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PubMed1969

Active calcium and strontium transport in human erythrocyte ghosts.

Both calcium and strontium could be transported actively from erythrocytes if adenosine triphosphate, guanosine triphosphate, or inosine triphosphate were included in the hypotonic medium used to infuse calcium or strontium into the cells. Acetyl phosphate and pyrophosphate were not energy sources for the transport of either ion. Neither calcium nor strontium transport was accompanied by magnesium exchange, and the addition of Mg(++) to the reaction medium in a final concentration of 3.0 mmoles/liter did not promote the transport of either ion. In the absence of nucleotide triphosphates, the addition of 1.5 mmoles/liter of Sr(++) to the reaction solution did not bring about active calcium transport and similarly 1.5 mmoles/liter of Ca(++) did not bring about active strontium transport. The inclusion of 1.5 mmoles/liter of Ca(++) or Sr(++) in the reaction medium did not interfere with the transport of the other ion when the erythrocytes were infused with adenosine triphosphate.

Adenosine Triphosphate↗
PubMed1986

Rapid RNA sequencing using double-stranded template DNA, SP6 polymerase, and 3'-deoxynucleotide triphosphates.

A simple and efficient nucleic acid sequencing method is described in which RNA transcription by the SP6 polymerase is specifically terminated using 3'-deoxynucleotide triphosphates. Initial difficulties in resolving the RNA ladder were overcome by replacing guanosine triphosphate by inosine triphosphate in the reaction mixture and electrophoresing gels at high temperature (50 degrees C). This method presents advantages over current sequencing techniques: Unprocessed plasmid DNA is the template and preparation of inserts and/or single-stranded templates is unnecessary. Use of the specific promoter for SP6 polymerase removes the need for a primer in sequencing reactions.

Base Sequence↗
PubMed1991

Modulation of acetylcholine-activated K+ channel function in rat atrial cells by phosphorylation.

1. In voltage-clamped whole cells dialysed with GTP, extracellular application of ACh elicits an inwardly rectifying K+ current which subsequently decreases to a steady-state level well below the maximally induced current (desensitization). The mechanism of desensitization of the acetylcholine (ACh)-activated K+ channel current was studied in rat neonatal atrial cells at the single-channel level using the patch-clamp technique. 2. In cell-attached patches with ACh in the pipette, a similar pattern of K+ channel current desensitization was present. Single-channel analyses revealed that the initial rapid decrease in channel activity was associated with progressive shortening of the mean open time (tau o) and prolongation of the mean closed time (tau c) of the K+ channel. 3. In excised, inside-out patches with ACh in the pipette, GTP activated K+ channels with a tau o of approximately 1.0 ms. Addition of ATP to the cytosolic surface resulted in progressive increases in tau o (from 1 to 5 ms) and channel activity. These changes are similar but opposite in direction to those observed during the early phase of ACh-induced channel desensitization in cell-attached patches. 4. The effect of ATP on the channel kinetics was abolished in Mg(2+)-free solution AMP-PNP (adenylyl-imidodiphosphate, a non-hydrolysable analogue of ATP), ADP, CTP (cytidine triphosphate), ITP (inosine triphosphate) or UTP (uridine triphosphate) did not alter the channel kinetics, suggesting that the ATP effect on channel gating probably occurs via phosphorylation by a membrane-bound kinase. H-8 (an isoquinolinesulphonamide derivative which inhibits protein kinases A and C) failed to prevent the action of ATP on the channel. 5. The increases in tau o and channel activity produced by ATP could be completely reversed by an elevation of cytosolic [Ca2+] to 3 x 10(-5) M or above. 6. The effect of Ca2+ on the ATP-induced changes in channel kinetics was blocked by sodium vanadate, a general phosphatase inhibitor. Okadaic acid, an inhibitor of protein phosphatase 1 and 2A, did not block the Ca2+ effect. Calmodulin antagonists, N-(6-aminohexyl)-5-chloro-1-naphthalenesulphonamide (W-7), trifluoroperazine, and calmidazolium, partially blocked the effect of Ca2+. 7. Alkaline phosphatase (20 units/ml) reversed the ATP-induced increases in tau o and channel activity. These results suggest that the ACh-activated K+ channel can be modulated by phosphorylation and dephosphorylation.(ABSTRACT TRUNCATED AT 400 WORDS)

Acetylcholine↗
PubMed1991

Inhibition of gluconeogenesis by extracellular ATP in isolated rat hepatocytes.

The aim of this study was to determine the effect of externally added ATP on gluconeogenesis by isolated hepatocytes from starved rats. High concentrations of extracellular ATP inhibited gluconeogenesis from lactate and pyruvate but not from glycerol or fructose. This inhibition was associated with an increase in intracellular adenosine contents. ADP, AMP, or adenosine but not guanosine 5'triphosphate, inosine 5' triphosphate, or adenine also inhibited gluconeogenesis. alpha, beta-Methylene-ATP, a nonmetabolizable structural analogue of ATP, did not affect the rate of gluconeogenesis. Intracellular ATP levels were increased by externally added ATP or adenosine, but ATP-to-ADP ratios in the cytosolic and mitochondrial compartments were diminished. Malate and phosphoenolpyruvate contents were decreased by extracellular ATP or adenosine. Our results show that inhibition of gluconeogenesis by high levels of extracellular ATP may be mediated by adenosine derived from ATP catabolism at the plasma membrane.

Adenosine Diphosphate↗
PubMed1998

Pharmacological characterization of ATP receptors in ampulla from frog semicircular canal.

Phosphoinositidase C activities sensitive to purine and pyrimidine nucleotides have been identified earlier in ampulla from Rana ridibunda semicircular canal. The aim of this study was to characterize the pharmacological properties of other P2 receptors borne by this structure. A microassay was developed to measure the binding of [35S]adenosine 5'-O-(2-thiodiphosphate) ([35S]ADPbetaS) to a few ampullas microdissected from frog semicircular canals. When determined at 4 degrees C in the absence of divalent cations, [35S]ADPbetaS binding was saturable with incubation time and reversible after elimination of free radioligand. The dissociation kinetics were biphasic and comprised a major component that was rapidly reversible and a minor component that dissociated slowly. [35S]ADPbetaS binding was competitively inhibited by unlabeled ADPbetaS with an apparent dissociation constant of 0.48 +/- 0.09 microM and a Hill coefficient of 0.70 +/- 0.06, and Scatchard analysis revealed a minor class of high-affinity binding sites (RT1 = 52 +/- 11 fmol [35S]ADPbetaS bound/ampulla and Kd1 = 0.15 +/- 0.04 microM) and a major class of low-affinity binding sites (RT2 = 436 +/- 79 fmol [35S]ADPbetaS bound/ampulla and Kd2 = 2.0 +/- 0.8 microM). The pattern of stereospecificity for recognition of unlabeled structural ATP analogs was ADPbetaS >/= alpha, beta-methyleneadenosine 5'-triphosphate = ADP = adenosine 5'-O-(3-thiotriphosphate) > ATP = diadenosine tetraphosphate = AMP > 2'- and 3'-O-(4-benzoylbenzoyl)-adenosine 5'-triphosphate >/= 2-methylthioadenosine 5'-triphosphate > 2-desoxythymidine 5'-triphosphate = guanosine 5'-triphosphate = inosine-5'-triphosphate = xanthosine 5'-triphosphate = cytosine 5'-triphosphate = uridine 5'-triphosphate = uridine-5'-diphosphate, whereas cAMP and adenosine were devoid of activity. For antagonists, suramin revealed competitive inhibitor potencies, whereas reactive blue 2 and DIDS acted as pure noncompetitive inhibitors. Results suggest that the population of labeled receptors is heterogeneous and contains a low number of P2Y-like receptors and a large number of P2X-like receptors whose molecular subtypes and functions in endolymph homeostasis remain to be defined.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1983

Potentiation of the antitumor effect of 5-fluorouracil by some nucleotides, and their possible role in the potentiation.

Of the various nucleotides tested, adenosine 5'-triphosphate (ATP), guanosine 5'-triphosphate, inosine 5'-triphosphate, and adenosine 5'-monophosphate potentiated the antitumor activity of 5-fluorouracil against Ehrlich carcinoma, while uridine 5'-triphosphate, cytidine 5'-triphosphate, deoxyadenosine 5'-triphosphate, and deoxyguanosine 5'-triphosphate were ineffective. The result of biochemical experiments suggests that ATP, which was used as a representative of the effective nucleotides, was ineffective until it was hydrolyzed to adenosine by nucleotide phosphohydrolases in the ascitic fluid of mice bearing Ehrlich ascites tumor cells, and that the adenosine functioned as a ribose donor for the increased formation of antineoplastic 5-fluoronucleotides. The possible role of the various effective nucleotides and nucleosides was discussed.

Adenosine↗
PubMed1983

In vitro synthesis of genome length complementary RNA of vesicular stomatitis virus in the presence of inosine 5'-triphosphate.

The virion-associated RNA polymerase of vesicular stomatitis virus (VSV) synthesizes RNA in vitro when GTP is replaced by inosine 5'-triphosphate (ITP). The synthesis is optimal at an ITP concentration of 200 microM and the extent of synthesis is between 15 to 20% compared to normal transcription in the presence of GTP. Analyses of the RNA products revealed that approximately 10% of the product RNA represented plus-strand complement of the genome RNA. Defective interfering particles of VSV were also capable of synthesizing complementary RNA in the presence of ITP, in addition to 46-base RNA, although in lesser amount (2%). Since I substitution facilitates read-through of the genome RNA, these results suggest that interaction of the product RNA with the genome template or ITP-mediated modification of a viral protein may be involved in the read-through process in vitro.

Defective Viruses↗
PubMed1998

Inosine 5'-triphosphate can dramatically increase the yield of NASBA products targeting GC-rich and intramolecular base-paired viroid RNA.

Nucleic acid sequence-based amplification (NASBA) according to the standard protocol failed to amplify cRNA of viroids, probably because of their GC-rich and intramolecular base-paired structure. However, NASBA in the presence of inosine 5'-triphosphate successfully amplified the cRNAs to viroids in total nucleic acid extracts from citrus plants. As sequence specificity of the cRNA to viroids was confirmed by northern analysis, the amplification and fidelity of cRNAs are sufficient for the sensitive and specific detection of viroids.

Base Composition↗
PubMed1990

Comparison of the self-association properties of the 5'-triphosphates of inosine (ITP), guanosine (GTP), and adenosine (ATP). Further evidence for ionic interactions in the highly stable dimeric [H2(ATP)]2(4-) stack.

The concentration dependence of the chemical shifts for the hydrogens H-2, H-8 and H-1' of ITP and for H-8 and H-1' of GTP has been measured in D2O at 25 degrees C under several degrees of protonation in the pD range 1.2-8.4. For reasons of comparison, inosine and guanosine have been included in the study The results are consistent with the isodesmic model of indefinite noncooperative stacking. The association constants for the nucleosides (Ns) inosine and guanosine decrease with increasing protonation: Ns greater than D(Ns)+/Ns in a 1:1 ratio greater than D(Ns)+. In contrast, a maximum is observed with ITP and GTP; the stacking tendency of GTP following the series: GTP4- less than or equal to D(GTP)3- (K approximately 0.7 M-1) less than D(GTP)3-/D2(GTP)2- in a 1:1 ratio (K approximately 2.9 M-1) greater than D2(GTP)2- greater than D3(GTP)- (K approximately 1.5 M-1). The order of the series with ITP corresponds to that with GTP, but the association constants are slightly smaller. At the maximum of the self-association tendency the triphosphate residue has only a minor influence; this follows from the fact that the association constants for the 1:1 ratios of Ino/D(Ino)+ and D(ITP)3-/D2(ITP)2- are identical within experimental error; this holds also for Guo/D(Guo)+ and D(GTP)3-/D2(GTP)2-; in all these pairs the K-7 site is 50% protonated. Comparison of the association constant for the deprotonated species shows that here charge effects, i.e. repulsion between the negatively charged triphosphate chains, are important: Ino (K approximately 3.3 M-1) greater than ITP4- (K approximately 0.4 M-1) and Guo (K approximately 8 M-1) greater than GTP4- (K approximately 0.8 M-1). In addition the series holds: Ado (K approximately 15 M-1) greater than Guo greater than Ino. However, most important is the comparison of the ITP and GTP series with previous data for ATP: ATP4- (K approximately 1.3 M-1) less than D(ATP)3- (2.1 M-1) less than 1:1 ratio of D(ATP)3-/D2(ATP)2- (6 M-1) much less than D2(ATP)2- (approximately 200 M-1) much greater than D3(ATP)- (K less than or equal to 17 M-1).(ABSTRACT TRUNCATED AT 400 WORDS)

Adenosine Triphosphate↗
PubMed1991

Acid-base properties of nucleosides and nucleotides as a function of concentration. Comparison of the proton affinity of the nucleic base residues in the monomeric and self-associated, oligomeric 5'-triphosphates of inosine (ITP), guanosine (GTP), and adenosine (ATP).

The acid-base properties of the nucleic base residues of ITP, GTP, and ATP, and for comparison also as far as possible of the corresponding nucleosides, were studied in dependence on their concentration, i.e. on the effect of self-association. From the dependence between the 1H-NMR chemical shifts of H-2 (where applicable), H-8, and H-1', and the pD of the solutions, the acidity constants for the deprotonation of the D+(N-7) site in D2(ITP)2-, D2(GTP)2-, D(Ino)+, and D(Guo)+, and of the D+(N-1) site in D2(ATP)2- and D(Ado)+ were calculated. Chemical shift/pD profiles for a whole series of varying concentrations of the nucleic base derivatives (= N) were constructed, including those for infinite dilution (delta o), which give the acidity constant for the monomeric N, and for infinitely concentrated solutions (delta infinity), which give the acidity constant of an N in an infinitely long stack. The acidity constants determined from the delta o/pD plots are in excellent agreement with the pKa values measured by potentiometric pH titrations of highly diluted solutions of N. The effects of self-association are striking; e.g. the pKa value of the D+(N-7) site in D2(GTP)2- is lowered by about 1 (as calculated from the delta o/pD and delta infinity/pD profiles), while the pKa value of the D+(N-1) site in D2(ATP)2- is increased by approximately 0.3; i.e. in the first case deprotonation is facilitated and in the second it is inhibited. The increasing inhibition of the H+(N-1) deprotonation with an increasing ATP concentration is due to the high stability of the dimeric [H2(ATP)]2(4-) stack for which the intermolecular H+(N-1)/gamma-P(OH)(O)2- ion pairs between the two ATP molecules are crucial. In those cases where no other significant interaction but aromatic-ring stacking in the self-association process occurs, the release of protons from protonated nitrogen-ring sites is facilitated with increasing stacking; this holds not only for D2(GTP)2- as indicated above, but also for D2(ITP)2-, D(Ino)+, and D(Ado)+. The latter example especially suggests that the situation for the D2(ATP)2- system is exceptional. Some consequences of the considered acid-base properties for biological systems are indicated.

Adenosine↗
PubMed2006

The ITPA c.94C>A and g.IVS2+21A>C sequence variants contribute to missplicing of the ITPA gene.

Inosine triphosphate pyrophosphatase (ITPase) catalyzes the conversion of inosine triphosphate (ITP) to the correspondent monophosphate. The ITPA c.94C>A and g.IVS2+21A>C allelic variants are associated with decreased red cell enzyme activity. The ITPA c.94C>A [P32T] sequence variant is associated with an increased risk of adverse drug reactions in patients treated with the thiopurine drug azathioprine. The aim of this study was to explore the molecular mechanisms of ITPase deficiency. ITPA mRNA was extracted from peripheral blood leukocytes (PBL), Epstein-Barr virus transformed lymphoblast cell cultures, reticulocytes, and cultured fibroblast from patients with known ITPA genotypes. ITPA mRNA was reversed transcribed, sequenced and the relative amounts of misspliced transcripts quantitated from three independent experiments. The ITPA g.IVS2+21A>C sequence variant resulted in missplicing of exon 3. The ITPA c.94C>A allelic variant resulted in missplicing of exons 2 and 3 representing, in PBL samples, 61% of the total mRNA expressed in ITPA c.94C>A homozygotes. We proposed that the ITPA c.94C>A allelic variant destroys an exonic splicing silencing (ESS) element in exon 2, resulting in the activation of two nearby upstream 5' splice sites and missplicing of the exons 2 and 3 cassette causing structural changes to the enzyme and contributing to ITPase deficiency.

Alleles↗
PubMed1996

Role of guanosine triphosphate in ferric ion-linked Fenton chemistry.

We measured the production of reactive hydroxyl radical (OH.) by Fe2+ itself or complexed with nucleotide triphosphates or tripolyphosphate (TPP). Coumarin-3-carboxylic acid (3-CCA) reacts with the OH. produced by Fe2+, Fe3+ or Cu2+ plus ascorbate and with various iron complexes. We measured in real time the increased fluorescence of 3-CCA after hydroxylation to 7-hydroxy-coumarin-3-carboxylic acid (7-OHCCA). Phosphate-buffered solutions do not affect the yield of Fe(2+)-linked OH. as do other organic buffer solutions. Our results show that guanosine triphosphate enhances the Fe(2+)-linked production of OH.. We also tested inosine triphosphate, adenosine triphosphate and xanthine triphosphate for their capacity to produce OH. with Fe2+. Inosine triphosphate is the most effective nucleotide in the production of OH.. However, the Fe(2+)-mediated yield of OH. is greater in the presence of TPP compared to the nucleotide triphosphates. Organic buffers as well as the purine and ribose portion of nucleotides compete for OH. and decrease the yield of fluorescent 7-OHCCA. We also decreased the yield of OH. by adding guanosine to the Fe2+/TPP-generating system. Adenosine, ribose and deoxyribose also react with Fe(2+)-generated OH.. The decreased yield of 7-OHCCA occurs because the ribose and purine part of the molecule reacts with OH.. The maximal production of reactive OH., compared to all nucleotides and phosphates tested, occurs with a ratio of 2 TPP/Fe2+ complex. In conclusion, the real-time measurement of the production of fluorescent 7-OHCCA provides a convenient means for measuring chemically generated OH.. The TPP/Fe(2+)-generating mixture, in the presence of 3-CCA, can be used to study the scavenging ability of other competing molecules.

Animals↗
PubMed1966

Cytochemistry of phosphatases of the sarcoplasmic reticulum. I. Biochemical studies.

A microsomal fraction was isolated from rabbit psoas muscle by a modification of Muscatello's method. The fraction contained a Mg-dependent ATPase which had a pH optimum of 7.5. Activity was further stimulated by addition of Na or K or other monovalent cations to the reaction mixture, but synergistic activation by Na and K, and ouabain inhibition, could not be demonstrated. The enzyme hydrolyzed only ATP (adenosine triphosphate) and ITP (inosine triphosphate) at appreciable rates, but Na or K stimulated activity only when ATP was used as substrate. Activity was inhibited by Ca and by low concentrations of Na deoxycholate, and was sensitive to inhibition by thiol group reagents. The enzyme could be distinguished from another enzyme, also present in the fraction, which was Ca-activated, and which exhibited a wider substrate specificity, different pH activation characteristics, lower specific activity, lack of stimulation by Na or K, and less sensitivity to inhibition by deoxycholate and by thiol group reagents. These findings formed the basis for demonstration of the Mg-dependent ATPase in situ.

Adenosine Triphosphatases↗
PubMed1981

Identification of a purine (P2) receptor linked to ion transport in a cultured renal (MDCK) epithelium.

1 Exogenous adenosine triphosphate (ATP) stimulates the short circuit current (SCC) in a cultured renal-derived epithelium (MDCK). Half-maximal stimulation is achieved at 1.91 X 10(-5) M ATP. 2 It is suggested that ATP interacts with a P2 purine receptor upon the basis of (a) agonist potency (ATP greater than adenosine diphosphate much greater than adenosine monophosphate, adenosine; ATP greater than uridine triphosphate greater than inosine triphosphate much greater than cytosine triphosphate, guanosine triphosphate); (b) the inhibition of the ATP response by quinidine (1 X 10(-3) M) but not by theophylline (1 X 10(-3) M). 3 Indomethacin (1 X 10(-5) M) inhibits the response of the cultured epithelium to ATP. 4 Prostaglandin E1 (PGE1) stimulates SCC but potentiates the effect of ATP on SCC. The divalent cationic ionophore A23187 (1 X 10(-6) M) transiently stimulates SCC itself and abolishes ATP-induced stimulation of the SCC.

Adenosine Triphosphate↗