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Separation of adenosine diphosphate-adenosine triphosphate–exchange activity from the cerebral microsomal sodium-plus-potassium ion-stimulated adenosine triphosphatase 下载免费PDF全文
1. A microsomal fraction from ox cerebral cortex catalysed [(14)C]ADP-ATP exchange at a speed similar to that at which it liberated P(i) from ATP in the presence of Na(+), K(+) and Mg(2+). 2. Repeated washing the fraction with MgATP solutions solubilized most of the exchange activity and left the adenosine triphosphatase insoluble and little changed in activity. The exchange activity was accompanied by negligible adenosine-triphosphatase activity and was enriched by precipitation at chosen pH and by DEAE-Sephadex. At no stage was its activity affected by Na(+), K(+) or ouabain. 3. The washed microsomal fraction was exposed to a variety of reagents; a sodium iodide-cysteine treatment increased both adenosine-triphosphatase and exchange activities, as also did a synthetic zeolite. Preparations were obtained with exchange activities less than 3% of their Na(+)-plus-K(+)-stimulated adenosine-triphosphatase activity. Some contribution to the residual exchange activity was made by an adenylate kinase. 4. Thus over 95% of the microsomal ADP-ATP-exchange activity does not take part in the Na(+)-plus-K(+)-stimulated adenosine-triphosphatase reaction. Participation of some of the residual 3% of the ADP-ATP-exchange activity has not been excluded, but there appears no firm evidence for its participation in the adenosine triphosphatase; the bearing of this conclusion on mechanisms proposed for the Na(+)-plus-K(+)-stimulated adenosine triphosphatase is indicated. 相似文献
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Although the substrate binding properties of adenylate kinase (AK) have been studied extensively by various biochemical and biophysical techniques, it remains controversial whether uncomplexed adenosine 5'-triphosphate (ATP) binds to the adenosine 5'-monophosphate (AMP) site of AK. We present two sets of experiments which argue against binding of ATP to the AMP site. (a) 31P nuclear magnetic resonance titration of ATP with AK indicated a 1:1 stoichiometry on the basis of changes in coupling constants and line widths. This ruled out binding of ATP to both sites. (b) ATP and MgATP were found to behave similarly by protecting AK from spontaneous inactivation while AMP showed only a small degree of protection. Such inactivation could also be protected or reversed by dithioerythritol and is most likely due to oxidation of sulfhydryl groups, one of which (cysteine-25) is located near the MgATP site. The results support binding of ATP to the MgATP site predominantly, instead of the AMP site, in the absence of Mg2+. 相似文献
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Two adenosine molecules are connected via their ribose moieties by transacetalation with 2,2,5,5-tetraethoxyhexane, yielding diastereoisomeric bis(isopropylidene adenosine) compounds with S,S- (1a) or R,S-configurated (1b) acetal carbons. The S,S isomer shows high hypochromicity and a pronounced positive Cotton effect, which implies strong stacking interactions. The stacking of 1b is less pronounced. Both isomers are substrates for mammalian adenosine deaminase (EC 3.5.4.4.). Whereas compound 1a is slowly deaminated due to steric hindrance and stacking interactions, the diastereoisomer 1b is a much better substrate for the enzyme. Because of the difference in configuration in 1b the adenosine moieties are processed stepwise. Moreover, isomer 1b is a strong competitive inhibitor for the deamination of adenosine by the enzyme. 相似文献
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1. K(+) did not affect the Mg(2+)-dependent transphosphorylation but markedly increased the Na(+)-stimulated ADP-ATP exchange rate mediated by a microsomal fraction from guinea-pig kidney. 2. Rb(+), Cs(+), NH(4) (+) and Li(+) were equally effective in stimulating the Na(+)-dependent ADP-ATP exchange activity. 3. Treatment of the microsomal fraction with N-ethylmaleimide or increased concentrations of Mg(2+) prevented stimulation of the Na(+)-dependent exchange reaction by K(+). 4. Ouabain (2.5mum) inhibited ATP hydrolysis by 33% but did not decrease the K(+)-stimulated Na(+)-dependent ADP-ATP exchange rate. 5. A possible mechanism for stimulation of exchange activity by K(+) is discussed. 相似文献
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Carbon monoxide (CO) is produced endogenously by heme oxygenase (HO) enzymes. HO-1 is highly expressed in many inflammatory
disease states, where it is broadly protective. The protective effects of HO-1 expression can be largely mimicked by the exogenous
application of CO and CO-releasing molecules (CORMs). Despite a dearth of pharmacological tools for their study, molecular
methodologies have identified P2X4 receptors as a potential anti-nociceptive drug target. P2X4 receptors are up-regulated
in animal models of inflammatory pain, and their knock-down reduces pain behaviours. In these same animal models, HO-1 expression
is anti-nociceptive, and we therefore investigated whether P2X4 was a target for CO and tricarbonyldichlororuthenium (II)
dimer (CORM-2). Using conventional whole-cell and perforated-patch recordings of heterologously expressed human P2X4 receptors,
we demonstrate that CORM-2, but not CO gas, is an inhibitor of these channels. We also investigated the role of soluble guanylate
cyclase and mitochondria-derived reactive oxygen species using pharmacological inhibitors but found that they were largely
unable to affect the ability of CORM-2 to inhibit P2X4 currents. A control breakdown product of CORM-2 was also without effect
on P2X4. These results suggest that P2X4 receptors are not a molecular target of endogenous CO production and are, therefore,
unlikely to be mediating the anti-nociceptive effects of HO-1 expression in inflammatory pain models. However, these results
show that CORM-2 is an effective antagonist at human P2X4 receptors and represents a useful pharmacological tool for the study
of these receptors given the current dearth of antagonists. 相似文献
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ATPace?, a novel injectable formulation of adenosine 5′-triphosphate (ATP), is developed by Cordex Pharma, Inc. (Cordex) as a diagnostic and therapeutic drug for the management of cardiac bradyarrhythmias. Extracellular ATP exerts multiple effects in various cell types by activating cell-surface receptors known as P2 receptors. In the heart, ATP suppresses the automaticity of cardiac pacemakers and atrioventricular (AV) nodal conduction via adenosine, the product of its degradation by ecto-enzymes, as well as by triggering a cardio-cardiac vagal reflex. ATP, given as a rapid intravenous bolus injection, has been used since the late 1940s as a highly effective and safe therapeutic agent for the acute termination of reentrant paroxysmal supraventricular tachycardia (PSVT) involving the AV node. In addition, preliminary studies have shown that ATP can also be used as a diagnostic agent for the identification of several cardiac disorders including sinus node dysfunction (sick sinus syndrome), dual AV nodal pathways, long QT syndrome, and bradycardic syncope. The US Food and Drug Administration has approved Cordex formulation for ATP as an Investigational New Drug and two pathways for its marketing approval; one therapeutic, i.e., acute termination of paroxysmal PSVT, and the other diagnostic, i.e., the identification of patients with bradycardic syncope who can benefit from pacemaker therapy. The scientific rationale for the development of ATPace? is discussed. 相似文献
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Shantanu Pal Won Jun Choi Seung Ah Choe Cara L. Heller Zhan-Guo Gao Moshe Chinn Kenneth A. Jacobson Xiyan Hou Sang Kook Lee Hea Ok Kim Lak Shin Jeong 《Bioorganic & medicinal chemistry》2009,17(10):3733-3738
On the basis of potent and selective binding affinity of truncated 4′-thioadenosine derivatives at the human A3 adenosine receptor (AR), their bioisosteric 4′-oxo derivatives were designed and synthesized from commercially available 2,3-O-isopropylidene-d-erythrono lactone. The derivatives tested in AR binding assays were substituted at the C2 and N6 positions. All synthesized nucleosides exhibited potent and selective binding affinity at the human A3 AR. They were less potent than the corresponding 4′-thio analogues, but showed still selective to other subtypes. The 2-Cl series generally were better than the 2-H series in view of binding affinity and selectivity. Among compounds tested, compound 5d (X = Cl, R = 3-bromobenzyl) showed the highest binding affinity (Ki = 13.0 ± 6.9 nM) at the hA3 AR with high selectivity (at least 88-fold) in comparison to other AR subtypes. Like the corresponding truncated 4′-thio series, compound 5d antagonized the action of an agonist to inhibit forskolin-stimulated adenylate cyclase in hA3 AR-expressing CHO cells. Although the 4′-oxo series were less potent than the 4′-thio series, this class of human A3 AR antagonists is also regarded as another good template for the design of A3 AR antagonists and for further drug development. 相似文献
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Summary Amino acids are activated by reaction with adenosine 5-phosphorimidazolide in aqueous imidazole buffers. If adenosine 5-(O-methylphosphate), an analogue of the 3-terminus of t-RNA is present, 2(3)-O-aminoacyladenosine 5-(O-methylphosphate) is formed. Fifteen percent of this compound accumulated at pH 5.8, but less was formed at higher pHs. The highest efficiency of utilization of ImpA attained in our experiments was about 24%. Analogous reactions occured with several other amino acids, including a number that have functional side-chains.Abbreviations pA
adenosine 5-monophosphate
- MepA
adenosine-5-(O-methylphosphate)
- ImpA
adenosine-5-phosphorimidazolide
- A
adenosine
- MepA-ala
2(3)-O-alanyl-adenosine-5-(O-methylphosphate)
- ala-N-pA
adenylyl-(5 N)-alanine
- ImH
imidazole
- DKP
diketopiperazine 相似文献
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Microsomes from guinea-pig cerebral cortex contain a system capable of exchanging ADP with ATP at rates of about 20mumoles/mg. of protein/hr. The ADP-ATP-exchange reaction requires Mg(2+) for activity. The reaction is not stimulated by Na(+) or K(+) and is not inhibited by ouabain, in contrast with the Na(+)-plus-K(+)-stimulated adenosine triphosphatase. The pH optimum also differs from that of the adenosine triphosphatase. The ADP-ATP-exchange reaction is stimulated two- to three-fold by non-ionic, anionic and cationic detergents, even when these agents are inhibiting the adenosine-triphosphatase reaction. This reaction may represent a component of the Na(+)-plus-K(+)-stimulated adenosine-triphosphatase reaction but is more likely to be due to other enzyme systems present in microsomal subfractions. 相似文献
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1. Bacilysin, a peptide which yields l-alanine and l-tyrosine on acid hydrolysis, was produced by a strain of Bacillus subtilis (A 14) in a chemically defined medium containing glucose, ammonium acetate or ammonium chloride, potassium phosphate and other inorganic salts, and ferric citrate. 2. Under the conditions used growth was diphasic. Bacilysin was formed during the second phase of slower growth, and there was little production during the stationary phase. Nevertheless, bacilysin production occurred when protein synthesis was inhibited by chloramphenicol. It thus appears that there is no obligatory coupling of protein synthesis and bacilysin synthesis. 3. When dl-[1-(14)C]alanine was added to a growing culture of B. subtilis, (14)C was incorporated into bacilysin, which contains an N-terminal alanine residue. 4. Under similar conditions virtually no (14)C was incorporated into bacilysin from dl-[2-(14)C]tyrosine, l-[U-(14)C]tyrosine or [1-(14)C]acetate, although these compounds were used by the cell for the biosynthesis of other substances. These results indicate that neither tyrosine nor acetate is a precursor of the fragment of bacilysin which yields tyrosine on hydrolysis with hot 6n-hydrochloric acid. 5. The tyrosine-yielding fragment of bacilysin was labelled with (14)C from [1,6-ring-(14)C(2)]shikimic acid. The biosynthesis of bacilysin thus appears to involve a diversion from the pathway leading to aromatic amino acids at the shikimic acid stage, or a subsequent one. 相似文献
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Ellen V. Langemeijer Dennis Verzijl Stefan J. Dekker Ad P. IJzerman 《Purinergic signalling》2013,9(1):91-100
The concept of functional selectivity offers great potential for the development of drugs that selectively activate a specific intracellular signaling pathway. During the last few years, it has become possible to systematically analyse compound libraries on G protein-coupled receptors (GPCRs) for this ‘biased’ form of signaling. We screened over 800 compounds targeting the class of adenosine A1 receptors using a β-arrestin-mediated signaling assay in U2OS cells as a G protein-independent readout for GPCR activation. A selection of compounds was further analysed in a G protein-mediated GTPγS assay. Additionally, receptor affinity of these compounds was determined in a radioligand binding assay with the agonist [3H]CCPA. Of all compounds tested, only LUF5589 9 might be considered as functionally selective for the G protein-dependent pathway, particularly in view of a likely overestimation of β-arrestin signaling in the U2OS cells. Altogether, our study shows that functionally selective ligands for the adenosine A1 receptor are rare, if existing at all. A thorough analysis of biased signaling on other GPCRs also reveals that only very few compounds can be considered functionally selective. This might indicate that the concept of functional selectivity is less common than speculated. 相似文献
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Kinetic studies of the reverse reaction catalysed by adenosine triphosphate–creatine phosphotransferase. The inhibition by magnesium ions and adenosine diphosphate 下载免费PDF全文
1. Kinetic investigations of the reaction catalysed by ATP–creatine phosphotransferase have been carried out. 2. No firm conclusions could be reached about the reaction of Mg2+ at the nucleotide-binding site of the enzyme. The value of the kinetic constant for this reaction depends on the value used for the apparent stability constant of the metal ion–nucleotide complex and, to a smaller extent, on the method of plotting the results. 3. At higher concentrations Mg2+ is a non-competitive inhibitor of the enzyme with respect to both MgADP− and phosphocreatine. 4. ADP3− is a competitive inhibitor of the enzyme with respect to MgADP− and a non-competitive inhibitor with respect to phosphocreatine. 5. The concentration of phosphocreatine has little, if any, effect on the kinetic constants for the nucleotide reactants. 相似文献
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Roots of spinach (Spinacia oleracea L.) seedlings contained only a very low activity of adenosine 5-phosphosulfate sulfotransferase compared to the cotyledons. Adenosine 5-phosphosulfate sulfotransferase activity increased about tenfold in cotyledons during greening. Preparation of organelle fractions from spinach leaves by a combination of differential and isopycnic density gradient centrifugation showed that adenosine 5-phosphosulfate sulfotransferase banded with NADP-glyceraldehyde-3-phosphate dehydrogenase, a marker enzyme for intact chloroplasts. In the fractions of peroxisomes, mitochondria and broken chloroplasts virtually no adenosine 5-phosphosulfate sulfotransferase activity was measured. Comparison with the chloroplast enzyme NADP-glyceraldehyde-3-phosphate dehydrogenase indicates that in spinach, adenosine 5-phosphosulfate sulfotransferase is localized almost exclusively in the chloroplasts.Abbreviations APS
Adenosine 5-phosphosulfate
- APSSTase
Adenosine 5-phosphosulfate sulfotransferase
- BSA
Bovine serum albumin
- BRIJ58
Polyethylene glycolmonostearylether
- DTE
Dithioerythritol
- DTT
Dithiothreitol
- EDTA
Ethylenediaminetetraacetic acid
- ME
2-Mercaptoethanol
- NADP-GPD
NADP-linked glyceraldehyde-3-phosphate dehydrogenase
- PAPS
Adenosine 3-phosphate 5-phosphate 5-phosphosulfate
- POPOP
1,4 Di [2-(5-phenyloxazolyl)]-benzene
- PPO
2,5-Diphenyloxazol
The results presented in this paper are taken from the Ph. D. thesis of H.F. 相似文献
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Do benzodiazepines bind at adenosine uptake sites in CNS? 总被引:6,自引:0,他引:6
Benzodiazepines inhibit adenosine uptake into rat cerebral cortical synaptosomes and their potency as inhibitors of adenosine uptake is closely correlated with therapeutic efficacy. Agents which possess “benzodiazepine like” activities such as CL218,872, zopiclone and fominoben and which displace benzodiazepine binding to brain cell membranes, are also inhibitors of adenosine uptake into brain synaptosomes. The IC50 values of all these compounds as inhibitors of adenosine uptake are in close agreement with the IC50 values obtained for the displacement of benzodiazepine binding to the brain receptors. Adenosine uptake inhibitors (dipyridamole, hexobendine, papaverine, 6-(2-hydroxy-5-nitrobenzyl)thioguanosine) which competitively inhibit adenosine uptake, presumably by blocking adenosine binding to its carrier-protein, are competitive inhibitors of diazepam binding to the brain membrane receptors. The finding of a pronounced correlation between inhibition of benzodiazepine binding and inhibition of adenosine uptake further supports the proposal that benzodiazepines may exert part of their pharmacological action through the inhibition of adenosine uptake. 相似文献
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31P NMR chemical shifts of salts of adenosine 5′-triphosphate and diphosphate: ATPH2?22(Me4N+) · H2O, ATPH2?22 Na+ · 3.5 H2O, ATPH2?2Mg2+ · 4 H2O, ATPH2?2Ca2+ · 2 H2O, ADPH2?2(Me4N+) · H2O and ADPH2?Mg2+ · 4 H2O have been measured in 0.02 M 2H2O solutions at 145.7 MHz (22° C) at constant p2H values (8.20 and 6.20). The results are compared with those obtained from salts of adenosine 5′-monophosphate and other simpler phosphomonoesters, e.g. AMP2?2(Me4N+), AMP2?Mg2+, AMPH?Me4N+ and (AMPH?)2Mg2+. It is concluded that the effects exerted by Mg2+ and Ca2+ on the 31P NMR shifts of dipoly- and tripolyphosphates relative to monovalent cations are due mainly to changes in conformation of the polyphosphate chain rather than to purely electronic factors associated with the binding of divalent cations to the phospho-oxyanions. The data are consistent with the existence of the following complexes at p2H 8.20: (MgPαPβ)ADP? and (MgPαPγ)ATP2?af (MgPαPβ)ATP2?af (MgPβPγ)ATP2? with the latter equilibrium relatively fast in the NMR time scale. Monoprotonation of the terminal phosphate appears to weaken the Mg2+-polyphosphate binding, particularly at Pβ of MgADPH and at Pβ and Pγ of MgATPH?. The Mg2+-polyphosphate binding weakens further at p2H 3.70, i.e. in MgATPH2. Possible implications of the results in the mechanism of actomyosin Mg2+-ATPase in muscle contraction are discussed. 相似文献
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1. A non-enzymic method for the preparation of adenosine 5′-diphosphate is described, in which the terminal phosphate of adenosine 5′-triphosphate is transferred to methanol in the presence of hydrochloric acid. The final purified product can be obtained in 60% yield. 2. Experiments with [14C]methanol showed that no methylation of the adenosine diphosphate occurs during the reaction. 3. Confirmation that the pyrophosphate moiety of the adenosine diphosphate produced was in the 5′-position was obtained by: (a) periodate oxidation; (b) treatment with apyrase and examination of the resulting adenylic acid isomer by paper chromatography. 4. The method appears to be generally applicable to the preparation of nucleoside 5′-diphosphates from the corresponding nucleoside 5′-triphosphates. 相似文献