Purification of chemically synthesised dinucleoside(5′,5′) polyphosphates by displacement chromatography

Jankowski, Joachim; Potthoff, Wilhelm; Zidek, Walter; Schlüter, Hartmut

doi:10.1016/s0378-4347(98)00408-3

Cited by 19 publications

(12 citation statements)

References 10 publications

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“…The final purification step is preparative reversed phase (RP) HPLC on standard RP-18 material. An HPLC method to purify dinucleotides by displacement chromatography has been developed [44].…”

Section: Purification Of Nucleotidesmentioning

confidence: 99%

P2 Receptors Activated by Uracil Nucleotides - An Update

Brunschweiger¹,

Müller²

2006

CMC

134

174

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Pyrimidine nucleotides, including UTP, UDP and UDP-glucose, are important signaling molecules which activate G protein-coupled membrane receptors (GPCRs) of the P2Y family. Four distinct pyrimidine nucleotide-sensitive P2Y receptor subtypes have been cloned, P2Y2, P2Y4, P2Y6 and P2Y14. P2Y2 and P2Y4 receptors are activated by UTP (the P2Y2, and the rat but not the human P2Y4 receptor are also activated by ATP), the P2Y6 receptor is activated by UDP, and the P2Y14 receptor by UDP-glucose. Furthermore, non-P2Y GPCRs, the cysteinylleukotriene receptors (CysLT1R and CysLT2R) have been described to be activated by UDP in addition to activation by cysteinylleukotrienes. While P2Y2, P2Y4, and P2Y6 receptor activation results in stimulation of phospholipase C, the P2Y14 receptor is coupled to inhibition of adenylate cyclase. Derivatives and analogs of the physiological nucleotides UTP, UDP and ATP have been synthesized and evaluated in order to obtain enzymatically stable, subtype-selective agonists. The P2Y2 receptor agonists diuridine tetraphosphate (diquafosol) and the uracil-cytosine dinucleotide denufosol are currently undergoing clinical trials for dry eye disease, retinal detachment disease, upper respiratory tract symptoms, and cystic fibrosis, respectively. The first antagonists for P2Y2 and P2Y6 receptors that appear to be selective versus other P2Y receptor subtypes have recently been described. Selective antagonists for P2Y4 and P2Y14 receptors are still lacking. Uracil nucleotide-sensitive P2Y receptor subtypes may constitute future targets for the treatment of certain cancer types, vascular diseases, inflammatory diseases, and immunomodulatory intervention. They have also been proposed to play a role in neurodegenerative diseases. This article is an updated version of "P2-Pyrimidinergic Receptors and Their Ligands" by C. E. Müller published in Curr. Pharm. Des. 2002, 8, 2353-2369.

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Section: Purification Of Nucleotidesmentioning

confidence: 99%

P2 Receptors Activated by Uracil Nucleotides - An Update

Brunschweiger¹,

Müller²

2006

CMC

134

174

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“…41 Briefly, Ap 8 A was synthesized by mixing adenosine tetraphosphate (50 mmol/L) with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (2.5 mol/L), HEPES (2 mol/L), and magnesium chloride (125 mmol/L). The substances were dissolved in water, thoroughly mixed with a vortex mixer, and incubated at 37°C at pH 6.5 for 48 hours.…”

Section: Synthesis and Chromatography Of Diadenosine Octaphosphate Asmentioning

confidence: 99%

Identification and Quantification of Diadenosine Polyphosphate Concentrations in Human Plasma

Jankowski

Laufer

et al. 2003

ATVB

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Objective-Diadenosine polyphosphates have been demonstrated to be involved in the control of vascular tone as well as the growth of vascular smooth muscle cells and hence, possibly, in atherogenesis. In this study we investigated the question of whether diadenosine polyphosphates are present in human plasma and whether a potential source can be identified that may release diadenosine polyphosphates into the circulation. Methods and Results-Plasma diadenosine polyphosphates (Ap n A with nϭ3 to 6) were purified to homogeneity by affinity-, anion exchange-, and reversed phase-chromatography from deproteinized human plasma. Analysis of the homogeneous fractions with matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) revealed molecular masses ([MϩH] ϩ ) of 757, 837, 917, and 997 d. Comparison of the postsource decay matrix-assisted laser desorption/ionization mass spectrometry mass spectra of these fractions with those of authentic diadenosine polyphosphates revealed that these isolated substances were identical to Ap 3 A, Ap 4 A, Ap 5 A, and Ap 6 A. Enzymatic analysis showed an interconnection of the phosphate groups with the adenosines in the 5Ј-positions of the ribose moieties. The mean total plasma diadenosine polyphosphate concentrations (mol/L; mean Ϯ SEM) in cubital veins of normotensive subjects amounted to 0.89Ϯ0.59 for Ap 3 A, 0.72Ϯ0.72 for Ap 4 A, 0.33Ϯ0.24 for Ap 5 A, and 0.18Ϯ0.18 for Ap 6 A. Cubital venous plasma diadenosine polyphosphate concentrations from normotensives did not differ significantly from those in the hypertensive patients studied. There was no significant difference between arterial and venous diadenosine polyphosphate plasma concentrations in 5 hemodialysis patients, making a significant degradation by capillary endothelial cells unlikely. Free plasma diadenosine polyphosphate concentrations are considerably lower than total plasma concentrations because approximately 95% of the plasma diadenosine polyphosphates were bound to proteins. There were no significant differences in the diadenosine polyphosphate plasma concentrations depending on the method of blood sampling and anticoagulation, suggesting that platelet aggregation does not artificially contribute to plasma diadenosine polyphosphate levels in significant amounts.The Ap n A (with nϭ3 to 6) total plasma concentrations in adrenal veins were significantly higher than the plasma concentrations in both infrarenal and suprarenal vena cava

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“…This procedure may be more effective to separate Ap n A from myocardial tissue, because displacement chromatography has been shown to be a powerful method for separation of dinucleotide polyphosphates. 26 From the presence of at least 5 different Ap n As in human myocardial tissue, it can be inferred that Ap n A may have specific functions in human heart. What is the significance of these findings for cardiac physiology and pathology?…”

Section: Discussionmentioning

confidence: 99%

Endogenous Diadenosine Tetraphosphate, Diadenosine Pentaphosphate, and Diadenosine Hexaphosphate in Human Myocardial Tissue

et al. 2004

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Abstract-Diadenosine polyphosphates have been characterized as extracellular mediators controlling numerous physiological effects. In this study, diadenosine tetraphosphate, diadenosine pentaphosphate, and diadenosine hexaphosphate were isolated and identified in human myocardial tissue. Human myocardial tissue was homogenized and fractionated by affinity chromatography, displacement chromatography, anion-exchange chromatography, and reversed-phase chromatography. In fractions purified to homogeneity, diadenosine tetraphosphate, diadenosine pentaphosphate, and diadenosine hexaphosphate were revealed by matrix-assisted laser desorption/ionization mass spectrometry and ultraviolet spectroscopy. These diadenosine polyphosphates were further identified by enzymatic analysis, which demonstrated an interconnection of the phosphate groups with the adenosines in the 5Ј positions of the riboses. Furthermore, diadenosine tetraphosphate, diadenosine pentaphosphate, and diadenosine hexaphosphate were found in human cardiac-specific granules, and the amount of diadenosine tetraphosphate, diadenosine pentaphosphate, and diadenosine hexaphosphate was estimated in the range of Ϸ500 mol/L. In conclusion, the experiments show that the diadenosine polyphosphates with 2 and 3 phosphate groups occur in human myocardial tissue, and so do the diadenosine polyphosphates with 4 to 6 phosphate groups. After being released by cholinergic stimulation, which is known to affect diadenosine polyphosphate release from secretory granules, diadenosine tetraphosphate, diadenosine pentaphosphate, and diadenosine hexaphosphate activate P2X purinoceptors in vascular smooth muscle; hence, they can act as vasoconstrictors. It may be inferred that the differential action of both predominantly vasodilator and vasoconstrictor diadenosine polyphosphates allow a fine-tuning of myocardial blood flow by locally released diadenosine polyphosphates. [1][2][3][4][5] The actions of the Ap n A within the cardiovascular system are mediated by the various purinoceptor subtypes. So far, 14 mammalian purinoceptor subtypes have been cloned, 6,7 and 6 Ap n As containing 2 to 7 phosphate groups have been identified in humans. 8 -11 The affinities of a given Ap n A to the various purinoceptor subtypes depends on the number of phosphate groups linking both adenosine moieties. 9,12,13 Moreover, the purinoceptor subtypes are very differently distributed within the cardiovascular system. Depending on the purinoceptor subtypes activated in a given tissue, the Ap n A are both vasoconstrictors and vasodilators, 14,15 inhibitors and stimulators of platelet aggregation, 8,9,11 and modulators of cell proliferation. 9,11,16 Given this diversity of Ap n A actions, it is not surprising that the Ap n A actions reported in literature widely differ among various species. Currently, it is difficult to decide to what extent species-dependent differences in Ap n A actions are caused by different purinoceptor distribution and to species-specificity of some of the known purinoceptor subtyp...

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Purification of chemically synthesised dinucleoside(5′,5′) polyphosphates by displacement chromatography

Cited by 19 publications

References 10 publications

P2 Receptors Activated by Uracil Nucleotides - An Update

P2 Receptors Activated by Uracil Nucleotides - An Update

Identification and Quantification of Diadenosine Polyphosphate Concentrations in Human Plasma

Endogenous Diadenosine Tetraphosphate, Diadenosine Pentaphosphate, and Diadenosine Hexaphosphate in Human Myocardial Tissue

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