Pedro Rotllán

Characterization and quantification of diadenosine hexaphosphate in chromaffin cells: Granular storage and secretagogue-induced release

The presence of diadenosine hexaphosphate (Ap6A) in chromaffin cells is described. The characterization of Ap6A has been accomplished by HPLC techniques, using three different elution conditions, rechromatography, and coelution with standards. Treatment with phosphodiesterase from Crotalus durissus produced AMP and adenosine pentaphosphate. The HPLC techniques described allowed the quantification of Ap6A in the picomole range. Chromaffin granules store Ap6A in a quantity of 48.5 +/- 9.7 nmol/mg protein, with a molar ratio ATP/Ap6A of 27. In chromaffin cells the Ap6A value was 1.46 +/- 0.32 nmol/10(6) cells. Diadenosine hexaphosphate was released from chromaffin cells by the action of carbachol and a value of 64 +/- 15 pmol/10(6) cells was obtained, which represents 4-5% of the total cellular content.

Suramin - a powerful inhibitor of neural ecto-diadenosine polyphosphate hydrolase

The neural ecto‐diadenosine polyphosphate hydrolase (ecto‐ApnAase) from plasma membranes of Torpedo synaptic terminals is inhibited by suramin. This study was carried out by discontinuous h.p.l.c. and continuous fluorometric methods. The concentration‐dependence studies showed a non‐competitive mechanism for suramin in the Dixon plot, with a Ki value of 1.79 ± 0.03 μm with respect to ɛ‐(Ap3A) as the substrate and 1.69 ± 0.05 μm and 1.86 ± 0.06 μm for ɛ‐(Ap4A) and ɛ‐(Ap5A) respectively. These results indicate that suramin could be a base compound inhibiting ecto‐ApnAase and providing an alternative way of studying the pharmacology of diadenosine polyphosphate receptors.

Specific dinucleoside polyphosphate cleaving enzymes from chromaffin cells: a fluorimetric study☆

This article presents a fluorimetric study of the main properties of the enzymes dinucleoside tetraphosphate (asymmetrical) hydrolase or dinucleoside tetraphosphatase (Ap4Aase, EC 3.6.1.17) and dinucleoside triphosphate hydrolase or dinucleoside triphosphatase (Ap3Aase, EC 3.6.1.29), both present in adrenal medulla cytosolic extracts. Diethenoadenosine polyphosphates, epsilon-(ApnA), are used as artificial fluorogenic substrates. Ap4Aase exhibits a molecular mass around 20 kDa and neutral optimum pH (7.0-7.5). It requires Mg2+ and preferentially hydrolyzes substrates with four phosphate groups. Km for epsilon-(Ap4A) is 1.3 microM and Ki for Ap4A and Gp4G are 1 and 0.2 microM respectively. Km for Ap4A determined by HPLC is 1.6 microM. epsilon-(Ap5A) and epsilon-(Ap6A) are hydrolyzed at reduced rates. This enzyme is inhibited by Zn2+, F- and very strongly by Ap4 and epsilon-Ap4. Ca2+ cannot replace Mg2+, but behaves as inhibitor in its presence. The substrate analogs dinucleoside triphosphates Ap3A, G;3G, m7Gp3G and m7Gp3A and the periodate-oxidized nucleotides o-(Ap4A), o epsilon-(Ap4A), o-Ap4 and o epsilon-Ap4 behave as inhibitors. Ap3Aase exhibits a molecular mass around 30 kDa and neutral optimum pH (7.0-7.5). It requires Mg2+ or Ca2+, but retains a low measurable activity around 10% in the absence of these divalent cations. It only hydrolyzes substrates with three phosphate groups. Km for epsilon-(Ap3A) is 11 microM and Ki for Ap3A and Gp3G are 20 and 22 microM, respectively. Km for Ap3A determined by HPLC is 16 microM. m7Gp3G and m7Gp3A are also good substrates for triphosphatase.

Receptor binding properties of di (1,N6-ethenoadenosine) 5′, 5′′′-P1, P4-tetraphosphate and its modulatory effect on extracellular glutamate levels in rat striatum

Our aim was to investigate the neuromodulatory role of diadenosine tetraphosphate (Ap(4)A). Ap(4)A-binding sites were detected in striatum and hippocampus membranes using [(35)S]-ADP beta S as radioligand and Ap(4)A and epsilon-(Ap(4)A), di-ethenoadenosine tetraphosphate, as displacers. Effects of epsilon-(Ap(4)A) on extracellular glutamate levels were studied using intracerebral perfusion. Both areas contain high-affinity binding sites for [(35)S]-ADP beta S with K(d) values in the low nM range. [(35)S]-ADP beta S binding was displaced by Ap(4)A and epsilon-(Ap(4)A). At 1 and 10 microM doses, epsilon-(Ap(4)A) markedly decreased glutamate levels in the striatum. The possibility of Ap(4)A acting as an endogenous modulator of excitatory neurotransmission is discussed.

Potent inhibition of specific diadenosine polyphosphate hydrolases by suramin

The cytosolic enzymes asymmetrical diadenosine tetraphosphate hydrolase (EC 3.6.1.17, Ap4Aase) and diadenosine triphosphate hydrolase (EC 3.6.1.29, Ap3Aase) are inhibited competitively by suramin. Ap4Aase and Ap3Aase were assayed in cytosolic rat brain extracts using fluorogenic analogues of the respective substrates diadenosine tetraphosphate (Ap4A) and diadenosine triphosphate (Ap3A). K i values for suramin as inhibitor of Ap4Aase and Ap3Aase were 5×10−6 M and 3×10−7 M, respectively. Results indicate that suramin or suramin‐like derivatives may be useful tools to investigate diadenosine polyphosphate cleaving enzymes and that the intracellular diadenosine polyphosphate metabolism may be a pharmacological target of suramin with biological and clinical implications.

Fluorimetric detection of enzymatic activity associated with the human tumor suppressor Fhit protein.

The human tumor suppressor Fhit protein exhibits diadenosine triphosphatase activity, hydrolyzing Ap(3)A to AMP and ADP. We report that Fhit protein efficiently cleaves the fluorogenic Ap(3)A analog diethenoadenosine triphosphate giving support to establish a simple fluorimetric assay for quantification of Fhit enzyme. Fluorimetric assays were initially tested to demonstrate that diethyl pyrocarbonate and suramin inhibit Fhit enzyme.

Salvage and interconversion of purines in developing Artemia

Incorporation of the radiolabelled purine bases adenine, guanine and hypoxanthine into acid soluble fraction, RNA and DNA nucleotides during the early larval development of Artemia sp. was studied. Adenine was the best precursor and guanine the poorest. The adenine phosphoribosyltransferase (APRT) activity was considerably higher than that of hypoxanthine-guanine phosphoribosyltransferase (HGPRT) and these activities did not significantly change throughout larval development. The pattern of purine interconversion was dependent on naupliar age. Conversion of [14C]adenine and [14C]hypoxanthine into guanine nucleotides increased with time of development. However, the conversion of [14C]guanine into [14C]adenine nucleotides was very low.