,gamma-methylene-adenosine 5'-tetraphosphate (ppCH2ppA), gamma,delta-methylene-adenosine 5'-tetraphosphate (pCH2pppA), alphabeta,gammadelta-bismethylene-adenosine 5'-tetraphosphate (pCH2ppCH2pA), alphabeta, betagamma-bismethylene-adenosine 5'-tetraphosphate (ppCH2pCH2pA) and betagamma, gammadelta-bis(dichloro)methylene-adenosine 5'-tetraphosphate (pCCl2pCCl2ppA), and tested them as potential substrates and/or inhibitors of three specific nucleoside tetraphosphatases. In addition, we employed these p4A analogues with two asymmetrically and one symmetrically acting dinucleoside tetraphosphatases. Of the six analogues, only pppCH2pA is a substrate of the two nucleoside tetraphosphatases (EC 3.6.1.14), from yellow lupin seeds and human placenta, and also of the yeast exopolyphosphatase (EC 3.6.1.11). Surprisingly, none of the six analogues inhibited these p4A-hydrolysing enzymes. By contrast, the analogues strongly inhibit the (asymmetrical) dinucleoside tetraphosphatases (EC 3.6.1.17) from human and the narrow-leafed lupin. ppCH2ppA and pCH2pppA, inhibited the human enzyme with Ki values of 1.6 and 2.3 nm, respectively, and the lupin enzyme with Ki values of 30 and 34 nm, respectively. They are thereby identified as being the strongest inhibitors ever reported for the (asymmetrical) dinucleoside tetraphosphatases. The three analogues having two halo/methylene bridges are much less potent inhibitors for these enzymes. These novel nucleotides should prove valuable tools for further studies on the cellular functions of mono- and dinucleoside polyphosphates and on the enzymes involved in their metabolism.

Calcium-stimulated guanosine--inosine nucleosidase from yellow lupin (Lupinus luteus).

Guanosine-inosine-preferring nucleoside N-ribohydrolase has been purified to homogeneity from yellow lupin (Lupinus luteus) seeds by ammonium sulfate fractionation, ion-exchange chromatography and gel filtration. The enzyme functions as a monomeric, 80kDa polypeptide, most effectively between pH 4.7 and 5.5. Of various mono- and divalent cations tested, Ca(2+) appeared to stimulate enzyme activity. The nucleosidase was activated 6-fold by 2mM exogenous CaCl(2) or Ca(NO(3))(2), with K(a)=0.5mM (estimated for CaCl(2)). The K(m) values estimated for guanosine and inosine were 2.7+/-0.3 microM. Guanosine was hydrolyzed 12% faster than inosine while adenosine and xanthosine were poor substrates. 2'-Deoxyguanosine, 2'-deoxyinosine, 2'-methylguanosine, pyrimidine nucleosides and 5'-GMP were not hydrolyzed. However, the enzyme efficiently liberated the corresponding bases from synthetic nucleosides, such as 1-methylguanosine, 7-methylguanosine, 1-N(2)-ethenoguanosine and 1-N(2)-isopropenoguanosine, but hydrolyzed poorly the ribosides of 6-methylaminopurine and 2,6-diaminopurine. MnCl(2) or ZnCl(2) inhibited the hydrolysis of guanosine with I(50) approximately 60 microM. Whereas 2'-deoxyguanosine, 2'-methylguanosine, adenosine, as well as guanine were competitive inhibitors of this reaction (K(i) values were 1.5, 3.6, 21 and 9.7 microM, respectively), hypoxanthine was a weaker inhibitor (K(i)=64 microM). Adenine, ribose, 2-deoxyribose, 5'-GMP and pyrimidine nucleosides did not inhibit the enzyme. The guanosine-inosine hydrolase activity occurred in all parts of lupin seedlings and in cotyledons it increased up to 5-fold during seed germination, reaching maximum in the third/fourth day. The lupin nucleosidase has been compared with other nucleosidases.

Synthesis and enzymatic characterization of methylene analogs of adenosine 5'-tetraphosphate (P4A).

A new methodology for synthesis of biologically important nucleoside tri- and tetraphosphates containing a bisphosphonate moiety instead of the terminal pyrophosphate bond is described. The series consists of tri- and tetraphosphate analogs of adenosine, guanosine and 7-methylguanosine (characteristic for mRNA cap). We have adopted a two-step procedure that allowed us to insert a methylene bridge into the phosphate chain. Nucleoside mono- or diphosphates were first activated (as imidazole derivatives) and then used in coupling reactions with organic salts of bisphosphonate. The resulting synthetic method enabled us to obtain the desired compounds with high yields and does not require any protective groups. This makes it very useful for the synthesis of labile compounds such as those containing the 7-methylguanosine ring. The structures of the synthesized compounds were confirmed by NMR spectroscopy. They were tested as potential substrates and inhibitors of several hydrolases.

Adenylylsulfate-ammonia adenylyltransferase activity is another inherent property of Fhit proteins.

Fhits (fragile histidine triad proteins) occur in eukaryotes but their function is largely unknown, although human Fhit is believed to act as a tumour suppressor. Fhits also exhibit dinucleoside triphosphatase, adenylylsulfatase and nucleoside phosphoramidase activities that in each case yield nucleoside 5'-monophosphate as a product. Due to the dinucleoside triphosphatase activity, Fhits may also be involved in mRNA decapping. In the present study, we demonstrate Fhit-catalysed ammonolysis of adenosine 5'-phosphosulfate, which results in the formation of adenosine 5'-phosphoramidate. This reaction has previously been associated with adenylylsulfate-ammonia adenylyltransferase (EC 2.7.7.51). Our finding shows that the capacity to catalyse ammonolysis is another inherent property of Fhits. Basic kinetic parameters and substrate specificity of this reaction catalysed by human Fhit are presented.

Exogenous adenosine 5'-phosphoramidate behaves as a signal molecule in plants; it augments metabolism of phenylpropanoids and salicylic acid in Arabidopsis thaliana seedlings.

Cells contain various congeners of the canonical nucleotides. Some of these accumulate in cells under stress and may function as signal molecules. Their cellular levels are enzymatically controlled. Previously, we demonstrated a signaling function for diadenosine polyphosphates and cyclic nucleotides in Arabidopsis thaliana and grape, Vitis vinifera. These compounds increased the expression of genes for and the specific activity of enzymes of phenylpropanoid pathways resulting in the accumulation of certain products of these pathways. Here, we show that adenosine 5'-phosphoramidate, whose level can be controlled by HIT-family proteins, induced similar effects. This natural nucleotide, when added to A. thaliana seedlings, activated the genes for phenylalanine:ammonia lyase, 4-coumarate:coenzyme A ligase, cinnamate-4-hydroxylase, chalcone synthase, cinnamoyl-coenzyme A:NADP oxidoreductase and isochorismate synthase, which encode proteins catalyzing key reactions of phenylpropanoid pathways, and caused accumulation of lignins, anthocyanins and salicylic acid. Adenosine 5'-phosphofluoridate, a synthetic congener of adenosine 5'-phosphoramidate, behaved similarly. The results allow us to postulate that adenosine 5'-phosphoramidate should be considered as a novel signaling molecule.

Uridine 5'-polyphosphates (p4U and p5U) and uridine(5')polyphospho(5')nucleosides (Up(n)Ns) can be synthesized by UTP:glucose-1-phosphate uridylyltransferase from Saccharomyces cerevisiae.

UTP:glucose-1-phosphate uridylyltransferase (EC 2.7.7.9) from Saccharomyces cerevisiae can transfer the uridylyl moiety from UDP-glucose onto tripolyphosphate (P(3)), tetrapolyphosphate (P(4)), nucleoside triphosphates (p(3)Ns) and nucleoside 5'-polyphosphates (p(4)Ns) forming uridine 5'-tetraphosphate (p(4)U), uridine 5'-pentaphosphate (p(5)U) and dinucleotides, such as Ap(4)U, Cp(4)U, Gp(4)U, Up(4)U, Ap(5)U and Gp(5)U. Unlike UDP-glucose, UDP-galactose was not a UMP donor and ADP was not a UMP acceptor. This is the first example of an enzyme that may be responsible for accumulation of dinucleoside tetraphosphates containing two pyrimidine nucleosides in vivo. Occurrence of such dinucleotides in S. cerevisiae and Escherichia coli has been previously reported (Coste et al., J. Biol. Chem. 262 (1987) 12096-12103).

Metabolism of diadenosine tetraphosphate (Ap4A) and related nucleotides in plants; review with historical and general perspective.

This review presents our knowledge of potential biochemical conversions of minor mononucleotides, such as adenosine-5'-tetraphosphate (p4A) and adenosine-5'-pentaphosphate (p5A), and dinucleotides, such as diadenosine-5',5"'-P1,P3-triphosphate (Ap3A) and diadenosine-5',5"'-P1,P4-tetraphosphate (Ap4A), in plants. Although the occurrence of p4A, Ap3A and/or Ap4A has been demonstrated in various bacteria, fungi and animals, identification of these compounds in plants has not been reported as yet. However, the ubiquity of both the compounds and enzymes that can synthesize them (certain ligases and transferases), the demonstration that certain plant ligases can synthesize pnAs and ApnNs in vitro, and the existence in plants of specific and nonspecific degradative enzymes strongly suggest that these various pnNs and NpnN's do indeed occur and play a biological role in plant cells. In fact, some of the plant enzymes involved in the synthesis and degradation of these minor mono- and dinucleotides have been studied even more thoroughly than their counterparts from other organisms.

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.

Selective splitting of 3'-adenylated dinucleoside polyphosphates by specific enzymes degrading dinucleoside polyphosphates.

Several 3'-[(32)P]adenylated dinucleoside polyphosphates (Np(n)N'p*As) were synthesized by the use of poly(A) polymerase (Sillero MAG et al., 2001, Eur J Biochem.; 268: 3605-11) and three of them, ApppA[(32)P]A or ApppAp*A, AppppAp*A and GppppGp*A, were tested as potential substrates of different dinucleoside polyphosphate degrading enzymes. Human (asymmetrical) dinucleoside tetraphosphatase (EC 3.6.1.17) acted almost randomly on both AppppAp*A, yielding approximately equal amounts of pppA + pAp*A and pA + pppAp*A, and GppppGp*, yielding pppG + pGp*A and pG + pppGp*A. Narrow-leafed lupin (Lupinus angustifolius) tetraphosphatase acted preferentially on the dinucleotide unmodified end of both AppppAp*A (yielding 90% of pppA + pAp*A and 10 % of pA + pppAp*A) and GppppGp*A (yielding 89% pppG + pGp*A and 11% of pG + pppGp*A). (Symmetrical) dinucleoside tetraphosphatase (EC 3.6.1.41) from Escherichia coli hydrolyzed AppppAp*A and GppppGp*A producing equal amounts of ppA + ppAp*A and ppG + ppGp*A, respectively, and, to a lesser extent, ApppAp*A producing pA + ppAp*A. Two dinucleoside triphosphatases (EC 3.6.1.29) (the human Fhit protein and the enzyme from yellow lupin (Lupinus luteus)) and dinucleoside tetraphosphate phosphorylase (EC 2.7.7.53) from Saccharomyces cerevisiae did not degrade the three 3'-adenylated dinucleoside polyphosphates tested.

New Tripodal, "Supercharged" Analogues of Adenosine Nucleotides: Inhibitors for the Fhit Ap3 A Hydrolase.

Methanetrisphosphonic acids provide a branch point for synthetic nucleotide analogues which can be exploited either to generate novel tripodal nucleotides or to incorporate additional negative charge into linear analogues relative to the parent nucleotide, as exemplified in the picture for ATP and diadenosine tetraphosphate (Ap4 A). These compounds show valuable discriminatory behavior as competitive inhibitors for the tumor suppressor protein Fhit and a second Apn A pyrophosphohydrolase. X=H, Cl, F.

Diadenosine triphosphate is a novel factor which in combination with cyclodextrins synergistically enhances the biosynthesis of trans-resveratrol in Vitis vinifera cv. Monastrell suspension cultured cells.

Dinucleoside polyphosphates are considered as signal molecules that may evoke response of plant cells to stress. Other compounds whose biological effects have been recognized are cyclodextrins. They are cyclic oligosaccharides that chemically resemble the alkyl-derived pectic oligosaccharides naturally released from the cell walls during fungal attack, and they act as true elicitors, since, when added to plant cell culture, they induce the expression of genes involved in some secondary metabolism pathways. Previously, we demonstrated that some dinucleoside polyphosphates triggered the biosynthesis of enzymes involved in the phenylpropanoid pathway in Arabidopsis thaliana. In Vitis vinifera suspension cultured cells, cyclodextrins were shown to enhance the accumulation of trans-resveratrol, one of the basic units of the stilbenes derived from the phenylpropanoid pathway. Here, we show that diadenosine triphosphate, applied alone or in combination with cyclodextrins to the grapevine suspension-cultured cells, increased the transcript level of genes encoding key phenylpropanoid-pathway enzymes as well as the trans-resveratrol production inside cells and its secretion into the extracellular medium. In the latter case, these two compounds acted synergistically. However, the accumulation of trans-resveratrol and its glucoside trans-piceid inside cells were stimulated much better by diadenosine triphosphate than by cyclodextrins.