92 results on '"Järv J"'
Search Results
2. Alumina coating of polyvinylidene fluoride (PVDF) surface in liquid phase.
- Author
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Liiv, J., Panov, D., Tenno, T., and Järv, J.
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- 2014
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3. Differentiating between drugs with short and long residence times.
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Kukk, S. and Järv, J.
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- 2016
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4. Role of water in determining organic reactivity in aqueous binary solvents
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Salmar Siim, Järv Jaak, Tenno Tiina, and Tuulmets Ants
- Subjects
solvent effects ,lfe relationships ,organic-aqueous solutions ,similarity coefficients ,hydrophobic interactions ,Chemistry ,QD1-999 - Published
- 2012
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5. Phosphorylation is switch of L-type pyruvate kinase allostery
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Faustova Ilona, Kuznetsov Aleksei, Juronen Erkki, Loog Mart, and Järv Jaak
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regulatory phosphorylation ,allostery ,l-type pyruvate kinase ,switch between allosteric and non-allosteric forms ,glycolysis regulation ,Biology (General) ,QH301-705.5 - Published
- 2010
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6. Ligand structure controlled allostery in cAMP-dependent protein kinase catalytic subunit
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Kuznetsov Aleksei and Järv Jaak
- Subjects
single-subunit allostery ,allosteric cooperativity ,enzyme kinetics ,enzyme inhibition ,ligand binding ,interaction factor ,peptide phosphorylation ,camp-dependent protein kinase catalytic subunit ,atp binding site ,peptide binding site ,Biology (General) ,QH301-705.5 - Published
- 2009
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7. Site-specificity of butyrylcholinesterase alkylation with N, N-dimethyl-2-phenylaziridinium ion
- Author
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Palumaa, P., Raba, R., and Järv, J.
- Published
- 1984
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8. Binding of specific ligands to muscarinic receptors alters the fluidity of membrane fragments from rat brain A fluorescence polarization study with lipid-specific probes
- Author
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Manevich, E.M., Köiv, A., Järv, J., Molotkovsky, J.G., and Bergelson, L.D.
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- 1988
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9. Two-step binding of green mamba toxin to muscarinic acetylcholine receptor
- Author
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Toomela, T., Jolkkonen, M., Rinken, A., Järv, J., and Karlsson, E.
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- 1994
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10. The Influence of Inorganic Salts on the Inhibition of Acetylcholinesterase by O,O-Diethylthiophosphates.
- Author
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Kesvatera, T., Järv, J., and Aaviksaar, A.
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- 1990
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11. The Mechanism of Cholinesterase Reactions with Organo-Phosphorus Quasisubstrates and Ester Substrates.
- Author
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Järv, J. and Sepp, A.
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- 1990
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12. ACE2 Peptide Fragment Interaction with Different S1 Protein Sites.
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Kuznetsov A, Arukuusk P, Härk H, Juronen E, Ustav M, Langel Ü, and Järv J
- Abstract
We study the effect of the peptide QAKTFLDKFNHEAEDLFYQ on the kinetics of the SARS-CoV-2 spike protein S1 binding to angiotensin-converting enzyme 2 (ACE2), with the aim to characterize the interaction mechanism of the SARS-CoV2 virus with its host cell. This peptide corresponds to the sequence 24-42 of the ACE2 α1 domain, which marks the binding site for the S1 protein. The kinetics of S1-ACE2 complex formation was measured in the presence of various concentrations of the peptide using bio-layer interferometry. Formation of the S1-ACE2 complex was inhibited by the peptide in cases where it was preincubated with S1 protein before the binding experiment. The kinetic analysis of S1-ACE2 complex dissociation revealed that preincubation stabilized this complex, and this effect was dependent on the peptide concentration as well as the preincubation time. The results point to the formation of the ternary complex of S1 with ACE2 and the peptide. This is possible in the presence of another binding site for the S1 protein beside the receptor-binding domain for ACE2, which binds the peptide QAKTFLDKFNHEAEDLFYQ. Therefore, we conducted computational mapping of the S1 protein surface, revealing two additional binding sites located at some distance from the main receptor-binding domain on S1. We suggest the possibility to predict and test the short protein derived peptides for development of novel strategies in inhibiting virus infections., Supplementary Information: The online version contains supplementary material available at 10.1007/s10989-021-10324-7., Competing Interests: Conflict of interestThe authors declare no competing interests., (© The Author(s), under exclusive licence to Springer Nature B.V. 2021.)
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- 2022
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13. In Vitro Ligand Binding Kinetics Explains the Pharmacokinetics of [ 18 F]FE-PE2I in Dopamine Transporter PET Imaging.
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Kukk S, Loog O, Hiltunen JV, and Järv J
- Abstract
Two of the most popular positron emission tomography (PET) tracers, [
11 C]PE2I and [18 F]FE-PE2I, used to quantify dopamine transporters (DAT), display dissimilar kinetic behavior in in vivo assays. This difference can be explained by comparing values of kinetic rate constants, which characterize interaction of these tracers with DAT sites in vitro . At the same time, this kinetic analysis showed that the overall binding mechanism is similar for these two tracers and includes a fast step of complex formation followed by a slow isomerization step of this complex. Comparison with previous PE2I data revealed that isomerization of the DAT complex with PE2I occurs three times faster than in the case of FE-PE2I, which leads to the slower onset of peak specific binding of the former tracer in the DAT-rich regions. Therefore, ligands with slower isomerization on-rate, including [18 F]FE-PE2I, seem to be better tracers in vivo , and their properties can be predicted in vitro ., Competing Interests: The authors declare no competing financial interest.- Published
- 2018
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14. Allosteric Effect of Adenosine Triphosphate on Peptide Recognition by 3'5'-Cyclic Adenosine Monophosphate Dependent Protein Kinase Catalytic Subunits.
- Author
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Kivi R, Solovjova K, Haljasorg T, Arukuusk P, and Järv J
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- 2-Naphthylamine analogs & derivatives, 2-Naphthylamine chemistry, Adenosine Triphosphate metabolism, Allosteric Regulation, Allosteric Site, Amino Acid Sequence, Binding Sites, Catalytic Domain, Cyclic AMP metabolism, Cyclic AMP-Dependent Protein Kinases metabolism, Fluorescent Dyes chemistry, Humans, Kinetics, Ligands, Peptides metabolism, Protein Binding, Protein Kinase Inhibitors metabolism, Staining and Labeling methods, Thermodynamics, Adenosine Triphosphate chemistry, Cyclic AMP chemistry, Cyclic AMP-Dependent Protein Kinases chemistry, Peptides chemistry, Protein Kinase Inhibitors chemistry
- Abstract
The allosteric influence of adenosine triphosphate (ATP) on the binding effectiveness of a series of peptide inhibitors with the catalytic subunit of 3'5'-cyclic adenosine monophosphate dependent protein kinase was investigated, and the dependence of this effect on peptide structure was analyzed. The allosteric effect was calculated as ratio of peptide binding effectiveness with the enzyme-ATP complex and with the free enzyme, quantified by the competitive inhibition of the enzyme in the presence of ATP excess, and by the enzyme-peptide complex denaturation assay, respectively It was found that the principle "better binding-stronger allostery" holds for interactions of the studied peptides with the enzyme, indicating that allostery and peptide binding with the free enzyme are governed by the same specificity pattern. This means that the allosteric regulation does not include new ligand-protein interactions, but changes the intensity (strength) of the interatomic forces that govern the complex formation in the case of each individual ligand. We propose that the allosteric regulation can be explained by the alteration of the intrinsic dynamics of the protein by ligand binding, and that this phenomenon, in turn, modulates the ligand off-rate from its binding site as well as the binding affinity. The positive allostery could therefore be induced by a reduction in the enzyme's overall intrinsic dynamics.
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- 2016
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15. Different States of Acrylodan-Labeled 3'5'-Cyclic Adenosine Monophosphate Dependent Protein Kinase Catalytic Subunits in Denaturant Solutions.
- Author
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Kivi R and Järv J
- Subjects
- 2-Naphthylamine analogs & derivatives, 2-Naphthylamine chemistry, Buffers, Catalytic Domain, Fluorescent Dyes chemistry, Humans, Hydrophobic and Hydrophilic Interactions, Protein Denaturation, Protein Unfolding, Spectrometry, Fluorescence, Staining and Labeling, Cyclic AMP-Dependent Protein Kinases chemistry, Guanidine chemistry, Morpholines chemistry, Urea chemistry
- Abstract
Fluorescence spectroscopy was used to differentiate between different states of acrylodan-labeled cAMP-dependent protein kinase catalytic subunits in urea, guanidine hydrochloride and 3-(N-morpholino)propanesulfonic acid solutions, by measuring changes in the emission spectrum of the protein-coupled dye, which is very sensitive to its microenvironment. Decomposition of the observed fluorescence spectra by a parameterized log-normal distribution function allowed the resolution of overlapping spectral bands and revealed the formation of three distinct protein states, denominated as native, denatured and unfolded structures. At low denaturant concentrations the formation of the denatured form from the native protein was observed, and this process was characterized by a blue-shift of the fluorescence spectrum of acrylodan, indicating that the dye was transferred into some water-deficit hydrophobic environment inside the protein molecule. Therefore, formation of a "dry molten globule" structure could be suggested in state. At high denaturant concentrations a red-shift of the emission spectrum of the protein-coupled probe was observed indicating significant extrusion of the dye molecule into water environment as a result of the unfolding of the protein structure.
- Published
- 2016
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16. Computational modeling of acrylodan-labeled cAMP dependent protein kinase catalytic subunit unfolding.
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Kuznetsov A, Kivi R, and Järv J
- Subjects
- 2-Naphthylamine chemistry, Catalytic Domain, 2-Naphthylamine analogs & derivatives, Cyclic AMP metabolism, Cyclic AMP-Dependent Protein Kinases chemistry, Models, Theoretical
- Abstract
Structure of the cAMP-dependent protein kinase catalytic subunit, where the asparagine residue 326 was replaced with acrylodan-cystein conjugate to implement this fluorescence reporter group into the enzyme, was modeled by molecular dynamics (MD) method and the positioning of the dye molecule in protein structure was characterized at temperatures 300K, 500K and 700K. It was found that the acrylodan moiety, which fluorescence is very sensitive to solvating properties of its microenvironment, was located on the surface of the native protein at 300K that enabled its partial solvation with water. At high temperatures the protein structure significantly changed, as the secondary and tertiary structure elements were unfolded and these changes were sensitively reflected in positioning of the dye molecule. At 700K complete unfolding of the protein occurred and the reporter group was entirely expelled into water. However, at 500K an intermediate of the protein unfolding process was formed, where the fluorescence reporter group was directed towards the protein interior and buried in the core of the formed molten globule state. This different positioning of the reporter group was in agreement with the two different shifts of emission spectrum of the covalently bound acrylodan, observed in the unfolding process of the protein., (Copyright © 2016 Elsevier Ltd. All rights reserved.)
- Published
- 2016
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17. NCAM-deficient mice show prominent abnormalities in serotonergic and BDNF systems in brain - Restoration by chronic amitriptyline.
- Author
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Aonurm-Helm A, Anier K, Zharkovsky T, Castrén E, Rantamäki T, Stepanov V, Järv J, and Zharkovsky A
- Subjects
- Animals, Brain metabolism, Disease Models, Animal, Electrochemical Techniques, Enzyme-Linked Immunosorbent Assay, Gene Expression Regulation drug effects, Gene Expression Regulation genetics, Mice, Mice, Inbred C57BL, Mice, Transgenic, Neural Cell Adhesion Molecules genetics, Phosphorylation drug effects, Phosphorylation genetics, Protein Binding drug effects, Protein Binding genetics, Receptor, trkB metabolism, Serotonin Plasma Membrane Transport Proteins metabolism, Adrenergic Uptake Inhibitors therapeutic use, Amitriptyline therapeutic use, Brain Diseases, Metabolic drug therapy, Brain Diseases, Metabolic genetics, Brain Diseases, Metabolic metabolism, Brain-Derived Neurotrophic Factor metabolism, Neural Cell Adhesion Molecules deficiency, Serotonin metabolism
- Abstract
Mood disorders are associated with alterations in serotonergic system, deficient BDNF (brain-derived neurotrophic factor) signaling and abnormal synaptic plasticity. Increased degradation and reduced functions of NCAM (neural cell adhesion molecule) have recently been associated with depression and NCAM deficient mice show depression-related behavior and impaired learning. The aim of the present study was to investigate potential changes in serotonergic and BDNF systems in NCAM knock-out mice. Serotonergic nerve fiber density and SERT (serotonin transporter) protein levels were robustly reduced in the hippocampus, prefrontal cortex and basolateral amygdala of adult NCAM(-)(/-) mice. This SERT reduction was already evident during early postnatal development. [(3)H]MADAM binding experiments further demonstrated reduced availability of SERT in cell membranes of NCAM(-)(/-) mice. Moreover, the levels of serotonin and its major metabolite 5-HIAA were down regulated in the brains of NCAM(-)(/-) mice. NCAM(-)(/-) mice also showed a dramatic reduction in the BDNF protein levels in the hippocampus and prefrontal cortex. This BDNF deficiency was associated with reduced phosphorylation of its receptor TrkB. Importantly, chronic administration of antidepressant amitriptyline partially or completely restored these changes in serotonergic and BDNF systems, respectively. In conclusion, NCAM deficiency lead to prominent and persistent abnormalities in brain serotonergic and BDNF systems, which likely contributes to the behavioral and neurobiological phenotype of NCAM(-/-) mice., (Copyright © 2015 Elsevier B.V. and ECNP. All rights reserved.)
- Published
- 2015
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18. Thermal Stability of Dopamine Transporters.
- Author
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Kukk S, Stepanov V, and Järv J
- Subjects
- Animals, Cell Line, Dopamine Plasma Membrane Transport Proteins metabolism, Mice, Protein Stability, Rats, Rats, Wistar, Dopamine Plasma Membrane Transport Proteins chemistry, Hot Temperature
- Abstract
The thermal stabilities of the rat and mouse dopamine transporter (DAT) proteins were studied within the temperature range of 0-37°C. The inactivation of the protein was followed by monitoring changes in radioligand-specific binding. We found that the process followed a rate equation with first-order kinetics and was characterized by having a single rate constant k inact. The activation energies (E a) that were calculated from the Arrhenius plots (ln k inact vs. 1/T) were 43 ± 5 and 45 ± 6 kJ/mol for the rat (rDAT) and mouse (mDAT) transporters, respectively, and 44 ± 7 kJ/mol for rDAT from PC-6.3 cell line. These E a values were similar to the E a values of thermal inactivation of the muscarinic receptor from rat brain cortex and to the thermal inactivation of other transmembrane proteins. However, all of these activation energy values were significantly lower than the E a values for soluble single-subunit proteins of similar size. These results therefore suggest that the thermal stability of transmembrane proteins may be governed to a significant extent by cell membrane properties and by interactions between the membrane components and the protein. In contrast, the stability of soluble proteins seems to be mostly governed by protein structure and size, which determine the sum of the stabilizing intramolecular interactions within the protein molecule. It is therefore not surprising that cell membrane properties and composition may have significant effects on the functional properties of transmembrane proteins.
- Published
- 2015
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19. Thermodynamic aspects of cAMP dependent protein kinase catalytic subunit allostery.
- Author
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Kivi R, Jemth P, and Järv J
- Subjects
- Animals, Catalytic Domain, Cyclic AMP-Dependent Protein Kinases genetics, Cyclic AMP-Dependent Protein Kinases metabolism, Escherichia coli, Kinetics, Mice, Protein Binding, Protein Subunits genetics, Protein Subunits metabolism, Recombinant Proteins chemistry, Recombinant Proteins genetics, Recombinant Proteins metabolism, Temperature, Thermodynamics, Allosteric Site, Cyclic AMP-Dependent Protein Kinases chemistry, Protein Subunits chemistry
- Abstract
Kinetics of thermal inactivation of acrylodan-labeled cAMP dependent protein kinase catalytic subunit, its binary complexes with ATP and peptide inhibitor PKI[5-24], respectively, and the ternary complex involving both of these ligands were studied at different temperatures (5-50 °C). The thermodynamic parameters ΔH and ΔS for ligand binding equilibria as well as for the allosteric interaction between the binding sites of these ligands were obtained by using the Van't Hoff analysis. The results indicated that more inter- and intra-molecular non-covalent bonds were involved in ATP binding with the protein when compared to the peptide binding. Similarly, nucleotide and peptide binding steps were accompanied with different entropy effects, while almost no entropy change accompanied PKI[5-24] binding, suggesting that the protein flexibility was not affected in this case. Differently from the binary complex formation the ternary complex formation was accompanied by a significant entropy change and with intensive formation of new non-covalent interactions (ΔH). At the same time both ligand binding steps as well as the allosteric interaction between ligand binding sites could be described by a common entropy-enthalpy compensation plot, pointing to a similar mechanism of these phenomena. It was concluded that numerous weak interactions govern the allostery of cAMP dependent protein kinase catalytic subunit.
- Published
- 2014
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20. Sonication effects on non-radical reactions. A sonochemistry beyond the cavitation?
- Author
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Tuulmets A, Piiskop S, Järv J, and Salmar S
- Abstract
The kinetics of pH-independent hydrolysis of 4-methoxyphenyl dichloroacetate were investigated under ultrasonic irradiation with an application of 10% of the maximum power of the equipment and without sonication in acetonitrile-water binary mixtures with a content of acetonitrile ranging from 0.008 to 35 wt.%. Similar kinetic investigations were performed at intensities corresponding to 10%, 20%, 30%, 40%, and 50% of the input energy in solvent mixtures containing 10 wt.% and 25 wt.% acetonitrile. In parallel, the responses of KI and terephthalic acid dosimeters at applied irradiation levels were registered under the same experimental conditions. Significant kinetic sonication effects were found at sound intensities presumably not inducing cavitation in the solution. This result provides an experimental evidence of kinetic effects of ultrasound in the absence of cavitation. A disturbing impact of cavitation on the ultrasonic acceleration of the reaction was found. The implications of these findings were discussed., (Copyright © 2013 Elsevier B.V. All rights reserved.)
- Published
- 2014
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21. Computational simulation of ligand docking to L-type pyruvate kinase subunit.
- Author
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Kuznetsov A, Faustova I, and Järv J
- Subjects
- Adenosine Diphosphate chemistry, Binding Sites, Computer Simulation, Fructosediphosphates chemistry, Ligands, Peptides chemistry, Phosphoenolpyruvate chemistry, Protein Structure, Tertiary, Protein Subunits chemistry, Molecular Docking Simulation, Pyruvate Kinase chemistry
- Abstract
Computational blind docking approach was used for mapping of possible binding sites in L-type pyruvate kinase subunit for peptides, RRASVA and the phosphorylated derivative RRAS(Pi)VA, which model the phosphorylatable N-terminal regulatory domain of the enzyme. In parallel, the same docking analysis was done for both substrates of this enzyme, phosphoenolpyruvate (PEP) and adenosine diphosphate (ADP), and for docking of fructose 1,6-bisphosphate (FBP), which is the allosteric activator of the enzyme. The binding properties of the entire surface of the protein were scanned and several possible binding sites were identified in domains A and C of the protein, while domain B revealed no docking sites for peptides or for substrates or the allosteric regulator. It was found that the docking sites of different ligands were partially overlapping, pointing to the possibility that some regulatory effects, observed in the case of L-type pyruvate kinase, may be caused by the competition of different ligands for the same binding sites., (Copyright © 2013 Elsevier Ltd. All rights reserved.)
- Published
- 2014
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22. Computer modeling of the dynamic properties of the cAMP-dependent protein kinase catalytic subunit.
- Author
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Izvolski A, Järv J, and Kuznetsov A
- Subjects
- Cyclic AMP-Dependent Protein Kinases metabolism, Models, Molecular, Protein Conformation, Protein Subunits metabolism, Cyclic AMP-Dependent Protein Kinases chemistry, Molecular Dynamics Simulation, Protein Subunits chemistry
- Abstract
The structural dynamics of the cAMP-dependent protein kinase catalytic subunit were modeled using molecular dynamics computational methods. It was shown that the structure of this protein as well as its complexes with ATP and peptide ligand PKI(5-24) consisted of a large number of rapidly inter-converting conformations which could be grouped into subsets proceeding from their similarity. This cluster analysis revealed that conformations which correspond to the "opened" and "closed" structures of the protein were already present in the free enzyme, and most surprisingly co-existed in enzyme-ATP and enzyme-PKI(5-24) complexes as well as in the ternary complex, which included both of these ligands. The results also demonstrated that the most mobile structure segments of the protein were located in the regions of substrate binding sites and that their dynamics were most significantly affected by the binding of the ATP and peptide ligand., (Copyright © 2013 Elsevier Ltd. All rights reserved.)
- Published
- 2013
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23. Kinetic sonication effects in aqueous acetonitrile solutions. Reaction rate levelling by ultrasound.
- Author
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Piiskop S, Salmar S, Tuulmets A, Kuznetsov A, and Järv J
- Subjects
- Kinetics, Solutions, Water chemistry, Acetonitriles chemistry, Sonication, Ultrasonics
- Abstract
The kinetics of the pH-independent hydrolysis of 4-methoxyphenyl dichloroacetate were investigated with and without ultrasonic irradiation in acetonitrile-water binary mixtures containing 0.008 to 35 wt.% of acetonitrile and the kinetic sonication effects (kson/knon) were calculated. Molecular dynamics (MD) simulations of the structure of the solutions were performed with ethyl acetate as the model ester. The ester is preferentially solvated by acetonitrile. The excess of acetonitrile over water in the solvation shell grows fast with an increase in the co-solvent content in the bulk solution. In parallel, the formation of a second solvation shell rich in acetonitrile takes place. Significant kinetic sonication effects for the hydrolysis were explained with facile destruction of the diffuse second solvation shell followed by a rearrangement of the remaining solvent layer under sonication. The rate levelling effect of ultrasound was discussed. In an aqueous-organic binary solvent, independent of the solvent composition, the ultrasonic irradiation evokes changes in the reaction medium which result in an almost identical solvation state of the reagent thus leading to the reaction rate levelling., (Copyright © 2013 Elsevier B.V. All rights reserved.)
- Published
- 2013
- Full Text
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24. Interaction of non-phosphorylated liver pyruvate kinase with fructose 1,6-bisphosphate and peptides that mimic the phosphorylatable N-terminus of the enzyme.
- Author
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Faustova I and Järv J
- Subjects
- Allosteric Regulation, Animals, Binding Sites, Cattle, Kinetics, Ligands, Liver metabolism, Peptides chemistry, Phosphorylation, Protein Structure, Tertiary, Rabbits, Rats, Fructose chemistry, Fructosediphosphates chemistry, Liver chemistry, Pyruvate Kinase chemistry
- Abstract
The interaction of non-phosphorylated L-type pyruvate kinase (L-PK) with fructose 1,6-bisphosphate (FBP), which is an allosteric activator of the phosphorylated enzyme, and peptides that mimic the phosphorylatable N-terminal regulatory domain of the enzyme, was studied. It was found that the catalytic activity of the enzyme was not enhanced in the presence of FBP, and this ligand acted as a relatively weak reversible inhibitor of the enzyme activity in the micromolar concentration range. The phosphorylation site analogue peptides RRASVA and RRAAVA had no effect on the activity of the enzyme, while the phosphorylated peptide RRAS(Pi)VA reversibly inhibited the enzyme and this process was characterised by the Ki value 47 μM. As the phosphorylated form of L-PK is a subject of significant allosteric regulation by FBP, it was concluded that phosphorylation should function as a molecular switch of the allosteric properties of this enzyme.
- Published
- 2013
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25. Novel systemically active galanin receptor 2 ligands in depression-like behavior.
- Author
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Saar I, Lahe J, Langel K, Runesson J, Webling K, Järv J, Rytkönen J, Närvänen A, Bartfai T, Kurrikoff K, and Langel Ü
- Subjects
- Amino Acid Sequence, Animals, Antidepressive Agents, Tricyclic pharmacology, Binding, Competitive drug effects, Cell Line, Tumor, Drug Design, Female, Galanin metabolism, Hindlimb Suspension, Humans, Imipramine pharmacology, Ligands, Male, Mice, Mice, Inbred BALB C, Molecular Sequence Data, Peptides chemical synthesis, Peptides pharmacology, Swimming psychology, Tissue Distribution, Behavior, Animal drug effects, Depression psychology, Receptor, Galanin, Type 2 drug effects
- Abstract
Neuropeptide galanin and its three G-protein coupled receptors, galanin receptor type 1-galanin receptor type 3 (GalR1-GalR3), are involved in the regulation of numerous physiological and disease processes, and thus represent tremendous potential in neuroscience research and novel drug lead development. One of the areas where galanin is involved is depression. Previous studies have suggested that activation of GalR2 leads to attenuation of depression-like behavior. Unfortunately, lack of in vivo usable subtype specific ligands hinders testing the role of galanin in depression mechanisms. In this article, we utilize an approach of increasing in vivo usability of peptide-based ligands, acting upon CNS. Thus, we have synthesized a series of novel systemically active galanin analogs, with modest preferential binding toward GalR2. We have shown that specific chemical modifications to the galanin backbone increase brain levels upon i.v. injection of the peptides. Several of the new peptides, similar to a common clinically used antidepressant medication imipramine, exerted antidepressant-like effect in forced swim test, a mouse model of depression, at a surprisingly low dose range (< 0.5 mg/kg). We chose one of the peptides, J18, for more thorough study, and showed its efficacy also in another mouse depression model (tail suspension test), and demonstrated that its antidepressant-like effect upon i.v. administration can be blocked by i.c.v. galanin receptor antagonist M35. The effect of the J18 was also abolished in GalR2KO animals. All this suggests that systemically administered peptide analog J18 exerts its biological effect through activation of GalR2 in the brain. The novel galanin analogs represent potential drug leads and a novel pharmaceutical intervention for depression., (© 2013 International Society for Neurochemistry.)
- Published
- 2013
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26. Kinetics of acrylodan-labelled cAMP-dependent protein kinase catalytic subunit denaturation.
- Author
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Kivi R, Loog M, Jemth P, and Järv J
- Subjects
- 2-Naphthylamine chemistry, Arginine chemistry, Cysteine chemistry, Kinetics, Protein Denaturation, Protein Subunits chemistry, Protein Subunits metabolism, Spectrometry, Fluorescence, 2-Naphthylamine analogs & derivatives, Cyclic AMP-Dependent Protein Kinases chemistry, Cyclic AMP-Dependent Protein Kinases metabolism
- Abstract
Fluorescence spectroscopy was used to study denaturation of cAMP-dependent protein kinase catalytic subunit labeled with an acrylodan moiety. The dye was covalently bound to a cystein residue introduced into the enzyme by replacement of arginine in position 326 in the native sequence, located near the enzyme active center. This labeling had no effect on catalytic activity of the enzyme, but provided possibility to monitor changes in protein structure through measuring the fluorescence spectrum of the dye, which is sensitive to changes in its environment. This method was used to monitor denaturation of the protein kinase catalytic subunit and study the kinetics of this process as well as influence of specific ligands on stability of the protein. Stabilization of the enzyme structure was observed in the presence of adenosine triphosphate, peptide substrate RRYSV and inhibitor peptide PKI[5-24].
- Published
- 2013
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27. Kinetic sonication effects in light of molecular dynamics simulation of the reaction medium.
- Author
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Salmar S, Kuznetsov A, Tuulmets A, Järv J, and Piiskop S
- Abstract
Molecular dynamics (MD) simulation of the structure of ethyl acetate solutions in two water-ethanol mixtures was performed at 280 and 330K. The MD simulations revealed that ethyl acetate was preferentially solvated by ethanol, water being mainly located in the next solvation layer. With increasing temperature ethanol was gradually replaced by water in the first solvation shell. These findings explain the decrease in the rate of ester hydrolysis with increasing molar ratio of ethanol in the solution as the reaction rate was linearly dependent on the relative ethanol content in the first solvation shell of the ester. Predominance of ethanol results in decreased polarity and water activity in the shell and accordingly in a decreased reaction rate. Based on the results of the MD simulations, the principal conclusion of this work is that ultrasound enhances the kinetic energy (the effective temperature) of species in the solution and, in this way, evokes shifts in the solvation equilibria thus affecting the reaction rate. It appears that ultrasound does not completely break down the solvent shells or clusters in the solution as previously believed. Phenomena of thermo-solvatochromism and reaction rate levelling by ultrasound in binary solvents are described., (Copyright © 2012 Elsevier B.V. All rights reserved.)
- Published
- 2013
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28. Novel galanin receptor subtype specific ligand in depression like behavior.
- Author
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Saar I, Runesson J, Järv J, Kurrikoff K, and Langel U
- Subjects
- Animals, CHO Cells, Cell Line, Tumor, Cricetinae, Cricetulus, Depression psychology, Female, Galanin metabolism, Humans, Ligands, Male, Mice, Mice, Inbred BALB C, Protein Binding physiology, Random Allocation, Receptor, Galanin, Type 2 agonists, Antidepressive Agents metabolism, Antidepressive Agents therapeutic use, Depression drug therapy, Depression metabolism, Receptor, Galanin, Type 2 metabolism
- Abstract
Neuropeptide galanin and its three receptors, galanin receptor type 1-galanin receptor type 3, are known to be involved in the regulation of numerous psychological processes, including depression. Studies have suggested that stimulation of galanin receptor type 2 (GalR2) leads to attenuation of the depression-like behavior in animals. However, due to the lack of highly selective galanin subtype specific ligands the involvement of different receptors in depression-like behavior is yet not fully known. In the present study we introduce a novel GalR2 selective agonist and demonstrate its ability to produce actions consistent with theorized GalR2 functions and analogous to that of the anti-depressant, imipramine.
- Published
- 2013
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29. Probing L-pyruvate kinase regulatory phosphorylation site by mutagenesis.
- Author
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Faustova I, Loog M, and Järv J
- Subjects
- Adenosine Diphosphate chemistry, Adenosine Diphosphate metabolism, Amino Acid Sequence, Amino Acid Substitution, Animals, Catalytic Domain, Kinetics, Molecular Sequence Data, Mutagenesis, Site-Directed, Phosphoenolpyruvate chemistry, Phosphoenolpyruvate metabolism, Phosphorylation, Pyruvate Kinase chemistry, Pyruvate Kinase genetics, Rats, Pyruvate Kinase metabolism
- Abstract
The activity of L-type pyruvate kinase (L-PK, ATP:pyruvate 2-O-phosphotransferase, EC 2.7.1.40) is regulated by phosphorylation of serine residue 12 of the N-terminal regulatory domain MEGPAGYLRR(10)AS ( 12 )VAQLTQEL(20)GTAFF of the protein. In this report we studied the effect of the point mutations around this phosphorylation site on the catalytic properties of this enzyme, by introducing amino acids A, L, K, Q and E into positions 9, 10 and 13 of this peptide sequence. It was found that some of these mutations in positions 9 and 10, although occurring at great distances from the enzyme's active site, affected the enzyme's activity by decreasing the effectiveness of phosphoenolpyruvate binding (PEP) with the enzyme, but had practically no influence on the binding effectiveness of the second substrate ADP. A similar asymmetric effect on the binding of these substrates was previously observed after phosphorylation of the enzyme regulatory N-domain peptide, and also after proteolytic truncation of the same N-terminal part of L-PK. All these results could be explained by the internal complex formation between the N-domain peptide and the enzyme's main body. The present study delineated the specificity of the internal binding site and revealed the possibility that the regulatory effect could be modulated by selecting mutation sites and amino acids introduced into the N-terminal domain structure.
- Published
- 2012
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30. Nβ-methylation changes the recognition pattern of aza-β3-amino acid containing peptidomimetic substrates by protein kinase A.
- Author
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Kisseljova K, Baudy-Floc'h M, Kuznetsov A, and Järv J
- Abstract
The protein kinase A (PKA)-catalyzed phosphorylation of peptide substrate RRASVA analogs, containing Nβ-Me-aza-β3-amino acid residues in all subsequent positions, was studied. This work follows along the lines of our previous research of the phosphorylation of aza-β3-analogs of RRASVA (the shortest active substrate of PKA) and allows characterizing the influence of Nβ-methylation of aza-β3-amino acid residues on substrate recognition by PKA on substrate binding and phosphorylation steps. It was found that the effect of Nβ-methylation was dependent upon the position of the structure alteration. Moreover, the presence of a single Nβ-methylation site in the substrate changed the recognition pattern of this series of peptidomimetics, strongly affecting the phosphorylation step. Structure modeling of aza-β3- and Nβ-Me-aza-β3-containing substrates revealed that Nβ-methylation of aza-β3-moieties changed the peptide bond geometry from trans- to cis-configuration in -CO-NMe- fragments, with an exception for the N-terminally methylated Nβ-Me-aza-β3-RRRASVA (with the N-terminal amino group not participating in the peptide bond) and RRAS-Nβ-Me-aza-β3-VA. As has been shown in literature, this conformational preference of the backbone has a significant influence on the flexibility of the peptide substrate chain. Following our results, this property seems to have significant influence on the recognition of the amino acid side groups by the enzyme binding site, and in the case of PKA this structural modification was decisive for the phosphate transfer step of the catalytic process.
- Published
- 2011
- Full Text
- View/download PDF
31. Effect of two simultaneous aza-β3-amino acid substitutions on recognition of peptide substrates by cAMP dependent protein kinase catalytic subunit.
- Author
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Kisseljova K, Kuznetsov A, Baudy-Floc'h M, and Järv J
- Subjects
- Amino Acid Sequence, Binding Sites, Catalytic Domain, Computer Simulation, Kinetics, Peptidomimetics, Phosphorylation, Substrate Specificity, Amino Acids chemistry, Aza Compounds chemistry, Cyclic AMP-Dependent Protein Kinases chemistry, Cyclic AMP-Dependent Protein Kinases metabolism, Peptides chemistry, Peptides metabolism
- Abstract
Peptidomimetic analogs of the hexapeptide RRASVA, containing simultaneously two aza-β(3)-amino acid residues in different positions of this sequence, except for the phosphorylatable serine residue, were synthesized and tested as substrates for the cAMP-dependent protein kinase catalytic subunit. All these peptidomimetics were phosphorylated by the enzyme and this reaction was characterized by the K(m) and k(cat) values as well as by the second-order rate constants k(II). Affinity and reactivity of all peptidomimetics was lower than that for the parent peptide RRASVA. The effect of backbone modification was dependent upon the positions where these two aza-β(3) residues were located, although the sequence of amino acid side groups remained the same in all compounds. It was found that the influence of two backbone modifications in the substrate structure was not described additively, i.e. the effect of each structural alteration was dependent upon the position of the second modification. The results were in agreement with the concept of specificity-determining clusters in the sequence of peptide and peptidomimetic ligands, which predominantly determine the molecular recognition of these ligands by their target sites and therefore serve as major modification points for the design of activity of peptidomimetic ligands., (Copyright © 2011 Elsevier Inc. All rights reserved.)
- Published
- 2011
- Full Text
- View/download PDF
32. Novel galanin receptor subtype specific ligands in feeding regulation.
- Author
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Saar I, Runesson J, McNamara I, Järv J, Robinson JK, and Langel U
- Subjects
- Amino Acid Sequence, Animals, Humans, Ligands, Male, Molecular Sequence Data, Rats, Rats, Sprague-Dawley, Receptors, Galanin chemistry, Tumor Cells, Cultured, Feeding Behavior, Receptors, Galanin metabolism
- Abstract
Galanin a 29/30-residue neuropeptide has been implicated in several functions in the central nervous system, including the regulation of food consumption. Galanin and its analogues administered intraventricularly or into the hypothalamic region of brain have been shown to reliably and robustly stimulate the consumption of food in sated rodents. Three galanin receptor subtypes have been isolated, all present in the hypothalamus, but little is known about their specific role in mediating this acute feeding response. Presently, we introduce several novel GalR2 selective agonists and then compare the most selective of these novel GalR2 subtype selective agonists to known GalR1 selective agonist M617 for their ability to stimulate acute consumption of several foods shown to be stimulated by central administration of galanin. GalR1 selective agonist M617 markedly stimulated acute consumption of high-fat milk, but neither GalR2 selective agonist affected either high-fat milk or cookie mash intake. The present results are consistent with the involvement of GalR1 in mediating the acute feeding consumption by galanin and suggest an approach applicable to exploring galanin receptor specificity in normal and abnormal behavior and physiology., (Copyright © 2011 Elsevier Ltd. All rights reserved.)
- Published
- 2011
- Full Text
- View/download PDF
33. Aza-beta(3)-amino acid containing peptidomimetics as cAMP-dependent protein kinase substrates.
- Author
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Kisseljova K, Kuznetsov A, Baudy-Floc'h M, and Järv J
- Subjects
- Amino Acids chemical synthesis, Amino Acids chemistry, Animals, Biomimetic Materials chemical synthesis, Biomimetic Materials chemistry, Mice, Peptides chemical synthesis, Peptides chemistry, Phosphorylation, Substrate Specificity, Amino Acids metabolism, Biomimetic Materials metabolism, Cyclic AMP-Dependent Protein Kinases metabolism, Peptides metabolism
- Abstract
Peptidomimetic analogs of the peptide RRASVA, known as the "minimal substrate" of the catalytic subunit of the cAMP-dependent protein kinase (PKA), were synthesized by consecutive replacement of natural amino acids by their aza-beta(3) analogs. The peptidomimetics were tested as PKA substrates and the kinetic parameters of the phosphorylation reaction were determined. It was found that the interaction of these peptidomimetics with the enzyme active center was sensitive to the location of the backbone modification, while the maximal rate of the reaction was practically not affected by the structure of substrates. The pattern of molecular recognition of peptidomimetics was in agreement with the results of structure modeling and also with the results of computational docking study of peptide and peptidomimetic substrates with the active center of PKA. It was concluded that the specificity determining factors which govern substrate recognition by the enzyme should be grouped along the phosphorylatable substrate, and such clustering might open new perspectives for pharmacophore design of peptides and peptide-like ligands., (2010 Elsevier Inc. All rights reserved.)
- Published
- 2010
- Full Text
- View/download PDF
34. Mechanism and stoichiometry of 2,2-diphenyl-1-picrylhydrazyl radical scavenging by glutathione and its novel alpha-glutamyl derivative.
- Author
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Viirlaid S, Mahlapuu R, Kilk K, Kuznetsov A, Soomets U, and Järv J
- Subjects
- Free Radical Scavengers metabolism, Glutathione analogs & derivatives, Glutathione metabolism, Indicators and Reagents chemistry, Kinetics, Peptides chemical synthesis, Peptides chemistry, Biphenyl Compounds chemistry, Free Radical Scavengers chemistry, Glutathione chemistry, Picrates chemistry
- Abstract
Kinetic mechanism and stoichiometry of scavenging the 2,2-diphenyl-1-picrylhydrazyl radical by glutathione and its novel analog, containing alpha-glutamyl residue in place of the gamma-glutamyl moiety, were studied using different ratios of reagents. At low concentrations of the peptides, the process was described as a bimolecular reaction obeying the stoichiometric ratio 1:1. However, at excess of peptides the formation of a non-covalent complex between the reagents was discovered and characterized by dissociation constants K = 0.61 mM for glutathione and K = 0.27 mM for the glutathione alpha-glutamyl analog, respectively. The complex formation was followed by a reaction step that was characterized by the similar rate constant k = 0.02 s(-1) for both peptides. Thus, the apparently different antioxidant activity of these two peptides, observed under common assay conditions, was determined by differences in the formation of this non-covalent complex.
- Published
- 2009
- Full Text
- View/download PDF
35. A novel GalR2-specific peptide agonist.
- Author
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Runesson J, Saar I, Lundström L, Järv J, and Langel U
- Subjects
- Amino Acid Sequence, Animals, Cell Line, Galanin, Humans, Inositol Phosphates metabolism, Peptides chemical synthesis, Protein Binding, Signal Transduction, Peptides pharmacology, Receptor, Galanin, Type 2 agonists
- Abstract
The galanin peptide family and its three receptors have with compelling evidence been implicated in several high-order physiological disorders. The co-localization with other neuromodulators and the distinct up-regulation during and after pathological disturbances has drawn attention to this neuropeptide family. In the current study we present data on receptor binding and functional response for a novel galanin receptor type 2 (GalR2) selective chimeric peptide, M1145 [(RG)(2)-N-galanin(2-13)-VL-(P)(3)-(AL)(2)-A-amide]. The M1145 peptide shows more than 90-fold higher affinity for GalR2 over GalR1 and a 76-fold higher affinity over GalR3. Furthermore, the peptide yields an agonistic effect in vitro, seen as an increase in inositol phosphate (IP) accumulation, both in the absence or the presence of galanin. The peptide design with a N-terminal extension of galanin(2-13), prevails new insights in the assembly of novel subtype specific ligands for the galanin receptor family and opens new possibilities to apply the galanin system as a putative drug target.
- Published
- 2009
- Full Text
- View/download PDF
36. Kinetic mechanism of dopamine transporter interaction with 1-(2-(bis-(4-fluorophenyl)methoxy)ethyl)-4-(3-phenylpropyl)piperazine (GBR 12909).
- Author
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Stepanov V and Järv J
- Subjects
- Animals, Binding, Competitive drug effects, Binding, Competitive physiology, Dopamine metabolism, Female, Isomerism, Kinetics, Molecular Structure, Protein Conformation drug effects, Radioligand Assay, Rats, Rats, Wistar, Subcellular Fractions, Time Factors, Brain drug effects, Brain metabolism, Dopamine Plasma Membrane Transport Proteins drug effects, Dopamine Plasma Membrane Transport Proteins metabolism, Dopamine Uptake Inhibitors pharmacology, Piperazines pharmacology
- Abstract
Interaction of piperazine-based dopamine transporter inhibitor GBR12909 with rat dopamine transporters has been studied by means of competition kinetics analysis, employing [(3)H]PE2I as the reporter ligand. It has been found that GBR12909 is capable of inducing so-called "slow isomerization step" upon binding to DAT, probably consisting of a conformational change in the transporter protein. The mechanism exhibited by GBR12909 appears to be similar to the mechanism of PE2I that has been reported earlier and also confirms previous observations for GBR12783 made by Do-Rego and co-workers using dopamine uptake data. It appears that the isomerization phenomenon previously described for PE2I is not limited to tropane-based DAT inhibitors, but is, in fact, a general property of dopamine transporter protein, similar to "isomerization" process reported previously for G-protein coupled receptors. The rapid first step of association of the GBR 12909 is characterized by the equilibrium constant K(L)=34+/-11nM and the second slow step by k(i)=0.033+/-0.005s(-1).
- Published
- 2008
- Full Text
- View/download PDF
37. Production of biosensors with exchangeable enzyme-containing threads.
- Author
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Rinken T, Järv J, and Rinken A
- Subjects
- Calibration, Catalysis, Diffusion, Glucose analysis, Kinetics, Membranes, Artificial, Sensitivity and Specificity, Biocompatible Materials chemistry, Biosensing Techniques methods, Enzymes, Immobilized, Glucose Oxidase chemistry, Nylons chemistry
- Abstract
We introduce a simple method for the construction of biosensors, based on coiling an enzyme-containing, thread-shaped material around a cylindrical signal transducer in the form of winding stairs with a variable length of step and so forming a variable biocatalytic membrane on the sensor surface, which can be easily modified for particular purposes. In the model system, we immobilized glucose oxidase (GO) on a nylon thread, formatted from a sheaf of numerous minor filaments and used as a biorecognition element integrated with a Clark-type oxygen sensor. The immobilized enzyme was evenly distributed throughout the thread, and the activity of the enzyme could be measured in units of length. Appropriate pieces of the enzyme-containing thread with a certain amount of GO could be cut for a definite biosensor or bioreactor. The enzyme amount and substrate diffusion parameters, which together control the sensor's working range and sensitivity, could be changed simultaneously with the change of the length of the thread. Besides glucose oxidase, experiments with other enzymes have confirmed the applicability of the proposed technological solution. Thus, the thread-type matrixes enable one to construct sensors with a required range of work, sensitivity, and selectivity, which can be easily customized within seconds.
- Published
- 2007
- Full Text
- View/download PDF
38. Ultrasonic evidence of hydrophobic interactions. Effect of ultrasound on benzoin condensation and some other reactions in aqueous ethanol.
- Author
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Tuulmets A, Hagu H, Salmar S, Cravotto G, and Järv J
- Subjects
- Benzaldehydes chemistry, Catalysis, Chemical Phenomena, Chemistry, Physical, Hydrolysis, Kinetics, Potassium Cyanide chemistry, Temperature, Thermodynamics, Ultrasonics, Water, Benzoin chemistry, Ethanol chemistry
- Abstract
The kinetics of KCN-catalyzed benzoin condensation of benzaldehyde in water and ethanol-water binary mixtures was investigated both under ultrasound at 22 kHz and without sonication. Thermodynamic activation parameters were calculated from kinetic data obtained at 35, 50, and 65 degrees C. Evidence that ultrasound can retard reactions is reported and hence a direct proof that sonochemical processes occur in the bulk solution. Former results and literature data for ester hydrolyses and tert-butyl chloride solvolysis are involved in the discussion. A quantitative relationship between sonication effects and the hydrophobicity of reagents is presented for the first time. Ultrasound affects hydrophobic interactions with the solvent, which are not manifested in conventional kinetics. When it suppresses the stabilization of the encounter complexes between reagents, sonication hinders the reaction but accelerates it when it perturbs the hydrophobic stabilization of the ground state of a reagent.
- Published
- 2007
- Full Text
- View/download PDF
39. Synthesis of 3H-labeled N-(3-iodoprop-2E-enyl)-2beta-carbomethoxy-3beta-(4-methylphenyl)nortropane (PE2I) and its interaction with mice striatal membrane fragments.
- Author
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Stepanov V, Schou M, Järv J, and Halldin C
- Subjects
- Animals, Cell Membrane metabolism, Female, Mice, Radiopharmaceuticals chemical synthesis, Radiopharmaceuticals metabolism, Corpus Striatum metabolism, Dopamine Plasma Membrane Transport Proteins metabolism, Isotope Labeling methods, Nortropanes chemical synthesis, Nortropanes metabolism, Tritium
- Abstract
PE2I is a high-affinity dopamine transporter (DA(T)) ligand that exhibits high selectivity for the DA(T) over the serotonin (5-HT(T)) and norephinephrine transporter (NE(T)) making its carbon-11 and iodine-125 labeled forms very useful for quantification of DA(T) binding sites in vivo. In this paper, we reported the synthesis of tritium-labeled PE2I, as well as improvements in the preparation of its acid precursor and PE2I itself with significant improvement in the total yield compared to previously published results. The new radioligand was evaluated as a tool for assessing DA(T) binding sites in vitro. The radioligand binding to mice striatal membranes demonstrated a homogeneous population of DA(T) binding sites with the K(d) value 9 nM.
- Published
- 2007
- Full Text
- View/download PDF
40. Slow isomerization step in the interaction between mouse dopamine transporter and dopamine re-uptake inhibitor N-(3-iodoprop-2E-enyl)-2beta-carbo-[3H]methoxy-3beta-(4'-methylphenyl)nortropane.
- Author
-
Stepanov V and Järv J
- Subjects
- Animals, Corpus Striatum cytology, Corpus Striatum metabolism, Dose-Response Relationship, Drug, Drug Interactions, Male, Mice, Protein Binding drug effects, Tritium pharmacology, Corpus Striatum drug effects, Dopamine Plasma Membrane Transport Proteins metabolism, Dopamine Uptake Inhibitors pharmacology, Nortropanes pharmacology
- Abstract
The kinetics of the association and dissociation of the tritium-labeled selective and potent dopamine transporter inhibitor N-(3-iodoprop-2E-enyl)-2beta-carbo-[3H]methoxy-3beta-(4'-methylphenyl)nortropane ([3H]PE2I) with the transporter of mouse striatal membranes was studied. The analysis revealed that the specific binding of [3H]PE2I occurs within a homogeneous population of binding sites in these membranes. The relatively slow binding process was characterized by the pseudo-first-order rate constant kobs. The plot of these rate constants versus free radioligand concentration was hyperbolic, demonstrating that at least two kinetically distinguishable steps can be identified in the interaction of dopamine transporter with this inhibitor. The fast and reversible binding step, characterized by dissociation constant KA = 51 +/- 23 nM, is followed by a slow but also reversible isomerization step of the complex, characterized by the isomerization rate constant ki = (7 +/- 2)10(-2) s(-1) and by the rate constant k(-i) = (3.9 +/- 0.5)10(-3) s(-1) for the reverse process. This isomerization step increases the apparent affinity of the ligand and probably consists of a conformational transition of the transporter protein, induced by the inhibitor molecule.
- Published
- 2006
- Full Text
- View/download PDF
41. Screening for the optimal specificity profile of protein kinase C using electrospray mass-spectrometry.
- Author
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Loog M, Ek B, Oskolkov N, Närvänen A, Järv J, and Ek P
- Subjects
- Amino Acid Sequence, Enzyme Inhibitors pharmacology, Kinetics, Molecular Sequence Data, Peptide Library, Phosphorylation, Protein Kinase C antagonists & inhibitors, Protein Kinase C chemistry, Substrate Specificity, Protein Kinase C metabolism, Spectrometry, Mass, Electrospray Ionization methods
- Abstract
A peptide library approach based on electrospray mass-spectrometric (ESI-MS) detection of phosphopeptides was designed for rapid and quantitative characterization of protein kinase specificity. The k(cat)/K(m) values for the protein kinase Cbeta (PKCbeta) were determined for a systematically varied set of individual substrate peptides in library mixtures by the ESI-MS method. The analysis revealed a complex structural specificity profile in positions around the phosphorylated serine with hydrophobic and/or basic residues being mostly preferred. On the basis of the kinetic parameters, a highly efficient peptide substrate for PKCbeta (K(m)value below 100 nM) FRRRRSFRRR and its alanine substituted pseudosubstrate-analog inhibitor (K(i) value of 76 nM) were designed. The quantitative specificity profiles obtained by the new approach contained more information about kinase specificity than the conventional substrate consensus motifs. The new method presents a promising basis for design of substrate-site directed peptide or peptidomimetic inhibitors of protein kinases. Second, highly specific substrates could be designed for novel applications such as high-throughput protein kinase activity screens on protein kinase chips.
- Published
- 2005
- Full Text
- View/download PDF
42. Comparison of cAMP-dependent protein kinase substrate specificity in reaction with proteins and synthetic peptides.
- Author
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Loog M, Oskolkov N, O'Farrell F, Ek P, and Järv J
- Subjects
- Catalysis, Electrophoresis, Polyacrylamide Gel, Mutagenesis, Site-Directed, Mutation, Peptides chemical synthesis, Phosphorylation, Pyruvate Kinase metabolism, Substrate Specificity, Cyclic AMP-Dependent Protein Kinases metabolism, Peptides metabolism
- Abstract
Mutants of L-type pyruvate kinase with modified peptide sequence around the Ser-12 phosphorylation site were prepared and kinetics of their phosphorylation by protein kinase A was studied. The profile of substrate specificity obtained for these proteins was compared with the kinetic data of phosphorylation of short peptide substrates. Alterations made in protein structure caused weaker effects than the corresponding alterations made in peptides, while the amino acid preferences and the overall specificity pattern remained similar in the both cases. Thus, similar consensus motif holds for both protein and peptide substrates, but is less critical for recognition of proteins if compared with short peptides.
- Published
- 2005
- Full Text
- View/download PDF
43. Similar dynamics of G-protein coupled receptors molecules in response to antagonist binding.
- Author
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Järv J and Oras A
- Subjects
- Adrenergic beta-Antagonists pharmacokinetics, Animals, Deoxyadenine Nucleotides pharmacokinetics, Dopamine Antagonists pharmacokinetics, Humans, Kinetics, Muscarinic Antagonists pharmacokinetics, Rats, Receptors, Adrenergic, beta metabolism, Receptors, Dopamine D2 metabolism, Receptors, Muscarinic metabolism, Receptors, Purinergic P2 metabolism, Receptors, Purinergic P2Y1, Thionucleotides pharmacokinetics, Brain physiology, Receptors, G-Protein-Coupled antagonists & inhibitors, Receptors, G-Protein-Coupled metabolism
- Abstract
In this communication we would like to draw attention on surprisingly similar transition of G-protein coupled receptors (GPCR) molecules, observed in these kinetic studies made with different receptors and their antagonists.
- Published
- 2005
- Full Text
- View/download PDF
44. Kinetic analysis of inhibition of cAMP-dependent protein kinase catalytic subunit by the peptide-nucleoside conjugate AdcAhxArg6.
- Author
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Kuznetsov A, Uri A, Raidaru G, and Järv J
- Subjects
- Adenosine Triphosphate chemistry, Catalysis, Enzyme Inhibitors chemistry, Kinetics, Molecular Structure, Oligopeptides chemistry, Phosphorylation, Protein Subunits antagonists & inhibitors, Protein Subunits chemistry, Structure-Activity Relationship, Cyclic AMP-Dependent Protein Kinases antagonists & inhibitors, Cyclic AMP-Dependent Protein Kinases chemistry, Enzyme Inhibitors pharmacology, Nucleosides chemistry, Peptides chemistry
- Abstract
Kinetic analysis of the inhibition of the phosphorylation of Kemptide, (LRRASLG), catalyzed by the catalytic subunit of cAMP-dependent protein kinase, by a peptide-nucleoside conjugate inhibitor AdcAhxArg6 was carried out over a wide range of ATP and peptide concentrations. A simple procedure was proposed for characterization of the interaction of this inhibitor with the free enzyme, and with the enzyme-ATP and enzyme-peptide complexes. The second-order rate constants, calculated from the steady-state reaction kinetics, were used for this analysis to avoid the complications related to the complex catalytic mechanism of the protein kinase catalyzed reaction.
- Published
- 2004
- Full Text
- View/download PDF
45. Kinetics of [35S]dATPalphaS interaction with P2Y1 purinoceptor in rat brain membranes.
- Author
-
Oras A and Järv J
- Subjects
- Adenosine Triphosphate metabolism, Adenosine Triphosphate pharmacokinetics, Animals, Animals, Newborn, Binding Sites drug effects, Binding Sites physiology, Binding, Competitive drug effects, Binding, Competitive physiology, Brain drug effects, Cell Line, Tumor, Cell Membrane drug effects, Isomerism, Kinetics, Ligands, Radioligand Assay, Rats, Receptors, Purinergic P2 drug effects, Receptors, Purinergic P2Y1, Subcellular Fractions, Sulfur Radioisotopes pharmacokinetics, Time Factors, Brain metabolism, Cell Membrane metabolism, Deoxyadenine Nucleotides pharmacokinetics, Receptors, Purinergic P2 metabolism, Thionucleotides pharmacokinetics
- Abstract
Kinetics of [35S]dATPalphaS (2'deoxyadenosine-5'-[alpha-35S]-thiotriphosphate) interaction with rat brain membrane fragments was studied at 25 degrees C and at radioligand concentrations from 2 to 250 nM. At least two different ways of [35S]dATPalphaS interaction with the membranes were distinguished on the basis of radioligand on-rate. Firstly, the binding sites characterized by 'fast' on-rate can be observed. Secondly, the 'slow' binding sites were kinetically identified and quantified. As in both cases the bound radioligand could be displaced by excess of ATP, all these binding sites can be defined as 'specific sites'. In the 'slow' binding sites isomerization of the receptor-ligand complex was observed, as is typical for interaction of antagonists with G-protein coupled receptors, and the kinetic parameters for this interaction were similar with the appropriate data for the hP2Y1 receptors expressed in 1321N1 astrocytoma cells Therefore these sites could be assigned to the same receptor subtype in brain membranes while the 'fast' binding sites belong to other membrane-bound proteins, also interacting with ATP and its analogues. The kinetic properties of the latter sites were not analysed in detail.
- Published
- 2004
- Full Text
- View/download PDF
46. Kinetics of the grignard reaction with silanes in diethyl ether and ether-toluene mixtures.
- Author
-
Tuulmets A, Nguyen BT, Panov D, Sassian M, and Järv J
- Abstract
Kinetics of the reactions of butylmagnesium chloride and phenylmagnesium bromide with tetraethoxysilane and methyltrichlorosilane was investigated in diethyl ether and diethyl ether-toluene mixtures. Replacement of ether by toluene significantly accelerates the reaction with alkoxysilanes, while no effect was found for the reaction with chlorosilanes. We established that the reaction with alkoxysilanes consists of replacement of a donor molecule at the magnesium center by the silane followed by subsequent rearrangement of the complex to products through a four-center transition state. Chlorosilanes react differently without solvent molecule replacement but also via a four-center transition state. Large negative activation entropies are consistent with formation of cyclic transition states. Small activation enthalpy values together with remarkable exothermicity point to early transition states of the reactions.
- Published
- 2003
- Full Text
- View/download PDF
47. Reversible and irreversible components of [(3)H]-N-propylnorapomorphine interaction with rat striatal membranes.
- Author
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Lepiku M, Järv J, Fuxe K, and Rinken A
- Subjects
- Animals, Antioxidants pharmacology, Ascorbic Acid pharmacology, Binding Sites, Binding, Competitive, Cell Membrane metabolism, Dithiothreitol pharmacology, Dopamine metabolism, Kinetics, Oxidation-Reduction drug effects, Radioactive Tracers, Rats, Receptors, Dopamine metabolism, Tritium, Apomorphine analogs & derivatives, Apomorphine metabolism, Neostriatum metabolism
- Abstract
The kinetics of L-(-)-[N-propyl-(3)H(N)]-norapomorphine ([(3)H]NPA) interactions with rat striatal membranes were studied. The analysis revealed that in addition to specific dopaminergic binding a substantial part of the radioligand was bound irreversibly to heterogeneous populations of non-specific binding sites of these membranes. The specific binding of [(3)H]NPA with dopamine receptors, determined from the differences of kinetic curves of total and non-specific binding, was fast, reversible, and revealed high affinity. The irreversible component was heterogeneous and seems to be related to oxidative degradation of the radioligand, as the rate of this process was substantially reduced by antioxidants like ascorbic acid and dithiothreitol.
- Published
- 2002
- Full Text
- View/download PDF
48. 'Strain effect' descriptors for ATP and ADP derivatives with modified phosphate groups.
- Author
-
Sak K, Järv J, and Karelson M
- Subjects
- Adenosine Diphosphate chemistry, Adenosine Diphosphate metabolism, Adenosine Triphosphate chemistry, Adenosine Triphosphate metabolism, Models, Molecular, Nucleic Acid Conformation, Quantitative Structure-Activity Relationship, Adenosine Diphosphate analogs & derivatives, Adenosine Triphosphate analogs & derivatives
- Abstract
Semiempirical AM1 calculations were carried out for quantum chemically optimized conformations of ATP and ADP and their modified phosphate derivatives with the oxygen atoms intervening between phosphorus atoms substituted by imido or methylene groups or the double-bonded oxygen atoms substituted by sulfur. In addition to the calculation of conventional geometric and energetic parameters, the effect of these substitutions was quantified in terms of conformational 'strain energy'. The latter has been defined as the energy of transformation of the parent nucleotide (ATP or ADP) from the optimum conformation to the conformation optimized for its phosphate-modified analog. The results of calculations revealed that conformational 'strain' of phosphate-modified nucleotides depends not only on the nature of the substituent but also on its position. The respective effect had the largest magnitude when the substitution was made between two terminal phosphorus atoms. Given that the 'strain energy' characterizes the geometrical aspects of the interaction of nucleotide molecules with receptors and enzymes, an attempt was made to correlate it with the corresponding biological activities. Such correlation was significant in the case of highly specific binding sites for universal ligands like ATP.
- Published
- 2002
- Full Text
- View/download PDF
49. Kinetic analysis of [35S]dATP alpha S interaction with P2y(1) nucleotide receptor.
- Author
-
Oras A, Kilk K, Kunapuli S, Barnard EA, and Järv J
- Subjects
- Binding Sites, Binding, Competitive, Cell Membrane metabolism, Humans, Kinetics, Models, Biological, Receptors, Purinergic P2Y1, Sulfur Radioisotopes, Time Factors, Tumor Cells, Cultured, Deoxyadenine Nucleotides metabolism, Receptors, Purinergic P2 metabolism, Thionucleotides metabolism
- Abstract
The kinetics of 2'-deoxyadenosine-5'-O-(1-thiotriphosphate) ([(35)S]dATP alpha S) interaction with membrane fragments of transfected astrocytoma 1321N1 cells, expressing human P2Y(1) receptors, and the same wild-type cells, not expressing P2Y receptors were studied. Binding of this radioligand was observed with both types of membranes, but sites showing slow on-rate were found only on the transfected cells. These "slow" binding sites behaved as a kinetically homogeneous population and their interaction with the radioligand was shown to occur in two steps, R+A(K(A))<==>RA(k(i))<==>(k(-i))(RA), including the relatively slow isomerization of the complex RA into (RA). Evidence was obtained to assign the isomerized ("slow") binding sites on the transfected cells as P2Y(1) receptor sites, differentiated from other binding sites of non-receptor origin by kinetic analysis, and characterised by the kinetic parameters K(A)=59 +/- 19 nM, k(i)=(9.0 +/- 0.8)10(-3)s(-1) and k(-i)=(3.9 +/- 0.7)10(-3)s(-1). [(35)S]dATP alpha S binding, with kinetic criteria, can be of value for differentiation of the receptor sites from non-receptor sites and thus provides solid basis for radioligand assay of P2Y(1) receptors.
- Published
- 2002
- Full Text
- View/download PDF
50. Kinetic evidence for different mechanisms of interaction of black mamba toxins MT alpha and MT beta with muscarinic receptors.
- Author
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Jolkkonen M, Oras A, Toomela T, Karlsson E, Järv J, and Akerman KE
- Subjects
- Animals, Brain drug effects, Brain metabolism, Drug Interactions, Kinetics, N-Methylscopolamine pharmacokinetics, Parasympatholytics pharmacokinetics, Radioligand Assay, Rats, Receptors, Muscarinic metabolism, Elapid Venoms metabolism, Elapid Venoms toxicity, Elapidae, N-Methylscopolamine metabolism, Parasympatholytics metabolism, Receptors, Muscarinic drug effects
- Abstract
By studying the influence of two toxins from the black mamba Dendroaspis polylepis on the kinetics of [3H]-N-methylscopolamine binding to muscarinic acetylcholine receptors from rat cerebral cortex, it was revealed that these toxins, MT alpha and MT beta, interact with the receptors via kinetically distinct mechanisms. MT beta bound to receptors in a one-step, readily reversible process with the dissociation constant K(d)=5.3 microM. The binding mechanism of MTalpha was more complex, involving at least two consecutive steps. A fast receptor-toxin complex formation (K(T)=3.8 microM) was followed by a slow process of isomerisation of this complex (k(i)=1.8 x 10(-2) s(-1), half-time 39 s). A similar two-step interaction mechanism has been established for a related toxin, MT2 from the green mamba D. angusticeps (K(T)=1.4 microM, k(i)=8.3 x 10(-4) s(-1), half-time 840 s). The slow isomerisation process delays the effect of MT alpha and MT2, but increases their apparent potency compared to toxins unable to induce the isomerisation process.
- Published
- 2001
- Full Text
- View/download PDF
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