Binding of 5'-O-(2-thiodiphosphate) to rat brain membranes is prevented by diadenosine tetraphosphate and correlates with ecto-nucleotide pyrophosphatase phosphodiesterase 1 (NPP1) activity.

The distribution of binding sites for [(35)S]5'-O-(2-thiodiphosphate) ([(35)S]ADPbetaS), a radioligand of P2Y(1,12,13) receptors, and of ecto-nucleotide pyrophosphatase phosphodiesterase activity were analyzed in the rat forebrain. Binding sites for the radilogand are widespreadly distributed in the rat forebrain, showing the highest density in hypothalamus. K(d) values were in the range 1-2 nM.

Biochemical analysis of ecto-nucleotide pyrophosphatase phosphodiesterase activity in brain membranes indicates involvement of NPP1 isoenzyme in extracellular hydrolysis of diadenosine polyphosphates in central nervous system.

Synaptosomes and plasma membranes obtained from rat brain display ectoenzymatic hydrolytic activity responsible for hydrolysis of the neurotransmitter/neuroregulatory nucleotides diadenosine polyphosphates. Intact synaptosomes and plasma and synaptic membranes isolated by sucrose-gradient ultracentrifugation from several brain regions (hypothalamus, hippocampus, temporal cortex, frontal cortex striatum and cerebellum) degraded the fluorogenic substrates diethenoadenosine polyphosphates up to ethenoadenosine as by-product. Purified ectoenzyme cleaved substrates always releasing the mononucleotide moieties ethenoadenosine 5'-monophosphate and the corresponding ethenoadenosine (n-1) 5'-phosphate.

Biochemical and immunochemical characterisation of human diadenosine triphosphatase provides evidence for its identification with the tumour suppressor Fhit protein.

We describe here the purification and characterisation of the human enzyme diadenosine triphosphatase isolated from human platelets and leukocytes, offering biochemical and immunochemical evidence to identify this enzyme with the novel tumour suppressor Fhit protein, a homodimer composed of approximately 17 kDa monomers. It catalyses the Mg(2+)-dependent hydrolysis of diadenosine triphosphate, Ap(3)A, to AMP+ADP. The fluorogenic substrate di-ethenoadenosine triphosphate, epsilon-(Ap(3)A), and Fhit antibodies were used for enzymatic and immunochemical characterisations, respectively.

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.

Ectoenzymatic breakdown of diadenosine polyphosphates by Xenopus laevis oocytes.

Xenopus laevis oocytes exhibit ectoenzymatic activity able to hydrolytically cleave extracellular diadenosine polyphosphates (Ap(n)A). The basic properties of this ectoenzyme were investigated using as substrates di-(1,N(6)-ethenoadenosine) 5',5"'-P(1),P(4)-tetraphospate [epsilon-(Ap(4)A)] and di-(1,N(6)-ethenoadenosine) 5',5"'-P(1),P(5)-pentaphospate [epsilon-(Ap(5)A)], fluorogenic derivatives of Ap(4)A and Ap(5)A, respectively. epsilon-(Ap(4)A) and epsilon-(Ap(5)A) are hydrolysed by folliculated oocytes according to hyperbolic kinetics with K(m) values of 13.

Human diadenosine triphosphate hydrolase: preliminary characterisation and comparison with the Fhit protein, a human tumour suppressor.

Human platelets diadenosine triphosphatase was characterised and compared with the Fhit protein, a human tumour suppressor with diadenosine triphosphatase activity. Both enzymes exhibit similar Km, are similarly activated by Mg2+, Ca2+ and Mn2+, and inhibited by Zn2+ and suramin. However, they are differentially inhibited by Fhit antibodies and exhibit differences in gel-filtration behaviour.

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.

Potent inhibition of specific diadenosine polyphosphate hydrolases by suramin.

The cytosolic enzymes asymmetrical diadenosine tetraphosphate hydrolase (EC 3.6.1.

Characterization of diadenosine polyphosphate transport into chromaffin granules from adrenal medulla.

The transport of diadenosine polyphosphates into chromaffin granules from bovine adrenal medulla has been studied by using the radiolabeled substrate [3H]Ap5A and the fluorescent substrate analog di(1,N6-ethenoadenosine)polyphosphate, epsilon-(Ap(n)A) (n=3-5). The vesicular concentration increase was time dependent and the substrates were not metabolized to any extent during the transport experiments. The saturation curve indicates the existence of kinetic and allosteric cooperativity during Ap(n)A (diadenosine polyphosphates) transport and could be the result of the presence of various affinity states of the transporter with K values of 16 +/- 1 microM and 75 +/- 6 microM, and corresponding Hill numbers of 2 and 4, when epsilon-(Ap4A) was the substrate.

Ecto-enzymatic hydrolysis of diadenosine polyphosphates by cultured adrenomedullary vascular endothelial cells.

We investigated the extracellular degradation of diadenosine polyphosphates (ApnA) by cultured adrenomedullary endothelial cells using fluorogenic analogs of ApnA, the di(1,N6-ethenoadenosine) 5',5"'-P1,Pn-polyphosphates [epsilon-(ApnA)]. Kinetic parameters of epsilon-(ApnA) cleavage and effects of pH, ions, and inhibitors were determined by continuous fluorometric assays, using suspensions of endothelial cells grown on Cytodex-1 microspheres. Ecto-enzyme kinetic parameters for epsilon-(Ap3A), epsilon-(Ap4A), and epsilon-(Ap5A) hydrolysis are as follows: Michaelis-Menten constants of 0.

Diadenosine polyphosphate hydrolase from presynaptic plasma membranes of Torpedo electric organ.

The diadenosine polyphosphate hydrolase present in presynaptic plasma membranes from the Torpedo electric organ has been characterized using fluorogenic substrates of the form di-(1, N6-ethenoadenosine) 5',5'''-P1,Pn-polyphosphate. The enzyme hydrolyses diadenosine polyphosphates (ApnA, where n=3-5), producing AMP and the corresponding adenosine (n-1) 5'-phosphate, Ap(n-1). The Km values of the enzyme were 0.

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.

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.

Use of fluorogenic substrates for detection and investigation of ectoenzymatic hydrolysis of diadenosine polyphosphates: a fluorometric study on chromaffin cells.

A set of procedures to assay and investigate ectoenzymatic hydrolysis of diadenosine polyphosphates (ApnA) in both intact cell or plasma membrane preparations is described. Procedures are based on the use of the fluorogenic ApnA analogs, epsilon-(ApnA), as artificial substrates. It is shown that these fluorogenic analogs behave as excellent substrates of the ectoenzyme present in cultured chromaffin cells.

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.

Extracellular hydrolysis of diadenosine polyphosphates, ApnA, by bovine chromaffin cells in culture.

An ectoenzyme hydrolyzing diadenosine polyphosphates (ApnA) to AMP and Ap(n-1) has been studied in cultured chromaffin cells from bovine adrenal medulla. The KM value for extracellular Ap4A hydrolysis was 2.90 +/- 0.

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.

De novo purine biosynthesis in the crustacean Artemia: influence of salinity and geographical origin.

In vivo studies of the incorporation of [U-14C]glycine into purine nucleotides have established the de novo pathway for purine biosynthesis in Artemia sp. during the early period of larval development. This pathway can be modified by the salt concentration of the incubation media.

De novo biosynthesis and base composition of total and ribosomal ribonucleic acids during early development in Artemia sp.

De novo synthesis of total and ribosomal ribonucleic acids has been studied during the early stages of Artemia sp. development. By in vivo incorporation studies of [14C]HCO3- an increase has been found in both total and ribosomal RNA synthesis post hatching, with a similar distribution of radioactivity and base composition.

Separation of major RNA-derived nucleotides by reversed-phase liquid chromatography.

The effects of pH, ionic strength and amount of methanol in the eluent on the retention of 5'-, 3'- and 2'-ribonucleoside monophosphates on a reversed-phase high-performance liquid chromatographic system are described. The data were used to develop suitable separation protocols for synthetic nucleotide mixtures and applied to the separation of RNA nucleotides derived by alkaline hydrolysis.

A set of procedures for resolving purine compounds by reversed-phase high performance liquid chromatography: application to the study of purine nucleotide and nucleic acid metabolism.

A set of simple procedures for the separation of major purine 5'-ribonucleotides including diguanosine polyphosphates, purine and pyrimidine bases, and 2'- and 3'-nucleotide monophosphates using reversed-phase high-performance liquid chromatography and isocratic elution study of purine nucleotide and nucleic acid biosynthesis in Artemia is presented.

Adenosine transport in bovine chromaffin cells in culture.

Bovine adrenal chromaffin cells in culture have a high capacity and affinity for adenosine uptake with Vmax = 14 +/- 2.4 pmol/10(6) cells/min (133 pmol/mg of protein/min) and Km = 1 +/- 0.2 microM.

Adenosine kinase from bovine adrenal medulla.

Adenosine kinase from bovine adrenal medulla was purified 1600-fold by using ammonium sulfate precipitation, gel filtration and affinity chromatography. Gel filtration yielded a relative molecular mass around 42000 and Michaelis constants were 0.2 microM for adenosine and 20 microM for MgATP.

Purine nucleotide synthesis in adrenal chromaffin cells.

The synthesis of purine nucleotides from the salvage precursors adenine and adenosine, and from the de novo precursors formate and glycine, was studied in isolated adrenal chromaffin cells. Both [8-14C]adenine and [8-14C]adenosine from extracellular medium are effectively incorporated into intracellular nucleotides. [14C]Formate and [U-14C]glycine are also incorporated, but de novo synthesis is clearly lower than synthesis from salvage precursors, although similar to de novo synthesis in liver.

Incorporation of adenosine into nucleotides of chromaffin cells maintained in primary cultures.

Extracellular adenosine was incorporated into nucleotides of bovine chromaffin cells maintained in primary culture. In intact chromaffin tissue, a very low incorporation was found (0.8 pmol/10(6) cells/h at an adenosine concentration of 11.