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Effects of growth state and amines on cytoplasmic and vacuolar pH, phosphate and polyphosphate levels in Saccharomyces cerevisiae: a 31P-nuclear magnetic resonance study.

The vacuoles of logarithmic and stationary stage cells were compared by 31P-NMR with regard to pH, orthophosphate (Pi) content and average size of polyphosphate. The vacuoles of stationary cells had lower pH, higher Pi content, and polyphosphates of longer average chain length, although total polyphosphate content was about the same as in logarithmic cells. The lower vacuolar pH in stationary cells was the major cause of a larger cytoplasmic-vacuolar pH gradient. Addition of NH4Cl, (NH4)2SO4, methylamine or amantadine at pH 8 to cells in either stage caused an increase in both cytoplasmic and vacuolar pH, with little or no change in the cytoplasmic-vacuolar pH gradient. However, the administration of ammonium salts to the cells at pH 8.0 resulted in rapid hydrolysis of the intravacuolar polyphosphate to tripolyphosphate and Pi, with attendant redistribution of Pi between the vacuolar and cytoplasmic compartments.

Amantadine↗

Inositol polyphosphates regulate the membrane interactions of the endosomal p100, G-protein-related protein.

The protein, p100, was previously identified as a G-protein related protein that cycles on and off the cytoplasmic face of the endosome membrane (Traub et al., Biochem. J. 280 (1991) 171-178). Here we present evidence that the inositol polyphosphates, inositol 1,4, 5-trisphosphate (IP3) and inositol hexakisphosphate (IP6), release p100 from light-density microsomal membranes and inhibit rebinding of p100 through receptors, which are specific for IP3 or for IP6. These receptors can be co-extracted with p100 from the microsomes by 0.5 M Tris-HCl and, in the soluble state, they exhibit similar binding activity towards the inositol polyphosphates as do untreated microsomes. Soluble p100 self-aggregates and this aggregation is blocked by both IP3 and IP6. Stimulation of permeabilized rat basophilic leukemia (RBL-2H3) cells with carbachol, via transfected muscarinic m1 receptors, results in increased levels of inositol polyphosphates and the quantitative release of p100 into the cytosol. This effect is reversible and cytosolic p100 rebinds to the membrane as the levels of inositol polyphosphates decline. These findings suggest that p100 may belong to a family of IP-binding proteins whose intracellular localization is determined by extracellular signals.

Animals↗

Diadenosine polyphosphate receptors. from rat and guinea-pig brain to human nervous system.

Diadenosine polyphosphates are a family of naturally occurring nucleotidic compounds present in secretory vesicles together with other chemical messengers. The exocytotic release of these compounds permits them to stimulate receptors termed "purinoceptors" or "ATP receptors." Purinoceptors for nucleotides are named P2 in contrast with those sensitive to nucleosides (P1). P2 receptors are further subdivided into metabotropic P2Y receptors, further divided into 5 subtypes, and ionotropic P2X receptors, with 7 different subtypes. Diadenosine polyphosphates can activate recombinant P2Y(1), P2Y(2), and P2Y(4) and recombinant homomeric P2X(1), P2X(2), P2X(3), P2X(4), and P2X(6). Heteromeric P2X receptors change their sensitivity to diadenosine polyphosphates when co-assembly between different subunits occurs. Diadenosine polyphosphates can activate specific receptors termed dinucleotide receptors or P4 receptors, which are insensitive to other nucleosides or nucleotides. The P4 receptor is a receptor-operated Ca(2)+ channel present in rat brain synaptic terminals, stimulated by diadenosine pentaphosphate and diadenosine tetraphosphate. This receptor is strongly modulated by protein kinases A and C and protein phosphatases. The dinucleotide receptor is present in different brain areas, such as midbrain (in rat and guinea-pig), cerebellum (in guinea-pig), and cortex (in human).

Animals↗

Diadenosine polyphosphate-mediated activation of phospholipase D in isolated rat liver cells.

Diadenosine polyphosphates (ApnAs) can, through interaction with appropriate purinoceptors, affect a range of cellular activities. Ap4A, the most prominent naturally occurring diadenosine polyphosphate, stimulates alterations in intracellular calcium homeostasis and subsequent activation of glycogen breakdown in isolated liver cells. Here we show that Ap4A, and other naturally occurring diadenosine polyphosphates, also stimulates phospholipase D (PLD) activity in isolated rat liver cells. The characteristics of Ap4A-mediated activation of PLD are similar to those for the activation of PLD by extracellular ATP. These results are discussed in the context of the relation between diadenosine polyphosphate- and adenine mononucleotide-mediated cellular signalling processes.

Animals↗

Spectroscopic study and local coordination of polyphosphate colloidal systems.

The interaction between metaphosphate chains and the metal ions Ca2+ and Eu3+ has been studied in water by Eu3+ luminescence, infrared absorption, and 31P NMR spectroscopy. Two main families of sites could be identified for the metal ions in the aqueous polyphosphate colloidal systems: (1) cagelike sites provided by the polyphosphate chain and (2) a family which arises following saturation of cagelike sites. Occupation of this second family leads to supramolecular interactions between polyphosphate chains and the consequent destabilization of the colloidal system. In the polyphosphate-Ca2+ system, this destabilization appears as a coacervation process. Equilibrium existing between colloidal species as a function of the compositions could be reasoned based on the spectroscopic measurements. The determination of coordination numbers and the correlation of the results with the observation of coacervates show that Eu3+ luminescence properties can be used to probe in a unique way the coacervation process.

Journal Article↗

The critical role of adenosine and guanosine in the affinity of dinucleoside polyphosphates to P(2X)-receptors in the isolated perfused rat kidney.

1. The activation of P(2x)-receptors in the rat renal vasculature by dinucleoside polyphosphates with variable phosphate group chain length (Xp(n)X; X=Adenin (A) /Guanin (G), n=4 - 6) was studied by measuring their effects on perfusion pressure of the isolated perfused rat kidney at constant flow in an open circuit. 2. Like Ap(4)A, Ap(5)A and Ap(6)A the dinucleoside polyphosphates Ap(4)G, Ap(5)G and Ap(6)G exerted a vasoconstriction which could be blocked by suramin and pyridoxal-phosphate-6-azophenyl-2; 4-disulphonic acid (PPADS). 3. Gp(4)G, Gp(5)G and Gp(6)G showed only very weak vasoconstriction at high doses. 4. Ap(6)A and alpha, beta-meATP could not be blocked by the selective P(2x1)-receptor antagonisten NF023 (30 microM), whereas Ap(4)A, Ap(4)G, Ap(5)A, Ap(5)G and Ap(6)G were partially blocked by NF023. 5. Inhibition of endothelial NO-synthase by N(omega)-nitro-L-arginine methyl ester (L-NAME) did not affect vasoconstrictions induced by dinucleosidepolyphosphates. 6. P(2x)-receptor can only be activated if at least one adenosine moiety is present in the molecule. 7. Ap(n)G show a weaker vasoconstrictive action than corresponding Ap(n)A, concluding that two adenosine moieties enhance the P(2x)-receptor binding and activation. 8. Xp(n)X containing five phosphate groups show the most pronounced vasoconstrictive effect whereas four phosphate groups show the less effect, therefore the number of phosphate groups critically changes receptor affinity. 9. Additional experiments using permanent perfusion with alpha, beta-methylene ATP (alpha,beta-meATP) and the selective P(2x1)-receptor antagonist NF023 showed that the newly discovered human dinucleoside polyphosphates activated the vascular P(2x1)-receptor and an recently identified new P(2x)-receptor subtype. 10. The differential effects of dinucleoside polyphosphates allow a fine tuning of local perfusion via composition of Xp(n)Xs.

Adenosine↗

Structure-activity relationships of diadenosine polyphosphates (Ap(n)As), adenosine polyphospho guanosines (Ap(n)Gs) and guanosine polyphospho guanosines (Gp(n)Gs) at P2 receptors in the rat mesenteric arterial bed.

1. Vascular effects of diadenosine polyphosphates (Ap(n)As), adenosine polyphospho guanosines (Ap(n)Gs) and guanosine polyphospho guanosines (Gp(n)Gs), novel families of naturally-occurring signalling molecules, were investigated in methoxamine preconstricted rat isolated perfused mesenteric arterial beds. 2. Three different types of response were elicited by Ap(n)As and Ap(n)Gs. Those with a short polyphosphate chain (n=2 - 3) elicited vasorelaxation. Ap(3)A was more potent than Ap(2)A, and both were more potent than the corresponding Ap(n)G. Relaxations to Ap(3)A and Ap(3)G, but not to Ap(2)A and Ap(2)G, were blocked by endothelium removal and pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid (PPADS), a P2 receptor antagonist. 3. Longer polyphosphate chain Ap(n)As and Ap(n)Gs (n=4 - 6) elicited dose-dependent vasoconstriction followed by prolonged vasorelaxation, with a potency order for both types of response of Ap(5)A> or =Ap(6)A>Ap(4)A. A similar order and potency was observed for Ap(n)Gs. Contractions and prolonged relaxations were blocked by PPADS and P2X(1) receptor desensitization with alpha,beta-methylene ATP (alpha,beta-meATP), and were largely endothelium-independent. 4. In the presence of alpha,beta-meATP rapid relaxations to contractile Ap(n)As and Ap(n)Gs (n=4 - 6) were revealed. 5. Gp(n)Gs were virtually inactive, except for Gp(2)G which elicited vasoconstriction via PPADS- and alpha,beta-meATP-sensitive smooth muscle P2X(1)-like receptors. 6. These data show that, as with Ap(n)As, the length of the polyphosphate chain (n) is an important determinant of the activity of Ap(n)Gs at P2 receptors in the rat mesenteric arterial bed. When the chain is short (n=2 - 3) the purines elicit rapid vasorelaxation, which for Ap(3)A and Ap(3)G is mediated via endothelial P2Y(1)-like receptors. When the chain is long (n=4 - 6) Ap(n)As and Ap(n)Gs elicit vasoconstriction via P2X(1)-like receptors, followed by prolonged endothelium-independent vasorelaxation. Rapid relaxation to contractile dinucleotides (n=4 - 6) is revealed by block of vasoconstriction. Regarding the purine moiety, one adenine is crucial and sufficient for vasoactivity as Gp(n)Gs were largely inactive, and Ap(n)As and Ap(n)Gs approximately equipotent.

Adenosine Triphosphate↗

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 0.543+/-0.015, 0.478+/-0.043 and 0. 520+/-0.026 microM, and the Vmax values were 633+/-4, 592+/-18 and 576+/-45 pmol/min per mg of protein, for the etheno derivatives of Ap3A (adenosine 5',5'''-P1,P3-triphosphate), Ap4A (adenosine 5',5"'-P1,P4-tetraphosphate) and Ap5A (adenosine 5',5'''-P1,P5-pentaphosphate) respectively. Ca2+, Mg2+ and Mn2+ are enzyme activators, with EC50 values of 0.86+/-0.11, 1.35+/-0.24 and 0.58+/-0.10 mM respectively. The fluoride ion is an inhibitor with an IC50 value of 1.38+/-0.19 mM. The ATP analogues adenosine 5'-tetraphosphate and adenosine 5'-[gamma-thio]triphosphate are potent competitive inhibitors and adenosine 5'-[alpha,beta-methylene]diphosphate is a less potent competitive inhibitor, the Ki values being 0.29+/-0.03, 0.43+/-0.05 and 7.18+/-0.8 microM respectively. The P2-receptor antagonist pyridoxal phosphate 6-azophenyl-2',4'-disulphonic acid behaves as a non-competitive inhibitor with a Ki value of 29.7+/-3.1 microM, and also exhibits a significant inhibitory effect on Torpedo apyrase activity. The effect of pH on the Km and Vmax values, together with inhibition by diethyl pyrocarbonate, strongly suggests the presence of functional histidine residues in Torpedo diadenosine polyphosphate hydrolase. The enzyme from Torpedo shows similarities with that of neural origin from neurochromaffin cells, and significant differences compared with that from endothelial vascular cells.

Acid Anhydride Hydrolases↗

Dinucleoside polyphosphates stimulate the primer independent synthesis of poly(A) catalyzed by yeast poly(A) polymerase.

Novel properties of the primer independent synthesis of poly(A), catalyzed by the yeast poly(A) polymerase are presented. The commercial enzyme from yeast, in contrast to the enzyme from Escherichia coli, is unable to adenylate the 3'-OH end of nucleosides, nucleotides or dinucleoside polyphosphates (NpnN). In the presence of 0.05 mm ATP, dinucleotides (at 0.01 mm) activated the enzyme velocity in the following decreasing order: Gp4G, 100; Gp3G, 82; Ap6A, 61; Gp2G, 52; Ap4A, 51; Ap2A, 41; Gp5G, 36; Ap5A, 27; Ap3A, 20, where 100 represents a 10-fold activation in relation to a control without effector. The velocity of the enzyme towards its substrate ATP displayed sigmoidal kinetics with a Hill coefficient (nH) of 1.6 and a Km(S0.5) value of 0.308 +/- 0.120 mm. Dinucleoside polyphosphates did not affect the maximum velocity (Vmax) of the reaction, but did alter its nH and Km(S0.5) values. In the presence of 0.01 mm Gp4G or Ap4A the nH and Km(S0.5) values were (1.0 and 0.063 +/- 0.012 mm) and (0.8 and 0.170 +/- 0.025 mm), respectively. With these kinetic properties, a dinucleoside polyphosphate concentration as low as 1 micro m may have a noticeable activating effect on the synthesis of poly(A) by the enzyme. These findings together with previous publications from this laboratory point to a potential relationship between dinucleoside polyphosphates and enzymes catalyzing the synthesis and/or modification of DNA or RNA.

Adenosine Triphosphate↗

Hydrolysis of diadenosine polyphosphates by nucleotide pyrophosphatases/phosphodiesterases.

Diadenosine polyphosphates (ApnAs) act as extracellular signaling molecules in a broad variety of tissues. They were shown to be hydrolyzed by surface-located enzymes in an asymmetric manner, generating AMP and Apn-1 from ApnA. The molecular identity of the enzymes responsible remains unclear. We analyzed the potential of NPP1, NPP2, and NPP3, the three members of the ecto-nucleotide pyrophosphatase/phosphodiesterase family, to hydrolyze the diadenosine polyphosphates diadenosine 5',5"'-P1,P3-triphosphate (Ap3A), diadenosine 5',5"'-P1,P4-tetraphosphate (Ap4A), and diadenosine 5',5"'-P1,P5-pentaphosphate, (Ap5A), and the diguanosine polyphosphate, diguanosine 5',5"'-P1,P4-tetraphosphate (Gp4G). Each of the three enzymes hydrolyzed Ap3A, Ap4A, and Ap5A at comparable rates. Gp4G was hydrolyzed by NPP1 and NPP2 at rates similar to Ap4A, but only at half this rate by NPP3. Hydrolysis was asymmetric, involving the alpha,beta-pyrophosphate bond. ApnA hydrolysis had a very alkaline pH optimum and was inhibited by EDTA. Michaelis constant (Km) values for Ap3A were 5.1 micro m, 8.0 micro m, and 49.5 micro m for NPP1, NPP2, and NPP3, respectively. Our results suggest that NPP1, NPP2, and NPP3 are major enzyme candidates for the hydrolysis of extracellular diadenosine polyphosphates in vertebrate tissues.

Adenine Nucleotides↗

Vascular smooth muscle cells (VSMC) proliferation of streptozotocin-diabetic animals induced by diadenosine polyphosphates.

UNLABELLED: Specific binding sites for diadenosine polyphosphates (Ap (4)A, Ap (5)A, Ap (6)A) exist in VSMC (cultured vascular smooth muscle cells). These compounds may regulate VSMC growth and proliferation which is a key event in atherogenesis. Since diabetes is a known risk factor for atherosclerosis, the proliferation of VSMC from normoglycemic (control) and hyperglycemic (diabetic) rats were compared and the possibly involved receptors for diadenosine polyphosphates inducing this effect were investigated. Diabetes was induced by streptozotocin (66 mg/kg i.p.) and VSMC were prepared from rat aorta (primary culture). ( (3)H)thymidine incorporation was a measure of cell proliferation. For all diadenosine polyphosphates tested a stimulatory effect was observed as a bell-shaped concentration-response curve and a maximum effect at 10 micro M (physiological concentration). Ap (6)A has the most prominent effect (247.8 +/- 33.2 % increase over basal). In VSMC of diabetic rats the effects were even more prominent (Ap (5)A: 430.1 +/- 62.7 %). ATP (a degradation product of Ap (6)A) is able to increase the maximum effect of 10 micro M Ap (6)A. UTP (P2Y (2) agonist) exhibits a weaker proliferation. 1 micro M suramin (P2 receptor antagonist) shifts the concentration response curve of ATP and of Ap (6)A to the right. In contrast, 10 micro M PPADS (P2 X receptor antagonist) has no effect. There is no difference between VSMC of normal and diabetic rats in this respect. ADP, AMP, and adenosine exhibit a dual proliferative effect. The effect of either of these 3 compounds is much higher in VSMC of diabetic rats than of controls. 2MeSATP (P2Y (1) agonist) and alpha,beta-Methylen-ATP (P2X agonist) were not effective in VSMC of both normoglycemic and diabetic rats. IN CONCLUSION: The proliferative effect of diadenosine polyphosphates and some degradation products is more pronounced in VSMC of diabetic than of normal rats. Ap (6)A acts maximally by itself and not by its degradation product ATP. Adenosine receptors or an unknown P2Y (ApxA) receptor may be involved in proliferative effects, but not P2X and P2Y (1) receptors irrespective of a diabetic situation.

Adenosine Triphosphate↗

Putative structure and functions of a poly-beta-hydroxybutyrate/calcium polyphosphate channel in bacterial plasma membranes.

A poly-beta-hydroxybutyrate complex extracted from the plasma membranes of genetically competent Escherichia coli contained polyhydroxybutyrate:polyphosphate:calcium in molar ratios approximating 1:1:0.5. The chain length of the polyhydroxybutyrate was estimated as 120-200 subunits, and that of the polyphosphate was estimated as 130-170 subunits. The extracted complex, when incorporated into liposomes, exhibited a lipid phase transition in the same temperature range as that of the membrane complex in whole cells as well as the same properties of irreversibility, lability, and sensitivity to chelating buffers. Space-filling molecular models and molecular energy minimization methods (Charmm) were used to develop and evaluate a plausible structure for the complex. It is proposed that the polyhydroxybutyrate forms an exolipophilic-endopolarophilic helix around an inner framework helix of calcium polyphosphate. The calcium ions link the two polymers by forming ionic bonds with phosphoryl oxygens of the polyphosphate and ion-dipole bonds with the ester carbonyl oxygens of the polyhydroxybutyrate. This symmetrical structure forms a channel through the membrane and may play a role in the transport of calcium, phosphate, and DNA.

Calcium Phosphates↗

Cell lines from kidney proximal tubules of a patient with Lowe syndrome lack OCRL inositol polyphosphate 5-phosphatase and accumulate phosphatidylinositol 4,5-bisphosphate.

The protein product of the gene that when mutated is responsible for Lowe syndrome, or oculocerebrorenal syndrome (OCRL), is an inositol polyphosphate 5-phosphatase. It has a marked preference for phosphatidylinositol 4,5-bisphosphate although it hydrolyzes all four of the known inositol polyphosphate 5-phosphatase substrates: inositol 1,4,5-trisphosphate, inositol 1,3,4,5-tetrakisphosphate, phosphatidylinositol 4,5-bisphosphate, and phosphatidylinositol 3,4,5-trisphosphate. The enzyme activity of this protein is determined by a region of 672 out of a total of 970 amino acids that is homologous to inositol polyphosphate 5-phosphatase II. Cell lines from kidney proximal tubules of a patient with Lowe syndrome and a normal individual were used to study the function of OCRL. The cells from the Lowe syndrome patient lack OCRL protein. OCRL is the major phosphatidylinositol 4,5-bisphosphate 5-phosphatase in these cells. As a result, these cells accumulate phosphatidylinositol 4,5-bisphosphate even though at least four other inositol polyphosphate 5-phosphatase isozymes are present in these cells. OCRL is associated with lysosomal membranes in control proximal tubule cell lines suggesting that OCRL may function in lysosomal membrane trafficking by regulating the specific pool of phosphatidylinositol 4,5-bisphosphate that is associated with lysosomes.

Binding Sites↗

An adjacent pair of human NUDT genes on chromosome X are preferentially expressed in testis and encode two new isoforms of diphosphoinositol polyphosphate phosphohydrolase.

Combinatorial expression of the various isoforms of diphosphoinositol synthases and phosphohydrolases determines the rates of phosphorylation/dephosphorylation cycles that have been functionally linked to vesicle trafficking, stress responses, DNA repair, and apoptosis. We now describe two new 19-kDa diphosphoinositol polyphosphate phosphohydrolases (DIPPs), named types 3alpha and 3beta, which possess the canonical Nudix-type catalytic motif flanked on either side by short Gly-rich sequences. The two enzymes differ only in that Pro-89 in the alpha form is replaced by Arg-89 in the beta form, making the latter approximately 2-fold more active in vitro. Another Nudix substrate, diadenosine hexaphosphate, was hydrolyzed less efficiently (k(cat)/K(m) = 0.2 x 10(5) m(-1) s(-1)) compared with diphosphoinositol polyphosphates (k(cat)/K(m) = 2-40 x 10(5) m(-1) s(-1)). Catalytic activity in vivo was established by individual overexpression of the human (h) DIPP3 isoforms in HEK293 cells, which reduced cellular levels of diphosphoinositol polyphosphates by 40-50%. The hDIPP3 mRNA is preferentially expressed in testis, accompanied by relatively weak expression in the brain, contrasting with hDIPP1 and hDIPP2 which are widely expressed. The hDIPP3 genes (NUDT10 encodes hDIPP3alpha; NUDT11 encodes hDIPP3beta) are only 152 kbp apart at p11.22 on chromosome X and probably arose by duplication. Transcription of both genes is inactivated on one of the X chromosomes of human females to maintain appropriate gene dosage. The hDIPP3 pair add tissue-specific diversity to the molecular mechanisms regulating diphosphoinositol polyphosphate turnover.

Acid Anhydride Hydrolases↗

A role for rat inositol polyphosphate kinases rIPK2 and rIPK1 in inositol pentakisphosphate and inositol hexakisphosphate production in rat-1 cells.

Over 30 inositol polyphosphates are known to exist in mammalian cells; however, the majority of them have uncharacterized functions. In this study we investigated the molecular basis of synthesis of highly phosphorylated inositol polyphosphates (such as inositol tetrakisphosphate, inositol pentakisphosphate (IP5), and inositol hexakisphosphate (IP6)) in rat cells. We report that heterologous expression of rat inositol polyphosphate kinases rIPK2, a dual specificity inositol trisphosphate/inositol tetrakisphosphate kinase, and rIPK1, an IP5 2-kinase, were sufficient to recapitulate IP6 synthesis from inositol 1,4,5-trisphosphate in mutant yeast cells. Overexpression of rIPK2 in Rat-1 cells increased inositol 1,3,4,5,6-pentakisphosphate (I(1,3,4,5,6)P5) levels about 2-3-fold compared with control. Likewise in Rat-1 cells, overexpression of rIPK1 was capable of completely converting I(1,3,4,5,6)P5 to IP6. Simultaneous overexpression of both rIPK2 and rIPK1 in Rat-1 cells increased both IP5 and IP6 levels. To reduce IPK2 activity in Rat-1 cells, we introduced vector-based short interference RNA against rIPK2. Cells harboring the short interference RNA had a 90% reduction of mRNA levels and a 75% decrease of I(1,3,4,5,6)P5. These data confirm the involvement of IPK2 and IPK1 in the conversion of inositol 1,4,5-trisphosphate to IP6 in rat cells. Furthermore these data suggest that rIPK2 and rIPK1 act as key determining steps in production of IP5 and IP6, respectively. The ability to modulate the intracellular inositol polyphosphate levels by altering IPK2 and IPK1 expression in rat cells will provide powerful tools to study the roles of I(1,3,4,5,6)P5 and IP6 in cell signaling.

Animals↗

Ascorbate polyphosphate is a bioavailable vitamin C source in juvenile rainbow trout: tissue saturation and compartmentalization model.

We studied the bioavailability of ascorbic acid ester, ascorbate polyphosphate, to juvenile rainbow trout (Oncorhynchus mykiss). Fish were fed molar equivalents of 0, 20, 40, 80, 160, 320 and 1280 mg ascorbic acid/kg diet in the form of ascorbate polyphosphate. During the 18 wk of the experiment, when body weight increase averaged 3.5-fold, we did not observe any deficiency symptoms in any group. Liver and kidney ascorbate concentrations differed significantly among groups after wk 9. The ascorbic acid concentrations in liver were significantly different in fish fed for 9 wk an equivalent of 0, 40 and 160 mg ascorbic acid/kg as ascorbate polyphosphate, values were 22.7 +/- 3.4, 93.7 +/- 17.0 and 368.0 +/- 60.8 nmol ascorbic acid/g. The ascorbic acid concentrations in kidney were significantly different in fish fed for 18 wk an equivalent of 0,20 and 40 mg ascorbic acid/kg as ascorbic polyphosphate (23.9 +/- 4.0, 72.1 +/- 13.6 and 254.4 +/- 22.7 nmol ascorbic acid/g, respectively). After wk 18, fish from groups fed 0, 20, 320 and 1280 mg ascorbic polyphosphate/kg were intraperitoneally injected with 25 mg/ascorbic acid/kg body wt. We observed differences in the profiles of tissue ascorbate concentration during the 96 h following the injection between groups with high and low tissue ascorbate concentration, i.e., fish fed 320 and 0 ascorbic acid/kg, respectively. We conclude that ascorbic acid metabolism in rainbow trout after intraperitoneal injection followed the three-compartmental model, with the intraperitoneal cavity as the first compartment, blood as the second, and tissues as the third.(ABSTRACT TRUNCATED AT 250 WORDS)

Aging↗

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. The saturation studies for [3H]Ap5A were performed in a broader concentration range; in this case a three-step curve was obtained with K values of 16 +/- 2 microM, 125 +/- 9 microM, and 545 +/- 11 microM; the corresponding Hill numbers were 2, 4, and 6. This kinetic behavior can be explained on the basis of a mnemonic model, as already demonstrated for the vesicular transport of ATP. The nonhydrolyzable adenine nucleotide analogs, ATPgammaS and ADPbetaS, inhibited the diadenosine polyphosphate transport at concentrations in the millimolar range. Ap(n)A transport was also inhibited by the P2 receptor antagonist suramin, the mitochondrial ATP/ADP exchange inhibitor atractyloside, the proton translocator FCCP, and N-ethylmaleimide.

Adrenal Medulla↗

4-Coumarate:coenzyme A ligase has the catalytic capacity to synthesize and reuse various (di)adenosine polyphosphates.

4-Coumarate:coenzyme A ligase (4CL) is known to activate cinnamic acid derivatives to their corresponding coenzyme A esters. As a new type of 4CL-catalyzed reaction, we observed the synthesis of various mono- and diadenosine polyphosphates. Both the native 4CL2 isoform from Arabidopsis (At4CL2 wild type) and the At4CL2 gain of function mutant M293P/K320L, which exhibits the capacity to use a broader range of phenolic substrates, catalyzed the synthesis of adenosine 5'-tetraphosphate (p(4)A) and adenosine 5'-pentaphosphate when incubated with MgATP(-2) and tripolyphosphate or tetrapolyphosphate (P(4)), respectively. Diadenosine 5',5''',-P(1),P(4)-tetraphosphate represented the main product when the enzymes were supplied with only MgATP(2-). The At4CL2 mutant M293P/K320L was studied in more detail and was also found to catalyze the synthesis of additional dinucleoside polyphosphates such as diadenosine 5',5'''-P(1),P(5)-pentaphosphate and dAp(4)dA from the appropriate substrates, p(4)A and dATP, respectively. Formation of Ap(3)A from ATP and ADP was not observed with either At4CL2 variant. In all cases analyzed, (di)adenosine polyphosphate synthesis was either strictly dependent on or strongly stimulated by the presence of a cognate cinnamic acid derivative. The At4CL2 mutant enzyme K540L carrying a point mutation in the catalytic center that is critical for adenylate intermediate formation was inactive in both p(4)A and diadenosine 5',5''',-P(1),P(4)-tetraphosphate synthesis. These results indicate that the cinnamoyl-adenylate intermediate synthesized by At4CL2 not only functions as an intermediate in coenzyme A ester formation but can also act as a cocatalytic AMP-donor in (di)adenosine polyphosphate synthesis.

Adenine Nucleotides↗