María A. Günther Sillero

Diadenosine tetraphosphate is co-released with ATP and catecholamines from bovine adrenal medulla.

Abstract: Secretion of adenosine(5′)tetraphospho(5′)adenosine (Ap4A) and ATP from perfused bovine adrenal glands stimulated with acetylcholine or elevated potassium levels was measured and compared with that of catecholamines. We have found a close correlation between the release of Ap4A and catecholamines elicited with all the secretagogues used in the presence of either Ca2+ or Ba2+, suggesting co‐release of both constituents from the chromaffin granules. By contrast, ATP secretion, as measured with luciferase, showed a significantly different time course regardless of the secretagogue used. ATP secretion consistently decreased after 1‐2 min of stimulation at a time when Ap4A and catecholamine secretions were still increasing. Measures of degradation of injected [3H]ATP to the gland during stimulation showed little difference in the level of uptake or decomposition of ATP throughout the pulse. However, a reexamination of ATP secretion by monitoring its products of degradation (AMP, adenosine, and inosine) by HPLC techniques showed that Ap4A, ATP, and catecholamines are indeed secreted in parallel from the perfused adrenal gland.

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

It was previously assumed that E·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·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·L‐AMP complex and yielding L‐CoA.

Diguanosinetetraphosphate guanylohydrolase in Artemia salina

Abstract 1. 1. The diguanosinetetraphosphate guanylohydrolase (EC 3.6.1.17) of Artemia salina has been found to be located in the cytosol while its substrate, diguanosine tetraphosphate, is in the 700 × g sediment. 2. 2. Two spectrophotometric methods have been developed to study this enzyme. One is based in the evaluation of one of the products, GTP, coupled to the auxiliary enzymes phosphoglycerate kinase/glyceraldehyde-3-phosphate dehydrogenase. The other method is based on the hyperchromicity observed at 252 nm by the splitting of diguanosine tetraphosphate. 3. 3. With a partially purified preparation, the following enzymatic properties have been found: K m for diguanosine tetraphosphate, 5 μM. GMP, GDP, GTP and ATP were competitive inhibitors of the reaction with K i values of 24, 56, 14 and 30 μM respectively. 4. 4. Even lower values were obtained with the guanosine 5′-tetraphosphate and adenosine 5′-tetraphosphate, which were 0.006 and 0.13 μM, respectively. These rather low K i values suggest a possible role for these compounds as metabolic regulators.

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

Abstract: 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 µM, 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), Ap4εA (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 µM, 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).

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 Pi 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 Vmax and Km 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–µm concentrations of ATP on the metabolism of AMP.

Uridine 5′-polyphosphates (p4U and p5U) and uridine(5′)polyphospho(5′)nucleosides (UpnNs) 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 (P3), tetrapolyphosphate (P4), nucleoside triphosphates (p3Ns) and nucleoside 5′‐polyphosphates (p4Ns) forming uridine 5′‐tetraphosphate (p4U), uridine 5′‐pentaphosphate (p5U) and dinucleotides, such as Ap4U, Cp4U, Gp4U, Up4U, Ap5U and Gp5U. 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).

Synthesis of bisphosphonate derivatives of ATP by T4 RNA ligase

T4 RNA ligase catalyzes the synthesis of ATP β,γ‐bisphosphonate analogues, using the following substrates with the relative velocity rates indicated between brackets: methylenebisphosphonate (pCH2p) (100), clodronate (pCCl2p) (52), and etidronate (pC(OH)(CH3)p) (4). The presence of pyrophosphatase about doubled the rate of these syntheses. Pamidronate (pC(OH)(CH2–CH2–NH2)p), and alendronate (pC(OH)(CH2–CH2–CH2–NH2)p) were not substrates of the reaction. Clodronate displaced the AMP moiety of the complex E‐AMP in a concentration dependent manner. The K m values and the rate of synthesis (k cat) determined for the bisphosphonates as substrates of the reaction were, respectively: methylenebisphosphonate, 0.26 ± 0.05 mM (0.28 ± 0.05 s−1); clodronate, 0.54 ± 0.14 mM (0.29 ± 0.05 s−1); and etidronate, 4.3 ± 0.5 mM (0.028 ± 0.013 s−1). In the presence of GTP, and ATP or AppCCl2p the relative rate of synthesis of adenosine 5′,5‴‐P1,P4‐tetraphosphoguanosine (Ap4G) was around 100% and 33%, respectively; the methylenebisphosphonate derivative of ATP (AppCH2p) was a very poor substrate for the synthesis of Ap4G. To our knowledge this report describes, for the first time, the synthesis of ATP β,γ‐bisphosphonate analogues by an enzyme different to the classically considered aminoacyl‐tRNA synthetases.

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.

Synthesis of (di)nucleoside polyphosphates by the ubiquitin activating enzyme E1

Previous work from this laboratory had shown that ligases may catalyze the synthesis of (di)nucleoside polyphosphates. Here, we show that one of the enzymes of the proteasome system (E1 or the ubiquitin (Ub) activating enzyme, EC 6.3.2.19) catalyzes very effectively (k cat = 0.29 ± 0.05 s−1) the transfer of AMP from the E–AMP–ubiquitin complex to tripolyphosphate or tetrapolyphosphate with formation of adenosine tetra‐ or pentaphosphate (p4A or p5A), respectively. Whereas the concomitant formation of AMP is stimulated by the presence of dithiothreitol in a concentration dependent manner, the synthesis of p4A is only slightly inhibited by this compound. Previous treatment of the enzyme (E1) with iodoacetamide inhibited only partially the synthesis of p4A. p4A can substitute for ATP as substrate of the reaction to generate the ubiquityl adenylate complex. A small amount of diadenosine pentaphosphate (Ap5A) was also synthesized in the presence of p4A.