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PubMed1994

Crystal structure of inositol polyphosphate 1-phosphatase at 2.3-A resolution.

Bovine inositol polyphosphate 1-phosphatase (1-ptase), M(r) = 44,000, is a Mg(2+)-dependent/Li(+)-sensitive enzyme that catalyzes the hydrolysis of the 1-position phosphate from inositol 1,4-bisphosphate and inositol 1,3,4-trisphosphate. We have determined the crystal structure of recombinant bovine 1-ptase in the presence of Mg2+ by multiple isomorphous replacement. The structure is currently refined to an R value of 0.198 for 15,563 reflections within a resolution range of 8.0-2.3 A. 1-Ptase is monomeric in the crystal, consistent with biochemical data, and folds into an alternatively layered alpha/beta/alpha/beta sandwich. The central core of 1-ptase consists of a six-stranded antiparallel beta sheet perpendicular to two parallel three-turn alpha-helices. The beta sheet is flanked by two antiparallel six-turn alpha-helices aligned parallel to the beta sheet, and the central helices are flanked by a five-stranded largely parallel beta sheet. Two neighboring metal binding sites are located in adjacent acidic pockets formed by the intersection of several secondary structure elements including an unusual kink structure formed by the "DPIDST" sequence motif. The fold of 1-ptase is similar to that of two other metal-dependent/Li(+)-sensitive phosphatases, inositol monophosphate phosphatase and fructose 1,6-bisphosphatase despite minimal amino acid identity. Comparison of the active-site pockets of these proteins will likely provide insight into substrate binding and the mechanisms of metal-dependent catalysis and Li+ inhibition.

Amino Acid Sequence↗
PubMed2004

Engineering human Fhit, a diadenosine triphosphate hydrolase, into an efficient dinucleoside polyphosphate synthase.

The putative human tumor suppressor gene FHIT encodes Fhit, the fragile histidine triad protein. Fhit is thought to participate in a signal transduction pathway involving dinucleoside polyphosphates. Fhit catalyzes the Mg2+-dependent hydrolysis of P1-5'-O-adenosine-P3-5'-O-adenosine triphosphate (Ap3A) to AMP and MgADP. Mutation of His96 to glycine disables Fhit as a catalyst for the hydrolysis of phosphoanhydrides such as Ap3A. However, the mutated enzyme H96G-Fhit efficiently catalyzes the synthesis of phosphoanhydride bonds in reactions of nucleoside-5'-phosphimidazolides with nucleoside di- and triphosphates. H96G-Fhit can be employed in the synthesis of a wide range of dinucleoside tri- and tetraphosphates. We here describe the use of H96G-Fhit to catalyze the synthesis of Ap3A, Ap3C, Ap3G, Ap3T, Ap3U, Cp3U, Tp3U, dAp3U, Ap4A, Ap4U, and the fluorescent Ap4etheno-C.

Acid Anhydride Hydrolases↗
PubMed1998

Three-dimensional matrices of calcium polyphosphates support bone growth in vitro and in vivo.

Novel macroporous calcium polyphosphate (CPP) scaffolds, with three-dimensional interconnected structure, were fabricated using a polyurethane sponge method. They were then employed in both in vitro and in vivo assays to examine their suitability as bone tissue engineering scaffolds. In the former, subcultured rat marrow cells were seeded on the scaffolds at 7.0 x 10(5) cells/sample and cultured for 2 wk. Cell-free controls were employed to monitor changes in the scaffold itself. In the in vivo assay, CPP rods were implanted in rat distal femur and recovered after 2 wk. Samples were examined by scanning electron microscopy following freeze-fracturing. Both in vitro and in vivo assays demonstrated the growth of bone within the scaffolds. In vitro, the bone/CPP interface was occupied by a morphologically distinguishable cement line, while in vivo non-mineralized fibrous tissue was seen at the interface together with bone ingrowth into the scaffold microporosity. The morphology of the individual surface grains of the CPP scaffolds employed in vivo changed to a more rounded form, while no change in geometry was observed in the in vitro cell-free group. These preliminary studies indicate that three-dimensional CPPs can be successfully used as scaffolds for bone tissue engineering.

Journal Article↗
PubMed2000

Diadenosine polyphosphates as antagonists of the endogenous P2Y(1) receptor in rat brain capillary endothelial cells of the B7 and B10 clones.

1. Diadenosine polyphosphates (Ap(n)As, n=2 - 7) are considered as stress mediators in the cardiovascular system. They act both via identified P2 purinoceptors and via yet to be characterized receptors. This study analyses the actions of Ap(n)As in clones of rat brain capillary endothelial cells that express P2Y(1) receptors (B10 cells) or both P2Y(1) and P2Y(2) receptors (B7 cells). 2. B10 cells responded to Ap(3)A with rises in intracellular Ca(2+) concentration ([Ca(2+)](i)). This response was prevented by adenosine-3'-phosphate-5'-phosphate, an antagonist of P2Y(1) receptors. It was largely suppressed by a treatment with apyrase VII or with creatine phosphokinase/creatine phosphate to degrade contaminating ADP. 3. Ap(n)As inhibited ADP induced increases in [Ca(2+)](i) mediated by P2Y(1) receptors by shifting ADP concentration-response curves to larger concentrations. Apparent K(i) values were estimated to be 6 microM for Ap(4)A, 10 microM for Ap(5)A and 47 microM for Ap(6)A. Ap(2)A and Ap(3)A were much less active. 4. Ap(n)As were neither agonists nor antagonists of the endogenous P2Y(2) receptor in B7 cells. 5. Ap(n)As are neither agonists nor antagonists of the G(i)-coupled, ADP receptor in B10 cells. 6. The results suggest that most actions of Ap(n)As in B7 and B10 cells can be accounted for by endogenous P2Y(1) receptors. Ap(4)A, Ap(5)A and Ap(6)A are specific antagonists of endogenous Ca(2+)-coupled P2Y(1) receptors.

Adenosine Diphosphate↗
PubMed1997

The phosphatidylinositol polyphosphate 5-phosphatase SHIP and the protein tyrosine phosphatase SHP-2 form a complex in hematopoietic cells which can be regulated by BCR/ABL and growth factors.

We report here that interleukin-3 (IL-3) and erythropoietin (EPO) induce formation of a complex composed of two SH2-containing phosphatases, the tyrosine phosphatase SHP-2 and the SH2 containing inositol 5-phosphatase (SHIP). Both SHP-2 and SHIP are known to be involved in growth factor signal transduction, but their potential interaction in the same pathway is novel. SHIP has previously been shown to associate with SHC, and potentially to be involved in regulating apoptosis. In contrast, in some model systems, SHP-2 has been demonstrated to positively regulate cell growth. Both phosphatases in the complex were tyrosine phosphorylated, and the amount of SHIP coprecipitating with SHP-2 was inversely related to the amount of SHIP coprecipitating with SHC. In hematopoietic cells transformed by the BCR/ABL oncogene, this phosphatase complex was found to be constitutively present with both components heavily tyrosine phosphorylated. Also, other proteins were detected in the complex, including BCR/ABL itself and c-CBL. However, transformation by BCR/ABL was associated with a reduced SHIP protein expression, which could further affect the accumulation of various inositol polyphosphates in these leukemic cells. These data suggest that the function of SHIP and SHP-2 in normal cells are linked and that BCR/ABL alters the function of this signaling complex.

Animals↗
PubMed1988

A novel metal-dye detection system permits picomolar-range h.p.l.c. analysis of inositol polyphosphates from non-radioactively labelled cell or tissue specimens.

A novel complexometric dye- and transition-metal-based post-column detection system for polyanions, called 'metal-dye detection' has been developed. This technique, combined with a new h.p.l.c. separation protocol, permits a direct highly-isomer-selective determination of bis- to poly-phosphorylated non-radioactively labelled compounds in the picomolar range, a sensitivity hitherto unknown for these substances. The application of the technique in the quantitative microanalysis of inositol polyphosphates from milligram amounts of cells or tissue specimens is described. The technique promises to answer hitherto unresolved questions about the role of inositol phosphates, especially those in intact tissues, which are not readily amenable to analysis by radioisotopic techniques.

Animals↗
PubMed1993

Inhibition of iron-catalysed hydroxyl radical formation by inositol polyphosphates: a possible physiological function for myo-inositol hexakisphosphate.

1. The ability of myo-inositol polyphosphates to inhibit iron-catalysed hydroxyl radical formation was studied in a hypoxanthine/xanthine oxidase system [Graf, Empson and Eaton (1987) J. Biol. Chem. 262, 11647-11650]. Fe3+ present in the assay reagents supported some radical formation, and a standard assay, with 5 microM Fe3+ added, was used to investigate the specificity of compounds which could inhibit radical generation. 2. InsP6 (phytic acid) was able to inhibit radical formation in this assay completely. In this respect it was similar to the effects of the high affinity Fe3+ chelator Desferral, and dissimilar to the effects of EDTA which, even at high concentrations, still allowed detectable radical formation to take place. 3. The six isomers of InsP5 were purified from an alkaline hydrolysate of InsP6 (four of them as two enantiomeric mixtures), and they were compared with InsP6 in this assay. Ins(1,2,3,4,6)P5 and D/L-Ins(1,2,3,4,5)P5 were similar to InsP6 in that they caused a complete inhibition of iron-catalysed radical formation at > 30 microM. Ins(1,3,4,5,6)P5 and D/L-Ins(1,2,4,5,6)P5, however, were markedly less potent than InsP6, and did not inhibit radical formation completely; even when Ins(1,3,4,5,6)P5 was added up to 600 microM, significant radical formation was still detected. Thus InsP5s lacking 2 or 1/3 phosphates are in this respect qualitatively different from InsP6 and the other InsP5s. 4. scyllo-Inositol hexakisphosphate was also tested, and although it caused a greater inhibition than Ins(1,3,4,5,6)P5, it too still allowed detectable free radical formation even at 600 microM. 5. We conclude that the 1,2,3 (equatorial-axial-equatorial) phosphate grouping in InsP6 has a conformation that uniquely provides a specific interaction with iron to inhibit totally its ability to catalyse hydroxyl radical formation; we suggest that a physiological function of InsP6 might be to act as a 'safe' binding site for iron during its transport through the cytosol or cellular organelles.

Ferric Compounds↗
PubMed1994

The intracellular distribution of inositol polyphosphates in HL60 promyeloid cells.

1. HL60 promyeloid cells contain high intracellular concentrations of inositol polyphosphates, notably inositol 1,3,4,5,6-pentakisphosphate (InsP5) and inositol hexakisphosphate (InsP6). To determine their intracellular location(s), we studied the release of inositol (poly)phosphates, of ATP, and of cytosolic and granule-enclosed enzymes from cells permeabilized by four different methods. 2. When cells were treated with digitonin, all of the inositol phosphates were released in parallel with the cytosolic constituents. Most of the InsP5 and InsP6 was released before significant permeabilization of azurophil granules. 3. Similar results were obtained from cells preloaded with ethylene glycol and permeabilized by osmotic lysis. 4. Electroporation at approximately 500 V/cm caused rapid release of free inositol. Higher field strengths provoked release of most of the ATP, InsP5 and InsP6, but only slight release of the intracellular enzymes. Multiple discharges released approximately 80-90% of total InsP5 and InsP6. In the absence of bivalent-cation chelators, InsP5 and InsP6 were released less readily than ATP. 5. Treatment of cells with Staphylococcus aureus alpha-toxin caused quantitative release of inositol and ATP, without release of intracellular enzymes. However, inositol phosphates were released much less readily than inositol or ATP. Even after prolonged incubation with a high concentration of alpha-toxin, only approximately 50-70% of InsP2, InsP3 and InsP4 and < or = 20% of InsP5 and InsP6 were released, indicating that the high charge or large hydrated radius of InsP5 and InsP6 might limit their release through small toxin-induced pores. 6. These results indicate that most intracellular inositol metabolites are either in, or in rapid exchange with, the cytosolic compartment of HL60 cells. However, they leave open the possibility that a small proportion of cellular InsP5 and InsP6 (< or = 10-20%) might be in some intracellular bound form.

Adenosine Triphosphate↗
PubMed1995

Comparison of the activities of a multiple inositol polyphosphate phosphatase obtained from several sources: a search for heterogeneity in this enzyme.

A multiple inositol polyphosphate phosphatase (formerly known as inositol 1,3,4,5-tetrakisphosphate 3-phosphatase) was purified approx. 22,000-fold from rat liver. The final preparation migrated on SDS/PAGE as a doublet with a mean apparent molecular mass of 47 kDa. Upon size-exclusion chromatography, the enzyme was eluted with an apparent molecular mass of 36 kDa. This enzyme was approximately evenly distributed between the 'rough' and 'smooth' subfractions of endoplasmic reticulum. There was a 20-fold range of specific activities of this phosphatase in CHAPS-solubilized particulate fractions prepared from the following rat tissues: liver, heart, kidney, testis and brain. However, each of these extracts contained different amounts of endogenous inhibitors of enzyme activity. After removal of these inhibitors by MonoQ anion-exchange chromatography, there was only a 2.5-fold range of specific activities; kidney contained the most and brain contained the least. We prepared and characterized polyclonal antiserum to the hepatic phosphatase, which immunoprecipitated 85-100% of both particulate and soluble phosphatase activities. The antiserum also immunoprecipitated, with equivalent efficacy, CHAPS-solubilized phosphatase activities from heart, kidney, testis, brain and erythrocytes (all prepared from rat). Our data strengthen the case that the function of the mammalian phosphatase is unrelated to the metabolism of Ca(2+)-mobilizing cellular signals. The CHAPS-solubilized phosphatase from turkey erythrocytes was not immunoprecipitated by the polyclonal antiserum, and is therefore an isoform that is structurally distinct, and possibly functionally unique.

Animals↗
PubMed1995

Effects of aluminium on the hepatic inositol polyphosphate phosphatase.

There is speculation that some of the toxic effects of Al3+ may originate from it perturbing inositol phosphate/Ca2+ signalling. For example, in permeabilized L1210 mouse lymphoma cells, 10-50 microM Al3+ activated Ins(1,3,4,5)P4-dependent Ca2+ mobilization and Ins(1,3,4,5)P4 3-phosphatase activity [Loomis-Husselbee, Cullen, Irvine and Dawson (1991) Biochem. J. 277, 883-885]. Ins(1,3,4,5)P4 3-phosphatase activity is performed by a multiple inositol polyphosphate phosphatase (MIPP) that also attacks Ins(1,3,4,5,6)P5 and InsP6 [Craxton, Ali and Shears (1995) Biochem. J. 305, 491-498]: 5-50 microM Al3+ increased MIPP activity towards both Ins(1,3,4,5)P4 (by 30%) and Ins(1,3,4,5,6)P5 (by up to 500%), without affecting metabolism of InsP6. Higher concentrations of Al3+ inhibited metabolism of all three substrates, and in the case of InsP6, Al3+ altered the pattern of accumulating products. When 1-50 microM Al3+ was present, InsP6 became a less effective inhibitor of Ins(1,3,4,5)P4 3-phosphatase activity; this effect did not depend on the presence of cellular membranes, contrary to a previous proposal. The latter phenomenon largely explains how, in a cell-free system where Ins(1,3,4,5)P4 3-phosphatase is inhibited by endogenous InsP6, the addition of Al3+ can apparently increase the enzyme activity. However, there was no effect of either 10 or 25 microM Al3+ (in either the presence or absence of apotransferrin) on inositol phosphate profiles in either Jurkat E6-1 lymphoma cells or AR4-2J pancreatoma cells.

Aluminum↗
PubMed1996

Thyroid-stimulating hormone rapidly stimulates inositol polyphosphate formation in FRTL-5 thyrocytes without activating phosphoinositidase C.

The thyroid-stimulating hormone (TSH) receptor is widely regarded as one of a limited number of G-protein-coupled receptors that activate both adenylate cyclase and phosphoinositidase C (PIC) via G-proteins, but the existing experimental evidence for TSH-stimulated PtdIns(4,5)P2 hydrolysis remains inconclusive. We have compared the effects of TSH and of ATP (acting via P2-purinergic receptors) on the inositol lipids and polyphosphates of [2-3H]inositol-labelled FRTL-5 rat thyroid cells. ATP initiated a rapid decrease in 3H-labelled PtdIns4P and PtdIns(4,5)P2, whereas TSH did not. Stimulation with ATP and, less consistently, with noradrenaline (acting via alpha-adrenergic receptors) provoked rapid formation of Ins(1,4,5)P3, Ins(1,3,4,5)P4, Ins(1,3,4)P3 and Ins(1,4)P2, confirming activation of PtdIns(4,5)P2 hydrolysis. No concentration of TSH provoked detectable accumulation of Ins(1,4,5)P3 or Ins(1,4)P2 during the first few minutes of stimulation. However, an InsP3 [with the chromatographic properties of Ins(1,3,4)P3] and two InsP4 isomers [neither of which was Ins(1,3,4,5)P4] accumulated quickly in TSH-stimulated cells. ATP immediately provoked a large increase in intracellular calcium concentration ([Ca2+]i) in Indo 1-AM-loaded cells. TSH provoked a small and delayed [Ca2+]i elevation in only some experiments. We therefore confirm that activation of P2-purinergic receptors and alpha 1-adrenergic receptors provokes PIC activation, an accumulation of Ins(1,4,5)P3 and its metabolites and rapid [Ca2+]i mobilization in FRTL-5 cells. By contrast, TSH provokes no rapid PIC-catalysed PtdIns(4,5)P2 hydrolysis or immediate [Ca2+]i mobilization. These results fail to support the widespread view that the TSH receptor of FRTL-5 cells signals, in part, through PIC activation. Our results suggest that TSH activates another, still undefined, mechanism that causes accumulation of an InsP3 and two isomers of InsP4.

Adenosine Triphosphate↗
PubMed1997

Diadenosine polyphosphate-stimulated gluconeogenesis in isolated rat proximal tubules.

Diadenosine polyphosphates released into the extracellular environment influence a variety of metabolic and other cellular activities in a wide range of target tissues. Here we have studied the impact of these novel nucleotides on gluconeogenesis in isolated rat proximal tubules. Gluconeogenesis was stimulated following exposure of isolated proximal tubules to a range of adenine-containing nucleotides including ADP, ATP, Ap3A, Ap4A, Ap5A and Ap6A. The concentration-dependence of ATP-, Ap3A- and Ap4A-mediated stimulation of gluconeogenesis was similar and was consistent with a role for these agents in the physiological control of renal metabolism. Nucleotide-stimulated gluconeogenesis was diminished in the presence of agents that interfere with phospholipase C activation or intracellular Ca2+ metabolism, indicative of a role for polyphosphoinositide-mediated Ca2+ mobilization in the mechanism of action of ATP, Ap3A and Ap4A. The characteristics of binding of [2-3H]Ap4A to renal plasma-membrane preparations suggest that Ap4A mediates its effects on proximal tubule gluconeogenesis via interaction with P2y-like purinoceptor(s) also recognized by extracellular ATP.

Adenine Nucleotides↗
PubMed2007

Inositol polyphosphate kinases: regulators of nuclear function.

Recent work has uncovered roles for inositide signalling pathways downstream of phospholipase C activation and inositol 1,4,5-trisphosphate in the regulation of nuclear processes including gene expression, mRNA export and DNA metabolism. The identification of several IPKs (inositol polyphosphate kinases) has renewed interest in the cellular roles of inositol tetra-, penta-, hexa- and pyro-phosphates. Discoveries of inositide receptors and novel mechanisms of inositide action have provided important insights into how such messengers couple to nuclear machinery. In this chapter, we discuss the IPK family members and the nuclear processes that their inositide products regulate.

Animals↗
PubMed1997

Diadenosine polyphosphates induce intracellular Ca2+ mobilization in human neutrophils via a pertussis toxin sensitive G-protein.

The diadenosine polyphosphates diadenosine 5',5"'-P1,P3-triphosphate (Ap3A), diadenosine 5',5"'-P1,P4-tetraphosphate (Ap4A), diadenosine 5',5"'-P1,P5-pentaphosphate (Ap5A) and diadenosine 5',5"'-P1,P6-hexaphosphate (Ap6A) all stimulated increases in intracellular Ca2+ in human neutrophils. Maximal increases in intracellular Ca2+ of 650 nM were obtained at dinucleotide concentrations of 500-700 microM. These increases in intracellular, Ca2+ were completely abolished by pre-treatment of the neutrophils with pertussis toxin and were hardly affected when the extracellular buffer was devoid of Ca2+. On the other hand, adenosine triphosphate (ATP) could stimulate much greater increases in intracellular Ca2+ (up to 1.1 microM) at much lower concentrations (half maximal responses obtained at around 5 microM ATP). Receptor de-sensitization experiments indicate that human neutrophils may possess two types of P2-purinoceptors. The first of these may bind ATP (but not the dinucleotides) with high affinity whilst the second may bind the dinucleotides with lower affinity and also bind ATP.

Adenosine Triphosphate↗
PubMed2003

Arg82p is a bifunctional protein whose inositol polyphosphate kinase activity is essential for nitrogen and PHO gene expression but not for Mcm1p chaperoning in yeast.

In Saccharomyces cerevisiae, the synthesis of inositol pyrophosphates is essential for vacuole biogenesis and the cell's response to certain environmental stresses. The kinase activity of Arg82p and Kcs1p is required for the production of soluble inositol phosphates. To define physiologically relevant targets of the catalytic products of Arg82p and Kcs1p, we used DNA microarray technology. In arg82delta or kcs1delta cells, we observed a derepressed expression of genes regulated by phosphate (PHO) on high phosphate medium and a strong decrease in the expression of genes regulated by the quality of nitrogen source (NCR). Arg82p and Kcs1p are required for activation of NCR-regulated genes in response to nitrogen availability, mainly through Nil1p, and for repression of PHO genes by phosphate. Only the catalytic activity of both kinases was required for PHO gene repression by phosphate and for NCR gene activation in response to nitrogen availability, indicating a role for inositol pyrophosphates in these controls. Arg82p also controls expression of arginine-responsive genes by interacting with Arg80p and Mcm1p, and expression of Mcm1-dependent genes by interacting with Mcm1p. We show here that Mcm1p and Arg80p chaperoning by Arg82p does not involve the inositol polyphosphate kinase activity of Arg82p, but requires its polyaspartate domain. Our results indicate that Arg82p is a bifunctional protein whose inositol kinase activity plays a role in multiple signalling cascades, and whose acidic domain protects two MADS-box proteins against degradation.

Gene Expression Regulation, Fungal↗
PubMed2000

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).

Adenosine Monophosphate↗
PubMed2001

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 13.4 and 12.0 microM and Vmax values of 4.8 and 5.5 pmol per oocyte per min, respectively. The ectoenzyme is activated by Ca(2+) and Mg(2+), reaches maximal activity at pH 8--9 and is inhibited by suramin. Defolliculated oocytes also hydrolyse both substrates with similar K(m) values but V(max) values are approximately doubled with respect to folliculated controls. Chromatographic analysis indicates that extracellular epsilon-(Ap(4)A) and epsilon-(Ap(5)A) are first cleaved into 1,N(6)-ethenoAMP (epsilon-AMP) + 1,N(6)-ethenoATP (epsilon-ATP) and epsilon-AMP + 1,N(6)-ethenoadenosine tetraphosphate (epsilon-Ap(4)), respectively, which are catabolized to 1,N(6)-ethenoadenosine (epsilon-Ado) as the end product by folliculated oocytes. Denuded oocytes, however, show a drastically reduced rate of epsilon-Ado production, epsilon-AMP being the main end-product of extracellular epsilon-(Ap(n)A) catabolism. Results indicate that, whereas the Ap(n)A-cleaving ectoenzyme appears to be located mainly in the oocyte, ectoenzymes involved in the dephosphorylation of mononucleotide moieties are located mainly in the follicular cell layer.

Adenosine↗
PubMed1995

Dopamine receptor blockade inhibits the amphetamine-induced release of diadenosine polyphosphates, diadenosine tetraphosphate and diadenosine pentaphosphate, from neostriatum of the conscious rat.

The diadenosine polyphosphates diadenosine tetraphosphate (Ap4A) and diadenosine pentaphosphate (Ap5A) are costored with ATP and released in a calcium-dependent manner from neural preparations in vitro. By means of a push-pull perfusion system, samples from conscious rat were collected from the caudate putamen area, and nucleotide compounds were analyzed by HPLC. The adenine dinucleotides were not detectable before systemic amphetamine injection. The maximal levels were reached 20 min after injection, independently of the dose. The EC50 values for amphetamine-induced release of dinucleotides were 2.04 +/- 0.15 and 2.43 +/- 0.36 mg/kg for Ap4A and Ap5A, respectively. Amphetamine doses higher than 5 mg/kg did not increase the dinucleotide release, the maximal values being 12.9 +/- 0.9 and 11.5 +/- 0.9 pmol/fraction for Ap4A and Ap5A, respectively, which corresponds with 64.5 and 57.5 nM in the samples. Adenosine and AMP were present in push-pull samples from rat brain under basal conditions. Their levels were 15 pmol/fraction (75 nM) and 50 pmol/fraction (250 nM) for adenosine and AMP, respectively. A significant increase was obtained for both compounds after amphetamine injection. The adenosine increase reached 45 pmol/sample (225 nM), which was 200% of the basal value 20 min after the stimulant administration. The increase at other times was not significant. The AMP levels increased significantly from 10 to 50 min. The maximal level was reached 20 min after amphetamine injection, with 150 pmol/fraction (750 nM), which represents a 200% increase with respect to the basal level. The adenine dinucleotide release was blocked by the dopamine receptor antagonist haloperidol, which returned the levels to the control basal values.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine↗