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.

Metabolic fate of AMP, IMP, GMP and XMP in the cytosol of rat brain: an experimental and theoretical analysis.

A systematic study of the metabolic fate of AMP, IMP, GMP and XMP (NMP) in the presence of cytosol from rat brain is here presented; the kinetics of both disappearance of NMP, and appearance of their degradation products was followed by HPLC. In the absence of ATP, AMP was preferentially degraded to adenosine with concomitant appearance of inosine and hypoxanthine. In the presence of ATP, AMP was preferentially degraded via IMP. The nucleosides generated in the course of the reactions are further degraded, almost exclusively, via nucleoside phosphorylase using as cofactor the P(i) generated in the reaction mixture. In order to quantify the effect of each one of the enzymes involved in the degradation of NMP, two complementary approaches were followed: (i) the V:(max) and K:(m) values of the enzymes acting in the intermediate steps of the reactions were determined; (ii) these data were introduced into differential equations describing the concentration of the nucleotides and their degradation products as a function of the time of incubation. Factors affecting kinetic parameters of the equation velocity as a function of ATP concentration were introduced when required. The differential equations were solved with the help of Mathematica 3.0. The theoretical method can be used to simulate situations not feasible to be carried out, such as to measure the influence of nM-microM concentrations of ATP on the metabolism of AMP.

Synthesis of dinucleoside polyphosphates catalyzed by firefly luciferase and several ligases.

The findings presented here originally arose from the suggestion that the synthesis of dinucleoside polyphosphates (Np(n)N) may be a general process involving enzyme ligases catalyzing the transfer of a nucleotidyl moiety via nucleotidyl-containing intermediates, with release of pyrophosphate. Within this context, the characteristics of the following enzymes are presented. Firefly luciferase (EC 1.12. 13.7), an oxidoreductase with characteristics of a ligase, synthesizes a variety of (di)nucleoside polyphosphates with four or more inner phosphates. The discrepancy between the kinetics of light production and that of Np(n)N synthesis led to the finding that E*L-AMP (L = dehydroluciferin), formed from the E*LH(2)-AMP complex (LH(2) = luciferin) shortly after the onset of the reaction, was the main intermediate in the synthesis of (di)nucleoside polyphosphates. Acetyl-CoA synthetase (EC 6.2.1.1) and acyl-CoA synthetase (EC 6.2.1. 8) are ligases that synthesize p(4)A from ATP and P(3) and, to a lesser extent, Np(n)N. T4 DNA ligase (EC 6.5.1.1) and T4 RNA ligase (EC 6.5.1.3) catalyze the synthesis of Np(n)N through the formation of an E-AMP complex with liberation of pyrophosphate. DNA is an inhibitor of the synthesis of Np(n)N and conversely, P(3) or nucleoside triphosphates inhibit the ligation of a single-strand break in duplex DNA catalyzed by T4 DNA ligase, which could have therapeutic implications. The synthesis of Np(n)N catalyzed by T4 RNA ligase is inhibited by nucleoside 3'(2'),5'-bisphosphates. Reverse transcriptase (EC 2.7.7.49), although not a ligase, catalyzes, as reported by others, the synthesis of Np(n)ddN in the process of removing a chain termination residue at the 3'-OH end of a growing DNA chain.

Several dinucleoside polyphosphates are acceptor substrates in the T4 RNA ligase catalyzed reaction.

Several dinucleoside polyphosphates accept cytidine-3', 5'-bisphosphate from the adenylylated donor 5'-adenylylated cytidine 5',3'-bisphosphate in the T4 RNA ligase catalyzed reaction. The 5'-adenylylated cytidine 5',3'-bisphosphate synthesized in a first step, from ATP and cytidine-3',5'-bisphosphate, is used as a substrate to transfer the cytidine-3',5'-bisphosphate residue to the 3'-OH group(s) of diguanosine tetraphosphate (Gp4G) giving rise to Gp4GpCp and pCpGp4GpCp in a ratio of approximately 10 : 1, respectively. The synthesized Gp4GpCp was characterized by treatment with snake venom phosphodiesterase and alkaline phosphatase and analysis (chromatographic position and UV spectra) of the reaction products by HPLC. The apparent Km values measured for Gp4G and 5'-adenylylated cytidine 5',3'-bisphosphate in this reaction were approximately 4 mM and 0.4 mM, respectively. In the presence of 0.5 mM ATP and 0.5 mM cytidine-3',5'-bisphosphate, the relative efficiencies of the following nucleoside(5')oligophospho(5')nucleosides as acceptors of cytidine-3',5'-bisphosphate from 5'-adenylylated cytidine 5', 3'-bisphosphate are indicated in parentheses: Gp4G (100); Gp5G (101); Ap4G (47); Ap4A (39). Gp2G, Gp3G and Xp4X were not substrates of the reaction. Dinucleotides containing two guanines and at least four inner phosphates were the preferred acceptors of cytidine-3', 5'-bisphosphate at their 3'-OH group(s).

Acyl-CoA synthetase catalyzes the synthesis of diadenosine hexaphosphate (Ap6A).

The synthesis of diadenosine hexaphosphate (Ap6A), a potent vasoconstrictor, is catalyzed by acyl-CoA synthetase from Pseudomonas fragi. In a first step AMP is transferred from ATP to tetrapolyphosphate (P4) originating adenosine pentaphosphate (p5A) which, subsequently, is the acceptor of another AMP moiety from ATP generating diadenosine hexaphosphate (Ap6A). Diadenosine pentaphosphate (Ap5A) and diadenosine tetraphosphate (Ap4A) were also synthesized in the course of the reaction. In view of the variety of biological effects described for these compounds the potential capacity of synthesis of diadenosine polyphosphates by the mammalian acyl-CoA synthetases may be relevant.

T4 RNA ligase catalyzes the synthesis of dinucleoside polyphosphates.

T4 RNA ligase has been shown to synthesize nucleoside and dinucleoside 5'-polyphosphates by displacement of the AMP from the E-AMP complex with polyphosphates and nucleoside diphosphates and triphosphates. Displacement of the AMP by tripolyphosphate (P3) was concentration dependent, as measured by SDS/PAGE. When the enzyme was incubated in the presence of 0.02 mm [alpha-32P] ATP, synthesis of labeled Ap4A was observed: ATP was acting as both donor (Km, microm) and acceptor (Km, mm) of AMP from the enzyme. Whereas, as previously known, ATP or dATP (but not other nucleotides) were able to form the E-AMP complex, the specificity of a compound to be acceptor of AMP from the E-AMP complex was very broad, and with Km values between 1 and 2 mm. In the presence of a low concentration (0.02 mm) of [alpha-32P] ATP (enough to form the E-AMP complex, but only marginally enough to form Ap4A) and 4 mm of the indicated nucleotides or P3, the relative rate of synthesis of the following radioactive (di)nucleotides was observed: Ap4X (from XTP, 100); Ap4dG (from dGTP, 74); Ap4G (from GTP, 49); Ap4dC (from dCTP, 23); Ap4C (from CTP, 9); Ap3A (from ADP, 5); Ap4ddA, (from ddATP, 1); p4A (from P3, 200). The enzyme also synthesized efficiently Ap3A in the presence of 1 mm ATP and 2 mm ADP. The following T4 RNA ligase donors were inhibitors of the synthesis of Ap4G: pCp > pAp > pA2'p.

Dehydroluciferyl-AMP is the main intermediate in the luciferin dependent synthesis of Ap4A catalyzed by firefly luciferase.

It was previously assumed that E x LH2-AMP was the intermediate complex in the synthesis of Ap4A catalyzed by firefly luciferase (EC 1.13.12.7), when luciferin (LH2) was used as cofactor. However, here we show that LH2 is partly transformed, shortly after the onset of the luciferase reaction, to dehydroluciferin (L) with formation of an E x L-AMP complex which is the main intermediate for the synthesis of Ap4A. Formation of three more derivatives of LH2 were also observed, related to the production of light by the enzyme. CoA, a known stimulator of light production, inhibits the synthesis of Ap4A by reacting with the E x L-AMP complex and yielding L-CoA.

T4 DNA ligase synthesizes dinucleoside polyphosphates.

T4 DNA ligase (EC 6.5.1.1), one of the most widely used enzymes in genetic engineering, transfers AMP from the E-AMP complex to tripolyphosphate, ADP, ATP, GTP or dATP producing p4A, Ap3A, Ap4A, Ap4G and Ap4dA, respectively. Nicked DNA competes very effectively with GTP for the synthesis of Ap4G and, conversely, tripolyphosphate (or GTP) inhibits the ligation of DNA by the ligase. As T4 DNA ligase has similar requirements for ATP as the mammalian DNA ligase(s), the latter enzyme(s) could also synthesize dinucleoside polyphosphates. The present report may be related to the recent finding that human Fhit (fragile histidine triad) protein, encoded by the FHIT putative tumor suppressor gene, is a typical dinucleoside 5',5''-P1,P3-triphosphate (Ap3A) hydrolase (EC 3.6.1.29).

IMP-GMP 5'-nucleotidase from rat brain: activation by polyphosphates.

IMP-GMP 5'-nucleotidase has been purified to homogeneity from total rat brain extracts. This preparation showed a unique band (Mr 54,000 +/- 1,509) in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme presented the following properties: optimal pH value, 6.5-6.8; relative velocity measured in the presence of MgCl2, MnCL2, CoCl2, and NiCl2 (2 mM), 100, 60, 11, and <1, respectively; preferred substrates, IMP and GMP; and activation constant (Ka) found for Ap4A, Ap5A, and Ap6A, 83 +/- 38, 77 +/- 32, and 57 +/- 12 microM, respectively. Under assay conditions where activation by Ap4A was fivefold, the activation produced by dinucleotides was as follows: Ap4G (4.0), Ap4I (2.9), Ap4X (3.3), Ap4C (0.7), Ap4U (1.1), Ap4epsilonA (1.5), Ap4ddA (1.7), Gp4G (2.2), Ap3A (1.1), and Ap2A (1.2). Polyphosphates P18, P19, P20, and P35 were activators of the reaction with calculated Ka values of 3.5 +/- 0.5, 0.9 +/- 0.2, 0.6 +/- 0.2, and 1.3 +/- 0.5 microM, respectively. The following compounds, at 0.1 mM, were effectors of the phosphotransferase reaction producing the fold activation indicated: Ap4A (8.3), Ap5A (10.2), Ap6A (10.1), Ap4G (7.7), Ap4X (7.6), Ap4U (2.1), glycerate 2,3-bisphosphate (3.9), and unpurified P15 (7.6). Two enzyme forms of IMP-GMP 5'-nucleotidase were detected when the extracts from rat tissues or from the crustacean Artemia were subjected to chromatography on a Dyematrex Green A column. The ratio of the hydrolytic activities under both peaks (peak I/peak II) was as follows: brain (1.5), heart (1.9), liver (1.6), lung (2.0), testis (3.8), and Artemia cysts (2.0).

Acyl coenzyme A synthetase from Pseudomonas fragi catalyzes the synthesis of adenosine 5'-polyphosphates and dinucleoside polyphosphates.

Acyl coenzyme A (CoA) synthetase (EC 6.2.1.8) from Pseudomonas fragi catalyzes the synthesis of adenosine 5'-tetraphosphate (p4A) and adenosine 5'-pentaphosphate (p5A) from ATP and tri- or tetrapolyphosphate, respectively. dATP, adenosine-5'-O-[gamma-thiotriphosphate] (ATP gamma S), adenosine(5')tetraphospho(5')adenosine (Ap4A), and adenosine(5')pentaphospho(5')adenosine (Ap5A) are also substrates of the reaction yielding p4(d)A in the presence of tripolyphosphate (P3). UTP, CTP, and AMP are not substrates of the reaction. The K(m) values for ATP and P3 are 0.015 and 1.3 mM, respectively. Maximum velocity was obtained in the presence of MgCl2 or CoCl2 equimolecular with the sum of ATP and P3. The relative rates of synthesis of p4A with divalent cations were Mg = Co > Mn = Zn >> Ca. In the pH range used, maximum and minimum activities were measured at pH values of 5.5 and 8.2, respectively; the opposite was observed for the synthesis of palmitoyl-CoA, with maximum activity in the alkaline range. The relative rates of synthesis of palmitoyl-CoA and p4A are around 10 (at pH 5.5) and around 200 (at pH 8.2). The synthesis of p4A is inhibited by CoA, and the inhibitory effect of CoA can be counteracted by fatty acids. To a lesser extent, the enzyme catalyzes the synthesis also of Ap4A (from ATP), Ap5A (from p4A), and adenosine(5')tetraphospho(5')nucleoside (Ap4N) from adequate adenylyl donors (ATP, ATP gamma S, or octanoyl-AMP) and adequate adenylyl acceptors (nucleoside triphosphates).

Synthesis of dehydroluciferin by firefly luciferase: effect of dehydroluciferin, coenzyme A and nucleoside triphosphates on the luminescent reaction.

The formation of dehydroluciferin (L) from luciferin (LH2) in the reaction catalyzed by firefly luciferase (EC 1.13.12.7) has been studied. The E.LH2-AMP complex may follow two different pathways: towards production of light and towards the synthesis of the E.L-AMP complex. This last step has an inhibitory effect on light emission as molecules of the enzyme are trapped in a light unproductive complex. The effects of CoA and nucleoside 5'-triphosphates (NTPs) on light emission are quantitatively different. CoA combines with the L moiety of the E.L-AMP complex, yielding L-CoA, promoting liberation of free luciferase, and increasing light yield. NTP reacts with the AMP moiety of the same complex, generating adenosine(5')tetraphospho(5')nucleoside (Ap4N) and, probably, the E. L complex and scarcely increasing light production. The results are discussed in relation to previous reports, by others, on luciferase.

2',3'-dideoxynucleoside triphosphates (ddNTP) and di-2',3'-dideoxynucleoside tetraphosphates (ddNp4ddN) behave differently to the corresponding NTP and Np4N counterparts as substrates of firefly luciferase, dinucleoside tetraphosphatase and phosphodiesterases.

2',3'-Dideoxynucleosides (ddN) and their derivatives are currently used as antiretroviral compounds. Their active agents are the corresponding 2',3'-dideoxynucleoside triphosphates (ddNTPs) generated inside the cell by host kinases. Dinucleoside tetraphosphates (Np4Ns) are molecules of interest in metabolic regulation; their synthesis in vitro can be catalyzed by firefly luciferase. The relative synthesis of diadenosine 5',5'''-P1,P4-tetraphosphate or adenosine(5')tetraphospho(5')adenosine (Ap4A) from ATP is about 100-fold faster than that of di-2',3'-dideoxyadenosine 5',5'''-P1,P4-tetraphosphate or 2',3'-dideoxyadenosine (5')tetraphospho (5')-2',3'-dideoxyadenosine (ddAp4ddA) from ddATP. In the presence of ATPgammaS and ddATP the yield of adenosine(5')tetraphospo(5')-2',3'-dideoxyadenosine (Ap4ddA) was similar to that attained for Ap4A in the presence of ATP. The findings of this work indicate that the presence of a 3'-hydroxyl group is essential for the formation of the luciferase-luciferin-AMP complex, and explains the very low yield of ddAp4ddA in the presence of luciferase, luciferin and ddATP. The absence of 3'-hydroxyl groups in ddAp4ddA greatly hindered their hydrolysis by snake venom phosphodiesterase, asymmetrical dinucleoside tetraphosphatase and by a purified membrane preparation from rat liver. The possibility of using di-2',3'-dideoxynucleoside tetraphosphate (ddNp4ddN) or nucleoside(5')tetraphospho(5')-2',3'-dideoxynucleoside (Np4ddN) as a source of the active retroviral agent ddNTP, for example in HIV infection, is outlined.

Time course of luciferyl adenylate synthesis in the firefly luciferase reaction.

The time course of luciferyl adenylate formation in the reaction catalyzed by firefly luciferase (EC 1.13.12.7) has been followed. The properties of luciferyl adenylate, enzymatically or chemically synthesized, as substrate of luciferase, have been compared. The potential use of luciferyl adenylate for luciferase detection is here proposed.

Labeled adenosine(5')tetraphospho(5')adenosine (Ap4A) and adenosine(5')tetraphospho(5')nucleoside (Ap4N). Synthesis with firefly luciferase.

Labeled dinucleoside polyphosphates are not commercially available, in spite of being important molecules in metabolic regulation. Firefly luciferase (EC 1.13.12.7) is a useful enzyme for the synthesis of adenosine(5')tetraphospho(5')adenosine (Ap4A). As luciferase behaves as a nucleotidase at low ATP concentration, adequate concentrations (higher than 0.1 mM ATP) should be used to obtain a good yield of labeled Ap4A. [32P]Ap4A has also been synthesized from ATP and [32P]PPi. In a first step, [beta, gamma-32P]ATP is generated in a ATP-[32P]PPi exchange reaction catalyzed by luciferase. In a second step, the reaction is supplemented with pyrophosphatase and 32P labeled Ap4A is obtained. Radioactive adenosine(5')tetraphospho(5')nucleoside (Ap4N) can also be synthesized from ATP gamma S and labeled NTP or from low concentrations of labeled ATP and high concentrations of cold NTP. The syntheses of radioactive ApnA and pnA (n > 4) can also be approached with luciferase.

Conversion of adenosine(5') oligophospho(5') adenosines into inosine(5') oligophospho(5') inosines by non-specific adenylate deaminase from the snail Helix pomatia.

Until now, the catabolism of adenosine(5')triphospho(5')adenosine (Ap3A) and adenosine(5')tetraphospho(5')adenosine (Ap4A) has been thought to commence with either hydrolytic or phosphorolytic cleavage of their oligophosphate chains, depending on the organism. Here, we show that in the extracts from the retractile 'foot' of the snail Helix pomatia deamination predominates; the adenosine moieties of these and other adenosine(5')oligophospho(5')adenosines (ApnAs) undergo successive deamination leading, via an inosine(5')oligophospho(5')adenosine (IpnA), to the corresponding inosine(5')oligophospho(5')inosine (IpnI). The reactions are catalyzed by the non-specific adenylate deaminase described earlier (Stankiewicz, A.J. (1983) Biochem. J. 215, 39-44). We describe TLC and HPLC systems which allow the separation of any of the deaminated derivatives from its parent compound; Ap2A, Ap3A, Ap4A or Ap5A. The Km values for these substrates are 20, 22, 32 and 39 microM, respectively, whereas the Km for 5'-AMP is 12 microM. Relative substrate specificities for these compounds amount to 25, 18, 14, 7 and 100. The enzyme was shown also to deaminate phosphonate and thiophosphate analogues of Ap3A.

Uric acid synthesis by rat liver supernatants from purine bases, nucleosides and nucleotides. Effect of allopurinol.

The synthesis of uric acid from purine bases, nucleosides and nucleotides has been measured in reaction mixtures containing rat liver supernatant and each one of the following compounds at 1 mM concentration (except xanthine, 0.5 mM and guanosine and guanine, 0.1 mM). The rates of the reaction, expressed as nanomoles of uric acid synthesized g-1 of wet liver min-1 were: ATP, 10; ADP, 37; AMP, 62; adenosine, 108; adenine 6; adenylosuccinate, 9; IMP 32; inosine, 112; hypoxanthine, 50; GTP, 19; GDP, 19; GMP, 27; guanosine, 34; guanine, 72; XMP, 10; xanthosine, 24; xanthine, 144. These figures divided by 55 correspond to nanomoles of uric acid synthesized min-1 per mg-1 of protein. The rate of synthesis of uric acid obtained with each one of those compounds at 0.1 and 0.05 mM concentrations was also determined. ATP (1 mM) strongly inhibited uric acid synthesis from 0.05 mM AMP (91 per cent) and from 0.05 mM ADP (88 per cent), but not from adenosine. CTP or UTP (1 mM) also inhibited (by more than 90 per cent) the synthesis of uric acid from 0.05 mM AMP. Xanthine oxidase was inhibited by concentrations of hypoxanthine higher than 0.012 mM. The results favour the view that the level of uric acid in plasma may be an index of the energetic state of the organism. Allopurinol, besides inhibiting uric acid synthesis, reduced the rate of degradation of AMP. The ability of crude extracts to catabolize purine nucleotides to uric acid is an important factor to be considered when some enzymes related to purine nucleotide metabolism, particularly CTP synthase, are measured in crude liver extracts.

Adenosine 5'-tetraphosphate and adenosine 5'-pentaphosphate are synthesized by yeast acetyl coenzyme A synthetase.

Yeast (Saccharomyces cerevisiae) acetyl coenzyme A (CoA) synthetase (EC 6.2.1.1) catalyzes the synthesis of adenosine 5'-tetraphosphate (P4A) and adenosine 5'-pentaphosphate (p5A) from ATP and tri- or tetrapolyphosphate (P3 or P4), with relative velocities of 7:1, respectively. Of 12 nucleotides tested as potential donors of nucleotidyl moiety, only ATP, adenosine-5'-O-[3-thiotriphosphate], and acetyl-AMP were substrates, with relative velocities of 100, 62, and 80, respectively. The Km values for ATP, P3, and acetyl-AMP were 0.16, 4.7, and 1.8 mM, respectively. The synthesis of p4A could proceed in the absence of exogenous acetate but was stimulated twofold by acetate, with an apparent Km value of 0.065 mM. CoA did not participate in the synthesis of p4A (p5A) and inhibited the reaction (50% inhibitory concentration of 0.015 mM). At pH 6.3, which was optimum for formation of p4A (p5A), the rate of acetyl-CoA synthesis (1.84 mumol mg-1 min-1) was 245 times faster than the rate of synthesis of p4A measured in the presence of acetate. The known formation of p4A (p5A) in yeast sporulation and the role of acetate may therefore be related to acetyl-CoA synthetase.

Diadenosine 5',5"-P1,P4-tetraphosphate (Ap4A), ATP and catecholamine content in bovine adrenal medulla, chromaffin granules and chromaffin cells.

The level of diadenosine 5',5"-P1-P4-tetraphosphate (diadenosine tetraphosphate or Ap4A), catecholamines, ATP and other nucleotides has been investigated in perchloric acid extracts of bovine adrenal medulla, chromaffin granules and cultured chromaffin cells. As a control, the amount of Ap4A and ATP has also been measured in human blood platelets. The following values (nmol/mg protein) were found in adrenal medulla: Ap4A, 0.019 +/- 0.004; ATP, 109 +/- 11; ADP, 23.8 +/- 5.8; AMP, 11.3 +/- 1.5; p4A, 0.18 +/- 0.08; catecholamines, 460 +/- 57. The level of Ap4A, catecholamines and ATP (nmol/mg protein) found in chromaffin granules and in chromaffin cells were, respectively: (0.15 +/- 0.07; 2175 +/- 99; 531 +/- 66) and (0.22 +/- 0.14; 1143 +/- 277; 222 +/- 53). In all the cases investigated, the ratio catecholamines/ATP and catecholamines/Ap4A were around 5 and in the order of 10(3), respectively. The amount of Ap4A found here, in bovine adrenal medulla, chromaffin granules and chromaffin cells, is two orders of magnitude lower than previously reported.

Effect of diets containing adenosine, guanosine, inosine or xanthosine on the nucleotide content of Artemia. Influence of mycophenolic acid.

Artemia uses the stored diguanosine tetraphosphate as a source of adenine and guanine nucleotides during development from the encysted gastrula to the free swimming larva. Further development of the larvae depends on a dietary source of purine rings. We have investigated the growth of Artemia in axenic cultures supplemented with 0.6 mg ml-1 of adenosine, guanosine, inosine or xanthosine. The total protein and soluble nucleotide content of Artemia grown in the presence of adenosine, guanosine or inosine was very similar, around (2 A260 units and 500 mg protein) and (4 A260 units and 1000 mg protein) after 4 and 6 days of postlarval development, respectively. The nucleotide pattern of those extracts subjected to HPLC were almost identical, the major peaks corresponding to ATP, ADP and AMP. Other nucleotides, not well characterized, were also present in those extracts. Mycophenolic acid (10 micrograms ml-1) inhibited the growth of Artemia (as measured by their protein and soluble nucleotide content) in the presence of adenosine and inosine as the purine source, and had no appreciable effect in the presence of guanosine. A quantitative analysis of the chromatographic peaks obtained from Artemia grown in the presence of any of the three nucleosides +/- mycophenolic acid showed that the effect of the antibiotic on each one of the chromatographic peaks was very similar, suggesting that Artemia, and probably other organisms as well, tend to maintain a balance between all nucleotides and to adjust the overall level to the limiting step(s) in their rates of synthesis/interconversion. Xanthosine was not able to support the development of Artemia.

Specific synthesis of adenosine(5')tetraphospho(5')nucleoside and adenosine(5')oligophospho(5')adenosine (n > 4) catalyzed by firefly luciferase.

Luciferase catalyzes the preferential synthesis of adenosine(5')tetraphospho(5')nucleoside (Ap4N) in the presence of luciferin (LH2), adenosine 5'-[gamma-thio]triphosphate (ATP[gamma S]) and NTP (other than ATP), with very low, or undetectable synthesis of Ap4A or Np4N, because ATP[gamma S] is a good adenylyl donor for the formation of the E-LH2-AMP complex, but a poor adenylyl acceptor from the complex, and NTP, other than ATP, are bad nucleotidyl donors, but good acceptors of the AMP moiety of the E-LH2-AMP complex. Synthesis of the corresponding Ap4N (or Ap5G in the case of p4G were obtained in the presence of ATP[gamma S] and GTP, UTP, CTP, XTP, dTTP, ITP, dGTP, dCTP, dITP, epsilon ATP (epsilon A, N6-ethenoadenosine) or p4G. The yield of synthesis of Ap4N was at least 50% of that theoretically expected. The process can be easily scaled-up, which allows synthesis of at least 1-5 mumol Ap4N. Further evidence for the synthesis of Ap4G from ATP[gamma S] and GTP was obtained by 1H-NMR and 31P-NMR spectroscopy. Synthesis of Ap4N, in yields lower than those above, can also be obtained in the presence of ADP and NTP; synthesis is due to the presence in commercial luciferase of enzymes (adenylate kinase and NDP kinase) that catalyze the synthesis of ATP from ADP and NTP. In the presence of ATP and polyphosphates, luciferase catalyzes the synthesis of a variety of compounds of adenosine 5'-polyphosphates (pnA; n = 3-20 and ApnA; n = 4-16). In the presence of P3 or P4, preferential synthesis of p4A and Ap5A or p5A and Ap6A were obtained, respectively, showing that both polyphosphates accept the adenylyl moiety of the E-LH2-AMP complex. Polyphosphates of chain length 5, 15 and 35 elicited the synthesis of a variety of PnA and ApnA. Ap4A is also split by luciferase in the presence of P3 or P4 (but not in the presence of P5) yielding preferential synthesis of p4A and Ap5A, or p5A and Ap6A, respectively.