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PubMed1979

Co-binding studies on Hb M Iwate. Allostery of a T state haemoglobin.

The mutant haemoglobin Hb M Iwate alpha 2Mmet87His leads to Tyr beta 2, is characterized by a stable T structure and a low ligand affinity. Sigmoidal CO-binding isotherms of symmetrical shape with Hill coefficients of n = 1.4 at pH 6 to n = 1.9 at pH 10 and the differences in the mean affinity (PCO(1/2)) and the affinity of the first ligand-binding beta subunit (1/L1 greater than Pco(1/2)) are the evidence for the cooperativity. The comparison of the Bohr effects of the two valency hybrid states (alpha 2Mmet beta met beta deoxy alpha 2Mmet beta 2deoxy) in the absence of and in the presence of polyphosphates leads to an indirect proof of pH-dependent subunit-subunit interaction. Inositol hexaphosphate-binding suppresses cooperativity in the pH range 5.5-8 (n = 1). Above pH 8 hte cooperativity increases to a final value of n = 1.9 at pH greater than 10, which is identical to that of stripped Hb M Iwate. The CO binding to the first binding site exhibits a Bohr effect. Polyphosphate anions have no influence on the CO binding of the first binding site. The heterotropic effects are discussed as intrachain effects (Bohr effect of the first binding site) and interchain effects (Bohr effect of Pco(1/2); influence of polyphosphates).

Allosteric Regulation↗
PubMed1993

Increase in inositol tris-, pentakis- and hexakisphosphates by high K+ stimulation in cultured rat cerebellar granule cells.

Effects of high K+ stimulation on inositol polyphosphate accumulations and intracellular free calcium concentration ([Ca2+]i) were investigated in cultured rat cerebellar granule cells. When the [3H]inositol-labelled cells were stimulated with KCl, concentration-dependent accumulations of [3H]Ins(1,4,5)P3, [3H]InsP5 and [3H]InsP6 were observed. Nifedipine (3 microM), a calcium channel antagonist, inhibited the high (KCl, 90 mM) K(+)-induced accumulations of these inositol polyphosphates. In Ca(2+)-depleted and EGTA-containing (0.1 mM) medium, the high K(+)-induced inositol polyphosphate accumulation were completely inhibited. Similar results were also observed in the case of [Ca2+]i. These results suggest that the rise in [Ca2+]i caused by activation of voltage-dependent calcium channels plays an important roles in the high K(+)-induced accumulation of [3H]Ins(1,4,5)P3, [3H]InsP5 and [3H]InsP6 in cultured rat cerebellar granule cells.

Animals↗
PubMed1987

Activation of polyphosphoinositide phospholipase C by guanosine 5'-O-(3-thio)triphosphate and fluoroaluminate in membranes prepared from a human T cell leukemia line, JURKAT.

Polyphosphoinositide hydrolysis was studied in membranes prepared from a human T cell leukemia line, JURKAT, prelabeled with myo-[2-3H]inositol. The formation of inositol bis- and trisphosphates was stimulated in a buffer with 110 nM free Ca2+ with a nonhydrolyzable GTP analogue, GTP gamma S, and NaF plus AlCl3 in a time- and concentration-dependent manner. GTP gamma S and NaF-AlCl3 had no significant effect on the inositol monophosphate level. AlCl3 enhanced the NaF-stimulated release of inositol polyphosphates. Optimum concentrations of NaF and AlCl3 produced 1.5-fold more inositol polyphosphates than that produced by optimum concentration of GTP gamma S. OKT3 monoclonal antibody, an antibody against the T-cell receptor complex, did not stimulate the inositol polyphosphate formation by JURKAT membranes even in the presence of GTP, although the antibody at the concentrations used markedly stimulated the hydrolysis of polyphosphoinositides in intact JURKAT cells.

Aluminum↗
PubMed1994

Phosphoinositide hydrolysis in Ki-ras-transformed fibroblasts stimulated by platelet-derived growth factor and bradykinin.

Signal transduction in response to platelet-derived growth factor (PDGF)-BB and bradykinin (BK) have been examined by measuring inositol polyphosphate formation in NIH3T3 fibroblast and v-Ki-ras-transformed NIH3T3 fibroblast (DT). The PDGF-induced inositol polyphosphate formation in NIH3T3 was greater than that in DT cells, in which autophosphorylation of PDGF receptor and tyrosine phosphorylation of phospholipase C (PLC)-gamma 1 were suppressed when examined by immunoblotting with anti-phosphotyrosine antibody. On the other hand, BK-stimulation produced a much higher level of inositol polyphosphate in DT cells which have a greater number of BK receptors. These results indicate that in Ki-ras transformed cells the decrease (caused by PDGF) and the increase (caused by BK) in phosphoinositide hydrolysis are due to the defective autophosphorylation of PDGF receptors leading to a reduction in PLC-gamma 1 tyrosine phosphorylation and the overexpression of BK receptors, respectively.

3T3 Cells↗
PubMed1990

A 31P nuclear magnetic resonance study of Crithidia luciliae.

31P NMR has been used to observe phosphorus-containing compounds in both perchloric acid and KOH extracts and in whole cell suspensions of Crithidia luciliae. Cells were grown in Bone and Steinert medium, or in a modified RPMI culture medium and harvested after about 72 h in mid- to late log phase. 31P NMR spectra of the perchloric acid extracts indicated that 3-phosphoglycerate, which is normally at low concentrations in most cells, was the dominant phosphorus-containing compound in the sugar phosphate region. 3-Phosphoglycerate is the end product of glycosomal glycolysis and our finding is consistent with previous observations of the failure to detect prior glycolytic intermediates. Other metabolites observed were ATP, ADP, NAD(P)+, phosphoenolpyruvate and low molecular weight polyphosphates (PPn, n less than 20). The presence of high-molecular-weight polyphosphates was established by spectra recorded on extracts obtained through subsequent treatment of the insoluble fraction with KOH. 31P NMR experiments on whole cells indicated that the average main internal pH of cells in late-log growth phase was approx. pH 7.2 +/- 0.1, using the orthophosphate resonance as an indicator. The cells responded to the addition of glucose (final concentration approx. 35 mM) with a decrease in pH, both internal (delta pH = -0.9 (55 min)-1) and external (delta pH = -1.3 (15 min)-1). Polyphosphates and ATP could not be observed in whole cell experiments, although perchloric acid extracts of identically treated cells showed no significant depletion of these compounds.

Animals↗
PubMed1991

The isolated 21 kDa N-terminal fragment of myosin binds to actin in an ATP and ionic strength-dependent manner.

Recently we reported that the isolated 23 kDa N-terminal fragment of myosin heavy chain, which contains the 'consensus' ATP binding site, binds to actin in an ATP-sensitive manner (Muhlrad, A. (1989) Biochemistry 28, 4002). In order to determine whether the 'consensus' ATP site has a role in the ATP-dependent actin binding of the fragment, we isolated a shorter 21 kDa N-terminal fragment, which contains only a part of the 'consensus' site. The 21 kDa fragment was obtained by photocleavage of myosin subfragment-1 in the presence of vanadate (Mocz, G. (1989) Eur. J. Biochem. 179, 373); the cleavage was followed by dissociation of the S-1 heavy chain fragments with guanidine hydrochloride and renaturation. The isolated 21 kDa fragment binds to F-actin, since it cosediments with actin, inhibits the actin-activated ATPase activity of myosin subfragment-1 and shows increase in light scattering upon titration by actin. The affinity of the binding is rather high (Kassoc = 0.83.10(7) M-1). The light scattering increase is reversed, e.g., the 21 kDa-actin complex is dissociated, upon addition of ATP both in the presence and absence of Mg, but less ATP is needed for dissociation when Mg is absent. Other polyphosphates, including inorganic triphosphate, pyrophosphate and ADP, also dissociate both the 21 kDa-actin and 23 kDa-actin complexes but the latter needs a higher concentration of polyphosphates for dissociation. However, these polyphosphates, except ATP, do not dissociate the (subfragment-1)-actin complex in the absence of Mg. The 21 kDa-actin and the 23 kDa-actin complexes are also dissociated by increasing ionic strength or by a low concentration of polyglutamate, which hardly affect the light scattering of the (subfragment-1)-actin complex. The results indicate that the binding of the N-terminal fragments of myosin to actin, unlike that of intact subfragment-1, is essentially of electrostatic nature. The polyanions dissociate the myosin fragment-actin complexes not by reacting with the 'consensus' ATP binding site, but by competing with actin for a positively charged binding site on the 21 kDa fragment. The only positively charged cluster in the amino acid sequence of this fragment is the 143-147 stretch, which may participate in forming the actin binding site.

Actins↗
PubMed1990

Pharmacological activity of adenine dinucleotides in the periphery: possible receptor classes and transmitter function.

1. The pharmacological actions of adenine dinucleotides, in particular beta-nicotinamide adenine dinucleotide (NAD), beta-nicotinamide adenine dinucleotide phosphate (NADP) and a homologous series of alpha,omega-adenine dinucleotide polyphosphates has been reviewed. 2. It is apparent that many actions of NAD can be explained in terms of activation of P1-purinoceptors, but actions of NADP cannot be explained in terms of activation of P1- or P2-purinoceptors. 3. Similarly, pharmacological activities of P1,P3-diadenosine triphosphate and P1,P4-diadenosine tetraphosphate are not in keeping with activation of P1- or P2-purinoceptors. 4. In the vas deferens and urinary bladder, P1,P4-diadenosine tetraphosphate, P1,P5-diadenosine pentaphosphate and P1,P6-diadenosine hexaphosphate act on P2x-purinoceptors and can cause desensitization of these receptors. 5. It is suggested that classes of receptors for adenine dinucleotides exist which are distinct from either P1- or P2-purinoceptors. 6. It is also suggested that in view of the finding of high concentrations of alpha,omega-adenine dinucleotide polyphosphates in adrenal medullary chromaffin cells, and of the involvement of the P2x-purinoceptor in the vas deferens and urinary bladder with purinergic neuromuscular transmission, that alpha,omega-adenine dinucleotide polyphosphates may yet be discovered in autonomic neurones and serve as neurotransmitters.

Adenosine Diphosphate↗
PubMed2005

Chitosan-alginate multilayer beads for controlled release of ampicillin.

The aim of this study is to develop multilayer beads with improved properties for controlled delivery of the antibiotic ampicillin. Ionotropic gelation was applied to prepare single and multilayer beads using various combinations of chitosan and Ca(2+) as cationic components and alginate and polyphosphate as anions. Beads prepared with higher concentrations of chitosan entrapped more ampicillin. During incubation in simulated gastric fluid, the beads swelled and started to float but did not show any sign of erosion. Single layer chitosan-alginate beads released 70% of the drug within 4 h. Multilayer beads released only 20-30% in the same period of time. During subsequent incubation in simulated intestinal fluid, both single and multilayer beads continued to release drug. At least part of this release is due to disintegration of the beads. The rate of release both in gastric and intestinal fluid and the kinetics of disintegration in intestinal fluid can be controlled by changing the chitosan concentration in the coagulation fluid. The release of the drug can also be controlled by the degree of cross-linking using polyphosphate. Cross-linked multilayer beads were prepared that released only 40% of the entrapped drug during 24 h. It is concluded that chitosan-alginate multilayer beads, cross-linked with polyphosphate offer an opportunity for controlled gastrointestinal passage of compounds with low molecular weight like ampicillin.

Alginates↗
PubMed2005

Invalidity of deriving interparticle distance in clay-water systems using the experimental structure factor maximum obtained by small-angle scattering.

Small-angle X-ray scattering (SAXS) has been widely used to investigate the organization of clay colloids in response to the particle concentration and ionic strength of the suspending medium. In such investigations, measuring the interparticle distance and/or spacing is usually attempted. In random or short-range ordered clay-water systems, the interparticle distances are often derived from the experimental structure factor maximum; however, the validity of such practice has never been theoretically or experimentally evaluated. The experimental structure factors of several clay-water systems with and without polyphosphate treatment to block the edge charges of clay particles were obtained from SAXS data in order to understand the physical meaning of this property. The results show that the polyphosphate treatment eliminated the experimental structure factor maximum and that the particle concentration effects were correlated with the depression on the curve in a random clay-water system (e.g., illite and laponite). For clay particles with greater anisotropy (i.e., montmorillonite), polyphosphate treatment enhanced the ordering of clay layers at high particle concentrations forming long-range ordered crystals showing Bragg reflections. In this ordered system, distinctive and symmetrical peaks representing the interparticle spacing were obtained by using a Fourier transform of the scattering curves. Thus, we conclude that the experimental structure factor maximum is induced by the edge-face oriented interactions, which may not be in direct contact as in a house-of-cards structure, and the position of the maximum should not be interpreted as an averaged interparticle distance in a clay-water system unless particles orient along the same direction.

Journal Article↗
PubMed2006

Endogenous processes during long-term starvation in activated sludge performing enhanced biological phosphorus removal.

In many biological wastewater treatment systems, bacterial growth and the amount of active biomass are limited by the availability of substrate. Under these low growth conditions, endogenous processes have a significant influence on the amount of active biomass and therefore, the overall system performance. In enhanced biological phosphorus removal (EBPR) systems endogenous processes can also influence the levels of the internal storage compounds of the polyphosphate accumulating organisms (PAO), directly affecting phosphorus removal performance. The purpose of this study was to evaluate the significance of different endogenous processes that occur during the long-term starvation of EBPR sludge under aerobic and anaerobic conditions. Activated sludge obtained from a laboratory sequencing batch reactor was used to perform a series of batch starvation experiments. Under aerobic starvation conditions we observed a significant decay of PAO (first-order decay rate of 0.15/d) together with a rapid utilization of polyhydroxyalkanoates (PHA) and a slower utilization of glycogen and polyphosphate to generate maintenance energy. On the other hand, anaerobic starvation was best described by maintenance processes that rapidly reduce the levels of polyphosphate and glycogen under starvation conditions while no significant decay of PAO was observed. The endogenous utilization of glycogen for maintenance purposes is currently not included in available EBPR models. Our experimental results suggest that mathematical models for in EBPR should differentiate between aerobic and anaerobic endogenous processes, as they influence active biomass and storage products differently.

Aerobiosis↗
PubMed1975

Implications of the reorganizational behavior of condensed phosphates for mitochondrial phosphorylation.

The composition of phosphorus-32 labeled polyphosphoric acid (32PPA) prepared from, (i) H3(32)PO4 and (82 percent w/w) P2O5 and, (ii) by incorporation of radioactivity from H3(32)PO4 into unlabeled polyphosphoric acid (PPA) was examined and found to be the same. The identity of these condensed phosphorate mixtures, as established, argues strongly in favor of a rapid and complete equilibration and provides direct evidence in support of Van Wazer's reorganization theory of condensed phosphates. No evidence for metaphosphates or label exchange in an aqueous environment was found. 32PPA was found to exist in metastable equilibrium with 32PPA as a crystalline solid of different composition. Agreement of hydrolytic rate data from the mixtures of condensed phosphates with literature values for individual species suggests that the hydrolysis of the pyro- and tripoly-phosphate species proceeds independently in the presence of other homologs. The rate of hydrolysis of the condensed phosphates (at pH5.0 and 65 degrees C) was found to be proportional to chain length, increasing about four-fold between pyro- and hexapoly-phosphate. The same correlation is also manifest in the adenosine 5' polyphosphates. A slight enhancement in the rate of hydrolysis of adenosine diphosphate, increasingly more pronounced in the case of adenosine tri- and tetra-phosphate, was also noted. This effect is attributed to the presence of the adenosine moiety. The most interesting aspect of the rate data is to be found in the similarity of the rates of hydrolysis of the adenosine 5' polyphosphates, relative to their inorganic polyphosphate counterparts. The significance of these findings is discussed and the viewpoint is presented that the properties of condensed phosphates are compatible with the requisite characteristics that must be predicated of any in situ phosphoryl precursor in mitochondrial phosphorylation.

Diphosphates↗
PubMed1998

The active-site arginine of S-adenosylmethionine synthetase orients the reaction intermediate.

S-Adenosylmethionine (AdoMet) synthetase catalyzes the formation of AdoMet and tripolyphosphate (PPPi) from ATP and L-methionine and the subsequent hydrolysis of the PPPi to PPi and Pi before product release. Little is known about the roles of active-site residues involved in catalysis of the two sequential reactions that occur at opposite ends of the polyphosphate chain. Crystallographic studies of Escherichia coli AdoMet synthetase showed that arginine-244 is the only arginine near the polyphosphate-binding site. Arginine-244 is embedded as the seventh residue in the conserved sequence DxGxTxxKxI which is also found at the active site of inorganic pyrophosphatases, suggesting a potential pyrophosphate-binding motif. Chemical modification of AdoMet synthetase by the arginine-specific reagents phenylglyoxal or p-hydroxyphenylglyoxal inactivates the enzyme. ATP and PPPi protect the enzyme from inactivation, consistent with the presence of an important arginine residue in the vicinity of the polyphosphate-binding site. Site-specific mutagenesis has been used to change the conserved arginine-244 to either leucine (R244L) or histidine (R244H). In the overall reaction, the R244L mutant has the kcat reduced approximately 10(3)-fold, with a 7 to 10-fold increase in substrate Km values; the R244H mutant has an approximately 10(5)-fold decrease in kcat. In contrast, the kcat values for hydrolysis of added PPPi by the R244L and R244H mutants have been reduced by less than 2 orders of magnitude. In contrast to the wild-type enzyme in which 98% of the Pi formed originates as the gamma-phosphoryl group of ATP, in the R244L mutant the orientation of the PPPi intermediate equilibrates at the active site yielding equal amounts of Pi from the alpha- and gamma-phosphoryl groups of ATP. Thus, the active-site arginine has a profound role in the cleavage of PPPi from ATP during AdoMet formation and in maintaining the orientation of PPPi in the active site, while playing a lesser role in the subsequent PPPi hydrolytic reaction.

Amino Acid Substitution↗
PubMed2004

DNA chip-based expression profile analysis indicates involvement of the phosphatidylinositol signaling pathway in multiple plant responses to hormone and abiotic treatments.

The phosphatidylinositol (PI) metabolic pathway is considered critical in plant responses to many environmental factors, and previous studies have indicated the involvement of multiple PI-related gene families during cellular responses. Through a detailed analysis of the Arabidopsis thaliana genome, 82 polypeptides were identified as being involved in PI signaling. These could be grouped into different families including PI synthases (PIS), PI-phosphate kinases (PIPK), phospholipases (PL), inositol polyphosphate phosphatases (IPPase), inositol polyphosphate kinases (IPK), PI transfer proteins and putative inositol polyphosphate receptors. The presence of more than 10 isoforms of PIPK, PLC, PLD and IPPase suggested that these genes might be differentially expressed during plant cellular responses or growth and development. Accordingly, DNA chip technology was employed to study the expression patterns of various isoforms. In total, 79 mRNA clones were amplified and used for DNA chip generation. Expression profile analysis was performed using samples that represented multiple tissues or cellular responses. Tested samples included normal leaf, stem and flower tissues, and leaves from plants treated with various hormones (auxin, cytokinin, gibberellin, abscisic acid and brassinosteroid) or environmental factors (temperature, calcium, sodium, drought, salicylic acid and jasmonic acid). Results showed that many PI pathway-related genes were differentially expressed under these experimental conditions. In particular, the different isoforms of each family were specifically expressed in many cases, suggesting their involvement in tissue specificity and cellular responses to environmental conditions. This work provides a starting point for functional studies of the relevant PI-related proteins and may help shed light onto the role of PI pathways in development and cellular responses.

1-Phosphatidylinositol 4-Kinase↗
PubMed1987

Adrenergic regulation of formation of inositol phosphates in rat submandibular acini.

Formation of inositol phosphates in response to adrenergic secretagogues was studied in rat submandibular acini labelled with myo-[2-3H]inositol. Noradrenaline rapidly (within 5 s) increased radioactivity incorporated into inositol 1,4,5-trisphosphate and inositol tetrakisphosphate, with less rapid (within 1 min) increases in inositol 1,3,4-trisphosphate being observed. Inositol polyphosphate formation was less sensitive to noradrenaline than was stimulation of mucin secretion and was mediated by stimulation of alpha- but not beta-adrenergic receptors. The beta-adrenergic agonist isoproterenol, which is a potent stimulator of mucin secretion [McPherson & Dormer (1984) Biochem. J. 224, 473-481] did not increase formation of inositol mono-, bis- or polyphosphates during a 15 min incubation. The results suggest that inositol phosphates do not mediate beta-adrenergic stimulation of mucin secretion in rat submandibular acini. In addition, rat submandibular acinar cells contain a Ca2+ pool which can be mobilized by isoproterenol [McPherson & Dormer (1984) Biochem. J. 224, 473-481], without involvement of inositol polyphosphates as second messengers.

Animals↗
PubMed1996

Ins(1,3,4,5)P4 is effective in mobilizing Ca2+ in mouse exocrine pancreatic acinar cells if phospholipase A2 is inhibited.

In enzymically isolated mouse pancreatic acinar cells, under conditions of whole-cell patch-clamp current recording, the effect of phospholipase C-coupled agonists can be mimicked by internal perfusion of the intracellular second messenger Ins(1,4,5)P3 (10 microM) or its analogue Ins(2,4,5)P3 (10 microM). The inositol trisphosphates mimic receptor activation by releasing Ca2+ from intracellular stores and by promoting Ca2+ influx across the surface membrane. This Ca(2+)-mobilizing role of inositol polyphosphates seems to be confined to the inositol trisphosphates because internal perfusion of Ins(1,3,4,5)P4 (10 microM) is not associated with any Ca(2+)-dependent current activation. In this study we investigate the effects of 4-bromophenacyl bromide (4BPB), a putative inhibitor of phospholipase A2 and arachadonic acid production, on inositol polyphosphate-induced Ca2+ signalling. At 10 microM, 4BPB has no effect on unstimulated Ca(2+)-dependent membrane currents. However, if 4BPB is applied to cells internally perfused with 10 microM Ins(1,4,5)P3 or Ins(2,4,5)P3 then the current responses are rapidly potentiated. In cells internally perfused with 10 microM Ins(1,3,4,5)P4, which has itself no effect on membrane currents, application of 4BPB resulted in the activation of Ca(2+)-dependent currents, seen either as repetitive spikes of current or as sustained current activations. The application of arachidonic acid blocks the current responses evoked by the inositol trisphosphates and by Ins(1,3,4,5)P4/4BPB. These results suggest that in enzymically isolated pancreatic acinar cells phospholipase A2 activity is exerting an inhibitory effect on inositol polyphosphate-mediated Ca2+ mobilization. 4BPB removes this inhibition and potentiates the responses to internally perfused inositol trisphosphates and, importantly, makes 10 microM Ins(1,3,4,5)P4 as effective as 10 microM Ins(1,4,5)P3 in mobilizing intracellular Ca2+ and in promoting Ca2+ influx.

Acetophenones↗
PubMed1997

Dinucleotide receptor modulation by protein kinases (protein kinases A and C) and protein phosphatases in rat brain synaptic terminals.

The diadenosine polyphosphates, diadenosine tetraphosphate and diadenosine pentaphosphate (Ap5A), can activate an ionotropic dinucleotide receptor that induces Ca2+ transients into synaptosomes prepared from rat brain. This receptor, also termed the P4 purinoceptor, is sensitive only to adenine dinucleotides and is insensitive to ATP. Studies on the modulatory role of protein kinase A (PKA), protein kinase C (PKC), and protein phosphatases on the response of diadenosine polyphosphate receptors were performed by measuring the changes in the intracellular Ca2+ levels with fura-2. Activation and inhibition of PKA were carried out by means of forskolin and the PKA inhibitory peptide (PKA-IP), respectively. The Ap5A response was inhibited by forksolin to 35% of control values, but PKA-IP induced an increase of 37%. The effect of PKC activation was similar to that observed for PKA. PKC stimulation with phorbol 12,13-dibutyrate produced an inhibition of 67%, whereas the PKC inhibitors staurosporine and PKC inhibitory peptide enhanced the responses elicited by Ap5A to 40% in both cases. Protein phosphatase inhibitors diminished the responses elicited by Ap5A to 17% in the case of okadaic acid, to 50% for microcystin, and to 45% in the case of cyclosporin A. Thus, the activity of dinucleotide receptors in rat brain synaptosomes appears to be modulated by phosphorylation/dephosphorylation. These processes could be of physiological significance in the control of transmitter release from neurons that are postsynaptic to nerves that release diadenosine polyphosphates.

Adenosine Triphosphate↗
PubMed1999

Identification and characterization of P(1), P(7)-Di(adenosine-5')-heptaphosphate from human platelets.

Diadenosine pentaphosphate and diadenosine hexaphosphate have been isolated in human platelets and have been postulated to play an important role in the control of vascular tone. Here we describe the isolation and identification of diadenosine heptaphosphate from human platelets. Dinucleoside polyphosphates were concentrated by affinity chromatography from a nucleotide-containing fraction from deproteinated human platelets. Dinucleoside polyphosphates were purified by anion-exchange and reversed phase high performance liquid chromatography to homogeneity. Analysis of one of these fractions with matrix-assisted laser desorption/ionization mass spectrometry revealed a molecular mass of 1076.4 (1077.4 = [M + H](+)) Da. UV spectroscopic analysis of this fraction showed the spectrum of an adenosine derivative. Comparison of the postsource decay matrix-assisted laser desorption/ionization mass spectrum of the fraction minus that of diadenosine heptaphosphate (Ap(7)A) demonstrated that the isolated substance was identical to Ap(7)A. The identity of the retention times of the authentic and the isolated compound confirmed this result. Enzymatic analysis demonstrated an interconnection of the phosphate groups with the adenosines in the 5'-positions of the riboses. With thrombin-induced platelet aggregation, Ap(7)A is released from the platelets into the extracellular space. The vasoconstrictive action of Ap(7)A on the vasculature of the isolated perfused rat kidney Ap(7)A was slightly less than that of Ap(6)A. The threshold of the vasoconstrictive action of Ap(7)A was 10(-5) mol/liter. The vasoconstrictive effect was abolished by suramin and pyridoxal phosphate 6-azophenyl-2', 4'-disulfonic acid, suggesting an activation of P(2x) receptors. Furthermore, Ap(7)A inhibits ADP-induced platelet aggregation. Thus, the potent vasoconstrictor Ap(7)A derived from human platelets, like other diadenosine polyphosphates, may play a role in the regulation of vascular tone and hemostasis.

Adenosine Diphosphate↗
PubMed2000

31P NMR spectroscopy of Trypanosoma brucei, Trypanosoma cruzi, and Leishmania major. Evidence for high levels of condensed inorganic phosphates.

High resolution (31)P nuclear magnetic resonance spectra at 303.6 MHz (corresponding to a (1)H resonance frequency of 750 MHz) have been obtained of perchloric acid extracts of Trypanosoma brucei, Trypanosoma cruzi, and Leishmania major, the causative agents of African sleeping sickness, Chagas' disease, and leishmaniasis. Essentially complete assignments have been made based on chemical shifts and by direct addition of authentic reference compounds. The results indicate the presence of high levels of short chain condensed polyphosphates: di-, tri-, tetra-, and pentapolyphosphate. (31)P NMR spectra of purified T. brucei, T. cruzi, and L. major acidocalcisomes, calcium and phosphorus storage organelles, indicate that polyphosphates are abundant in these organelles and have an average chain length of 3.11-3.39 phosphates. In the context of the recent discovery of several pyrophosphate-utilizing enzymes in trypanosomatids, the presence of these inorganic polyphosphates implies a critical role for these molecules in these parasites and a potential new route to chemotherapy.

Animals↗