Number Of Phosphate Groups Research Articles

Fate of low-molecular-weight organic phosphorus compounds in the P-rich and P-poor paddy soils

Continuous application of organic fertilizers can cause accumulation of organic phosphorus (P) in soil, especially in the low-molecular-weight organic phosphorus (LMWOP) forms. This organic P pool represents a potentially important source of P for both plants and microorganisms. To understand the effect of long-term fertilization (30 years) (P-rich soil) vs. fallowing (P-poor soil) on the bioavailability and fate of LMWOP in subtropical paddy soils, we determined the sorption and mineralization of 14C-labeled adenosine, adenosine monophosphate (AMP), adenosine diphosphate (ADP), and adenosine triphosphate (ATP) in each soil. The contents of carbon, nitrogen, and P in the P-rich soil were more than two times greater than those in the P-poor soil. The mineralization rates of the LMWOP compounds were faster in the P-rich soil compared to the P-poor soil, and followed the order AMP>ADP>ATP. Using sterilized soil, all forms of adenosine-P were strongly sorbed to the solid phase and reached saturation in a short time, with the adsorbance increasing with the number of phosphate groups. We concluded that the mineralization of LMWOP compounds was repressed slightly by sorption to the solid phase, but only in the short term. Thus, LMWOP compounds serve as readily available sources of C for microorganisms, making P available for themselves as well as for the plants. However, P accumulation and the progressive saturation of the P sorption sites in highly fertile soils may increase the potential risk of P runoff.

Critical phosphate salt concentrations leading to altered micellar casein structures and functional intermediates

We investigated the effect of different phosphate salts on the structural integrity of micellar casein (MC) at pH 7.0. With the increase of salt concentration, a reduction in turbidity was observed for the MC solutions, and it was modeled using an exponential decay function. The inflection point of the model was defined as the first critical salt concentration (C*), and it is suggested that the salt concentration initiates the disintegration of MC. For linear polyphosphates, C* decreased with the number of phosphate groups. Apparent viscosity (ηapp) of MC solutions increased with the increase of salt concentration, and they recorded a peak while the turbidity decreased to a minimum. The salt concentration that resulted in the highest ηapp was identified as the second critical salt concentration (C**). It is hypothesized that the interactions among protein species present in the mixtures are at an optimum state at C**. Both C* and C** were found to be dependent on the MC concentration. The work presented herein supports an understanding of the concentration effect of phosphate salts on MC for structuring dairy products.

Interaction of Nucleotides with a Trinuclear Terbium(III)-Dizinc(II) Complex: Efficient Sensitization of Terbium Luminescence by Guanosine Monophosphate and Application to Real-Time Monitoring of Phosphodiesterase Activity.

An in-depth study of the interaction of a trinuclear terbium(III)-dizinc(II) complex with an array of nucleotides differing in the type of nucleobase and number of phosphate groups, as well as cyclic versus acyclic variants, is presented. The study examined the nature of the interaction and the efficiency at which guanine was able to sensitize terbium(III) luminescence. Competitive binding and titration studies were performed to help establish the nature/mode of the interactions. These established that (1) interaction occurs by the coordination of phosphate groups to zinc(II) (in addition to uridine in the case of uridine monophosphate), (2) acyclic nucleotides bind more strongly than cyclic counterparts because of their higher negative charge, (3) guanine-containing nucleotides are able to sensitize terbium(III) luminescence with the efficiency of sensitization following the order guanosine monophosphate (GMP) > guanosine diphosphate > guanosine triphosphate because of the mode of binding, and (4) nucleoside monophosphates bind to a single zinc(II) ion, whereas di- and triphosphates appear to bind in a bridging mode between two host molecules. Furthermore, it has been shown that guanine is a sensitizer of terbium(III) luminescence. On the basis of the ability of GMP to effectively sensitize terbium(III)-based luminescence while cyclic GMP (cGMP) does not, the complex has been utilized to monitor the catalytic conversion of cGMP to GMP by a phosphodiesterase enzyme in real time using time-gated luminescence on a benchtop fluorimeter. The complex has the potential to find broad application in monitoring the activity of enzymes that process nucleotides (co)substrates, including high-throughput drug-screening programs.

Unexpected catalytic activity of Pd(II)-coordinated nucleotides in hydrogenation reduction

Controlled synthesis of noble metal nanocatalysts with desirable catalytic activities remains a great challenge. Inspired by electron-rich functional groups and supramolecular assembly properties of natural nucleotides, herein we propose a versatile approach to construct Pd nanocatalysts through employing seven nucleotides as both electron donors and stabilizers. The catalytic performance of nucleotide-Pd complexes are greatly associated with the chemical structures of nucleobases as well as the number of phosphate groups. Guanosine 5′-triphosphate-Pd complexes possess high activities in the hydrogenation reduction of 4-nitrophenol assisted by NaBH4, with the relative rate constant of 7770 min−1 mM−1 at 25 °C. Pd(II)-coordinated nucleotides are also promising in NaBH4-mediated degradation of organic dyes. This work demonstrates a green and facile process to construct Pd nanocatalysts containing abundant active sites under the assistance by natural nucleotides.

Analysis of mononucleotides by tandem mass spectrometry: investigation of fragmentation pathways for phosphate- and ribose-modified nucleotide analogues

Synthetic nucleotide and nucleic acid analogues are useful research tools and modern therapeutics. Hence, methods for the rapid and unambiguous identification of mononucleotides derived from organic syntheses or biological materials are of broad interest. Here, we analysed over 150 mononucleotides (mostly nucleoside 5′-mono-, 5′-di-, and 5′-triphosphates) and their structurally related nucleobase-, phosphate-, and ribose-modified analogues by electrospray tandem mass spectrometry (ESI/MS/MS), identifying characteristic fragmentation ions that may be helpful in structure determination. While positive-ion mode yielded fragments derived mainly from nucleobases, negative-ion mode provided insight into the structures of phosphoryl and phosphoribosyl moieties, enabling the determination of structural features such as the number of phosphate groups and the presence of ribose or phosphate substitutions. Based on these data, we proposed fragmentation pathways that were confirmed by experiments with [18O]-isotopologues. We demonstrated the utility of ESI(−)/MS/MS in the analysis of structurally related compounds by analysing isomeric and isobaric nucleotides and applying ESI(−)/MS/MS to rapid identification of nucleotide synthesis products. We formulated general rules regarding nucleotide structure–fragmentation pattern relationships and indicating characteristic fragmentation ions for the interpretation of ESI(−)/MS/MS spectra of nucleotides and their analogues. The ESI(−)/MS/MS spectra of all nucleotides are available in an on-line database, msTide, at www.msTide-db.com.

Inositol phosphates and phosphoinositides activate insulin-degrading enzyme, while phosphoinositides also mediate binding to endosomes

Insulin-degrading enzyme (IDE) hydrolyzes bioactive peptides, including insulin, amylin, and the amyloid β peptides. Polyanions activate IDE toward some substrates, yet an endogenous polyanion activator has not yet been identified. Here we report that inositol phosphates (InsPs) and phosphatdidylinositol phosphates (PtdInsPs) serve as activators of IDE. InsPs and PtdInsPs interact with the polyanion-binding site located on an inner chamber wall of the enzyme. InsPs activate IDE by up to ∼95-fold, affecting primarily Vmax The extent of activation and binding affinity correlate with the number of phosphate groups on the inositol ring, with phosphate positional effects observed. IDE binds PtdInsPs from solution, immobilized on membranes, or presented in liposomes. Interaction with PtdInsPs, likely PtdIns(3)P, plays a role in localizing IDE to endosomes, where the enzyme reportedly encounters physiological substrates. Thus, InsPs and PtdInsPs can serve as endogenous modulators of IDE activity, as well as regulators of its intracellular spatial distribution.

High-Confidence Sequencing of Phosphopeptides by Electron Transfer Dissociation Mass Spectrometry Using Dinuclear Zinc(II) Complex.

Phosphorylation is the most abundant protein modification, and tandem mass spectrometry (MS2) with electron transfer dissociation (ETD) has proven to be a promising method for phosphoproteomic applications owing to its ability to determine phosphorylation sites on proteins. However, low precursor charge states hinder the ability to obtain useful information through peptide sequencing by ETD, and the presence of acidic phosphate groups contributes to a low charge state of peptide ions. In the present report, we used a dinuclear zinc complex, (Zn2L)3+ (L = alkoxide form of 1,3-bis[bis(pyridin-2-ylmethyl)amino]propan-2-ol) for electrospray ionization (ESI), followed by ETD-MS2 analysis. Since (Zn2L)3+ selectively bound to phosphopeptide with addition of a positive charge per phosphate group, the use of (Zn2L)3+ for ESI improved the ionization yield of phosphopeptides in phosphoprotein digest. Additionally, an increase in the charge state of phosphopeptides were observed by addition of (Zn2L)3+, facilitating phosphopeptide sequencing by ETD-MS2. Since the binding between (Zn2L)3+ and the phosphate group was retained during the ETD process, a comparison between the ETD mass spectra obtained using two dinuclear zinc complex derivatives containing different zinc isotopes, namely (64Zn2L)3+ and (68Zn2L)3+, provided information about the number of phosphate groups in each fragment ion, allowing the phosphorylation site to be unambiguously determined. The details of the fragmentation processes of the (Zn2L)3+-phosphopeptide complex were investigated using a density functional theory calculation. As in the case of protonated peptides, ETD induced peptide backbone dissociation in the (Zn2L)3+-phosphopeptide complex proceeded through an aminoketyl radical intermediate.

Characterization of complex, heterogeneous lipid A samples using HPLC-MS/MS technique I. Overall analysis with respect to acylation, phosphorylation and isobaric distribution.

We established a new reversed phase-high performance liquid chromatography method combined with electrospray ionization quadrupole time-of-flight tandem mass spectrometry for the simultaneous determination and structural characterization of different lipid A types in bacteria (Escherichia coli O111, Salmonella adelaide O35 and Proteus morganii O34) showing serological cross-reactivity. The complex lipid A mixtures (obtained by simple extraction and acid hydrolysis of the outer membrane lipopolysaccharides) were separated and detected without phosphate derivatization. Several previously unidentified ions were detected, which differed in the number and type of acyl chains and number of phosphate groups. In several cases, we observed the different retention of isobaric lipid A species, which had different secondary fatty acyl distribution at the C2' or the C3' sites. The fragmentation of the various, C4' monophosphorylated lipid A species in deprotonated forms provided structural assignment for each component. Fragmentation pathways of the tri-acylated, tetra-acylated, penta-acylated, hexa-acylated and hepta-acylated lipid A components and of the lipid A partial structures are suggested. As standards, the hexa-acylated ion at m/z 1716 with the E. coli-type acyl distribution and the hepta-acylated ion at m/z 1954 with the Salmonella-type acyl distribution were used. The results confirmed the presence of multiple forms of lipid A in all strains analyzed. In addition, the negative-ion mode MS permitted efficient detection for non-phosphorylated lipid A components, too. Copyright © 2016 John Wiley & Sons, Ltd.

Transitions from simple electrolyte to polyelectrolyte in a series of polyphosphates

The dependence of polyelectrolyte characteristics on the number of phosphate groups in a series of polyphosphates was clarified by potentiometric titration and 23Na nuclear magnetic resonance using the Donnan model. Ca2+ ion binding to various polyphosphates was consistently inhibited as supporting electrolyte concentration increased. The electrostatic interactions between polyions and counterions, which is peculiar to polyelectrolytes, was enhanced by an increase in the number of phosphate groups. The polyelectrolyte characteristics increased non-monotonically with the number of phosphate groups that constituted the molecules, that is, (1) gradual emergence with three or four phosphate groups, (2) marked strengthening from four to eight phosphate groups, and (3) saturation with eight or more phosphate groups. The characteristics of polyelectrolyte molecules comprising eight or more phosphate groups were independent of the degree of polymerization. These interesting behaviors were observed to be closely similar using two quite different experimental techniques, electrochemical titration and nuclear magnetic resonance.

Cytosolic Nucleotides Block and Regulate the Arabidopsis Vacuolar Anion Channel AtALMT9

The aluminum-activated malate transporters (ALMTs) form a membrane protein family exhibiting different physiological roles in plants, varying from conferring tolerance to environmental Al(3+) to the regulation of stomatal movement. The regulation of the anion channels of the ALMT family is largely unknown. Identifying intracellular modulators of the activity of anion channels is fundamental to understanding their physiological functions. In this study we investigated the role of cytosolic nucleotides in regulating the activity of the vacuolar anion channel AtALMT9. We found that cytosolic nucleotides modulate the transport activity of AtALMT9. This modulation was based on a direct block of the pore of the channel at negative membrane potentials (open channel block) by the nucleotide and not by a phosphorylation mechanism. The block by nucleotides of AtALMT9-mediated currents was voltage dependent. The blocking efficiency of intracellular nucleotides increased with the number of phosphate groups and ATP was the most effective cellular blocker. Interestingly, the ATP block induced a marked modification of the current-voltage characteristic of AtALMT9. In addition, increased concentrations of vacuolar anions were able to shift the ATP block threshold to a more negative membrane potential. The block of AtALMT9-mediated anion currents by ATP at negative membrane potentials acts as a gate of the channel and vacuolar anion tune this gating mechanism. Our results suggest that anion transport across the vacuolar membrane in plant cells is controlled by cytosolic nucleotides and the energetic status of the cell.

Analysis of decapping scavenger cap complex using modified cap analogs reveals molecular determinants for efficient cap binding

Decapping scavenger (DcpS) assists in precluding inhibition of cap-binding proteins by hydrolyzing cap species remaining after mRNA 3'→5' degradation. Its significance was reported in splicing, translation initiation and microRNA turnover. Here we examine the structure and binding mode of DcpS from Caenorhabditiselegans (CeDcpS) using a large collection of chemically modified methylenebis(phosphonate), imidodiphosphate and phosphorothioate cap analogs. We determine that CeDcpS is a homodimer and propose high accuracy structural models of apo- and m(7) GpppG-bound forms. The analysis of CeDcpS regioselectivity uncovers that the only site of hydrolysis is located between the β and γ phosphates. Structure-affinity relationship studies of cap analogs for CeDcpS reveal molecular determinants for efficient cap binding: a strong dependence on the type of substituents in the phosphate chain, and reduced binding affinity for either methylated hydroxyl groups of m(7) Guo or an extended triphosphate chain. Docking analysis of cap analogs in the CeDcpS active site explains how both phosphate chain mobility and the orientation in the cap-binding pocket depend on the number of phosphate groups, the substituent type and the presence of the second nucleoside. Finally, the comparison of CeDcpS with its well known human homolog provides general insights into DcpS-cap interactions.

Impact of phosphorylation on the encapsulation of nucleoside analogues within porous iron(iii) metal–organic framework MIL-100(Fe) nanoparticles

Encapsulation of azidothymidine (AZT) or its phosphorylated derivatives (AZT-MP and AZT-TP) has been performed using nanoparticles of the porous crystalline iron(iii) trimesate metal-organic framework MIL-100(Fe). The number of phosphate groups per nucleoside analogue has a high impact on the drug loading capacity, and their interaction with the Lewis acid sites from the nanoMOFs is also discussed through a combination of techniques such as UV-vis absorption, circular dichroism, isothermal titration calorimetry, HPLC and molecular simulations. Finally, the effect of the differences in terms of host-guest interactions is discussed through the release in physiological buffers of AZT, AZT-MP and AZT-TP. New perspectives for the nanoencapsulation of monophosphorylated nucleoside analogues for effective anti-cancer and anti-viral therapies are then discussed.

Label-free, nucleotide-mediated dispersion of magnetic nanoparticles for “non-sandwich type” MRI-based quantification of enzyme

In the present work, we demonstrate that nucleotide can adsorb efficiently on the surface of carboxylic acid-functionalized nanoparticles and stabilize the particles against aggregation. In the present study we take magnetic nanoparticles (MNPs), manganese oxide nanoparticles (MnO), and upconversion nanophosphors (UCNPs) as models. The result shows that not only MNPs, but also other kinds of nanoparticles that have similar surface properties can be dispersed and stabilized by nucleotides. Interestingly, adenosine bearing different numbers of phosphate groups has distinct stabilizing effect. On the basis of this observation, we developed a magnetic relaxation-based enzyme assay for quantitative analysis of alkaline phosphatase. A detection limit of 0.002U/μL for calf intestine alkaline phosphatase (CIAP) could be obtained, which is lower than the gold nanoparticle-based colorimetric method. In contrast to the conventional magnetic relaxation switches (MRSw), this assay was achieved without covalent modification and separation steps, sandwich type binding was not required as well, which would potentially expand the application of magnetic relaxation-based analysis.

Nanodiamond-based two-step sampling of multiply and singly phosphorylated peptides for MALDI-TOF mass spectrometry analysis

Simultaneous detection of multiply and singly phosphorylated peptides using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) is challenging because of suppression effects during ionization. In oder to overcome this problem, this study presents a new approach to improve the detection of phosphopeptides by stepwise enrichment using polyarginine-coated (PA-coated) and titanium dioxide-coated (TiO(2)-coated) nanodiamonds for fractionation of multiply and singly phosphorylated peptides prior to on-probe MALDI MS analysis. The feasibility of this approach was demonstrated using synthetic peptides containing different numbers of phosphate groups, tryptic digests of α-casein, β-casein, and complex protein mixtures. The high specificity of the approach is shown in its effective enrichment and fractionation of phosphopeptides from the digest of β-casein and bovine serum albumin at a molar ratio as low as 1 : 1000, which out-performs the commercial Fe(3+)-IMAC and TiO(2) isolation kits. It offers a simple and effective alternative for the fractionation and identification of multiply and singly phosphorylated peptides by MALDI MS and allows for deduction of more information from limited starting materials.

Highly Sensitive Analysis of the Interaction between HIV-1 Gag and Phosphoinositide Derivatives Based on Surface Plasmon Resonance

Human immunodeficiency virus type 1 (HIV-1) Gag protein is the principal structural component of the HIV particle. Localization of the Pr55(Gag) protein to the plasma membrane initiates virus assembly. Recent studies indicated that d-myo-phosphatidylinositol (PI) 4,5-bisphosphate (PI(4,5)P2) regulates Pr55(Gag) localization and assembly. We determined the binding affinity between Pr55(Gag) or its N-terminal MA domain and various phosphoinositide derivatives using a highly sensitive surface plasmon resonance (SPR) sensor and biotinylated inositol phosphate. The equilibrium dissociation constants obtained using this approach reflected the distinct magnitude of acyl group-based and phosphate group-based interactions. The dissociation constant (K(D)) for Pr55(Gag) complexed with 1,4,5-IP3 (an inositol with divalent phosphate groups and devoid of lipid groups) was 2170 microM, while the K(D) for di-C(8)-PI (a lipid-containing inositol devoid of divalent phosphate groups) was 186 microM, and the K(D) for di-C(8)-PI(4,5)P2 (an inositol with both lipid and divalent phosphate groups) was 47.4 microM. The same trend in affinity was observed when these phosphoinositides were complexed with MA. Our results suggest that the contribution of hydrophobic acyl chains is greater than negatively charged inositol phosphates in Pr55(Gag)/MA binding. Furthermore, each inositol phosphate (devoid of lipid groups) tested showed a distinct Pr55(Gag)-binding affinity depending on the position and number of phosphate groups. However, the position and number of phosphate groups had no effect on MA-binding affinity.

Diadenosine polyphosphates in cultured vascular smooth-muscle cells and endothelium cells--their interaction with specific receptors and their degradation.

The role of diadenosine polyphosphates (ApnA, where "A" denotes "adenosine" and "n" denotes the number of phosphate groups "p") as vasoconstrictors of smooth-muscle cells and as blood-pressure regulating and insulin-releasing compounds has been described. It was the aim of this study to investigate whether specific receptors for these compounds, mediating the above mentioned effects, occur in cultured vascular smooth-muscle cells (VSMC) and in endothelium cells, and whether these compounds are degraded during incubation. Saturable binding sites for diadenosine polyphosphate [3H]Ap4A with an extremely quick saturation equilibrium, even at low temperature (4 degrees C), are present in vascular smooth-muscle cells. Diadenosine polyphosphates at micromolar concentrations displaced [3H]Ap4A from binding sites; the ranking order was Ap4A > Ap3A > Ap5A approximately Ap6A. Compounds interacting with purinergic P2X receptors such as suramin, alpha,beta-methylene ATP and pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid (PPADS), albeit at high concentrations, displaced [3H]Ap4A from its binding sites. Surprisingly, at low concentrations the compounds tested increased the binding of [3H]Ap4A, which might imply the occurrence of positive receptor cooperativity or inhibition of [3H]Ap4A degradation. By use of thin-layer chromatography it was observed that [3H]Ap4A was quickly degraded (half-life approx. 12 min) in the extracellular medium to (mainly) adenosine and inosine. [3H]Ap4A and its degradation products were quickly taken up by the cells. Degradation can be inhibited by Ap6A, alpha,beta-methylene ATP or PPADS. Rather similar degradation and uptake results were also obtained when endothelium cells were used. These data indicate that specific binding sites for [3H]Ap4A are present in vascular smooth-muscle cells and that diadenosine polyphosphates at physiological concentrations displace binding. The receptors involved might be distinct diadenosine polyphosphate receptors, although the involvement of others, such as P2X receptors, is also possible. Ap4A is quickly degraded in the extracellular space and compounds that inhibit degradation result in an increase in [3H]Ap4A binding. It should be remembered that when diadenosine polyphos-phates are being investigated in physiological and pathophysiological studies of their impact on smooth-muscle cell proliferation and on vasoconstriction (blood-pressure regulation), results obtained from long-term incubations might be critical.

The desorption and reactivity of butanol adsorbed on lithium iron phosphate (LISICON) activated in a hydrogen plasma

The reactivity and desorption of butanol-2 adsorbed on Li3Fe2(PO4)3 not subjected and subjected to treatment in a glow discharge hydrogen plasma were studied under flow conditions with a gas chromatographic analysis of products. X-ray photoelectron spectroscopy data showed that the number of phosphate groups on the surface of the phosphate was two times larger than the stoichiometric number and increased after plasma chemical treatment. The strength of butanol-phosphate bonds also increased, and the selectivity of alcohol decomposition with the formation of an olefin (dehydration) and ketone (dehydrogenation) changed. After plasma treatment, dehydrogenation centers were deactivated. The selectivities of alcohol transformations in the adsorbed state and under vapor phase conditions were different. Ketone was formed from adsorbed alcohol because the activation energies of dehydrogenation were equal for the two reaction variants.

Symmetry of electrostatic interaction between pyrophosphate DNA molecules

We study chiral electrostatic interaction between artificial ideal homopolymer DNA-like molecules in which a number of phosphate groups of the sugar-phosphate backbone are exchanged for the pyrophosphate ones. We employ a model in which the DNA is considered as a one-dimensional lattice of dipoles and charges corresponding to base pairs and (pyro)phosphate groups, respectively. The interaction between molecules of the DNA is described by a pair potential U of electrostatic forces between the two sets of dipoles and charges belonging to respective lattices describing the molecules. Minima of the potential U indicate orientational ordering of the molecules and thus liquid crystalline phases of the DNA. We use numerical methods for finding the set of minima in conjunction with symmetries verified by the potential U . The symmetries form a non-commutative group of 8th order, S . Using the group S we suggest a classification of liquid crystalline phases of the DNA, which allows several cholesteric phases, that is polymorphism. Pyrophosphate forms of the DNA could clarify the role played by charges in their liquid crystalline phases, and open experimental research, important for nano-technological and bio-medical applications.

Separation of phosphoprotein isotypes having the same number of phosphate groups using phosphate‐affinity SDS‐PAGE

Herein, we demonstrate the separation of phosphoprotein isotypes having the same number of phosphate groups using phosphate-affinity SDS-PAGE. The phosphate-affinity site is a polyacrylamide-bound Phos-tag that enables the mobility shift detection of phosphoproteins from their nonphosphorylated counterparts. As the first practical example of the separation, we characterized the monophosphorylated Tau isotypes by each of three tyrosine kinases, c-Abl, MET, and Fyn. Each monophosphoisotype phosphorylated at the Tyr-394, Tyr-197, or Tyr-18 was detected as three distinct migration bands. As a further application, we extended this technique to the mobility shift analysis of His and Asp phosphoisotypes in the Sinorhizobium meliloti FixL/FixJ two-component system. FixL is autophosphorylated at the His-285 with ATP, and the phosphate group is transferred to the Asp-54 of FixJ and subsequently removed by the FixL phosphatase activity. Using this method, we first performed simultaneous detection of the phosphorylated and nonphosphorylated isotypes of FixL and FixJ generated in their phosphotransfer reaction in vitro. As a result, a monophosphoisotype of FixL containing the phosphorylated His residue was confirmed. As for FixJ, on the other hand, two monophosphoisotypes were detected as two distinct migration bands. One is a well-known isotype phosphorylated at the Asp-54. The other is a novel isotype phosphorylated at the His-84.

Diadenosine polyphosphates Ap3A and Ap4A, but not Ap5A or Ap6A, induce proliferation of vascular smooth muscle cells

Depending on the number of phosphate groups, diadenosine polyphosphates (ApnA, Ap3A, Ap4A, Ap5A and Ap6A) differ in properties such as proliferation, apoptosis, vasoconstriction and vasodilatation of vascular smooth muscle cells (VSMCs). Possible signaling pathways leading to effects such as proliferation are still unknown. This study examined the proliferative effects of diadenosine polyphosphates on VSMCs and their intracellular pathways. Proliferation of VSMCs was measured by the cell count and [ 3H] thymidine incorporation. Phosphorylation of the MAP kinases ERK1/2 was determined by Western blotting. Single-cell [Ca 2+] i measurements were done to determine the influence of [Ca 2+] i on intracellular signaling. Stress fiber formation was assessed by fluorescence microscopy to detect an influence of Gα 12. Ap3A and Ap4A, but not Ap5A or Ap6A, were shown to increase proliferation of VSMCs by activating P2Y receptors, which leads to stimulation of the Ras-Raf-MEK-ERK1/2 cascade. Ap3A- and Ap4A-induced activation of the MAP kinases ERK1/2 was dependent on a signaling pathway that included the EGF receptor, PKC, PLCβ and the increase of [Ca 2+] i. In conclusion, Ap3A and Ap4A, but not Ap5A or Ap6A, induce proliferation of VSMCs by a signaling pathway that begins with activation of P2Y receptors and leads to stimulation of the MAP kinases ERK1/2.