Academic literature on the topic 'Dissociation constant of inhibitor'
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Journal articles on the topic "Dissociation constant of inhibitor"
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Björk, I., E. Pol, E. Raub-Segall, M. Abrahamson, A. D. Rowan, and J. S. Mort. "Differential changes in the association and dissociation rate constants for binding of cystatins to target proteinases occurring on N-terminal truncation of the inhibitors indicate that the interaction mechanism varies with different enzymes." Biochemical Journal 299, no. 1 (April 1, 1994): 219–25. http://dx.doi.org/10.1042/bj2990219.
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The importance of the N-terminal region of human cystatin C or chicken cystatin for the kinetics of interactions of the inhibitors with four cysteine proteinases was characterized. The association rate constants for the binding of recombinant human cystatin C to papain, ficin, actinidin and recombinant rat cathepsin B were 1.1 x 10(7), 7.0 x 10(6), 2.4 x 10(6) and 1.4 x 10(6) M-1.s-1, whereas the corresponding dissociation rate constants were 1.3 x 10(-7), 9.2 x 10(-6), 4.6 x 10(-2) and 3.5 x 10(-4) s-1. N-Terminal truncation of the first ten residues of the inhibitor negligibly affected the association rate constant with papain or ficin, but increased the dissociation rate constant approx. 3 x 10(4)- to 2 x 10(6)-fold. In contrast, such truncation decreased the association rate constant with cathepsin B approx. 60-fold, while minimally affecting the dissociation rate constant. With actinidin, the truncated cystatin C had both an approx. 15-fold lower association rate constant and an approx. 15-fold higher dissociation rate constant than the intact inhibitor. Similar results were obtained for intact and N-terminally truncated chicken cystatin. The decreased affinity of human cystatin C or chicken cystatin for cysteine proteinases after removal of the N-terminal region is thus due to either a decreased association rate constant or an increased dissociation rate constant, or both, depending on the enzyme. This behaviour indicates that the contribution of the N-terminal segment of the two inhibitors to the interaction mechanism varies with the target proteinase as a result of structural differences in the active-site region of the enzyme.
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Hofsteenge, J., H. Taguchi, and S. R. Stone. "Effect of thrombomodulin on the kinetics of the interaction of thrombin with substrates and inhibitors." Biochemical Journal 237, no. 1 (July 1, 1986): 243–51. http://dx.doi.org/10.1042/bj2370243.
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Thrombomodulin decreased by 20-30% the Michaelis constant of two tripeptidyl p-nitroanilide substrates of thrombin. Thrombomodulin increased the rate of inactivation of thrombin by two peptidyl chloromethane inhibitors by a similar amount. This effect appeared to be due to a decrease in the dissociation constants of the inhibitors. An improved method for the separation of fibrinopeptides A and B by h.p.l.c. was developed, and this method was used to study the effect of thrombomodulin on the thrombin-catalysed cleavage of fibrinogen. In this reaction, thrombomodulin was a competitive inhibitor with respect to the A alpha-chain of fibrinogen. The release of fibrinopeptide B was also inhibited by thrombomodulin. Analysis of the inhibition caused by thrombomodulin with respect to fibrinopeptides A and B yielded the same dissociation constant for the thrombin-thrombomodulin complex. In the presence of thrombomodulin, the rate of inactivation of thrombin by antithrombin III was stimulated 4-fold. This stimulation showed saturation kinetics with respect to thrombomodulin. Thrombomodulin was found to compete with hirudin for a binding site on thrombin. As a result of this competition, hirudin became a slow-binding inhibitor of thrombin at high thrombomodulin concentrations. Estimates of the dissociation constant for thrombomodulin were obtained in several of the above experiments, and the weighted mean value was 0.7 nM.
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Boudier, C., and J. G. Bieth. "Oxidized mucus proteinase inhibitor: a fairly potent neutrophil elastase inhibitor." Biochemical Journal 303, no. 1 (October 1, 1994): 61–68. http://dx.doi.org/10.1042/bj3030061.
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N-chlorosuccinimide oxidizes one of the methionine residues of mucus proteinase inhibitor with a second-order rate constant of 1.5 M-1.s-1. Cyanogen bromide cleavage and NH2-terminal sequencing show that the modified residue is methionine-73, the P′1 component of the inhibitor's active centre. Oxidation of the inhibitor decreases its neutrophil elastase inhibitory capacity but does not fully abolish it. The kinetic parameters describing the elastase-oxidized inhibitor interaction are: association rate constant kass. = 2.6 x 10(5) M-1.s-1, dissociation rate constant kdiss. = 2.9 x 10(-3) s-1 and equilibrium dissociation constant Ki = 1.1 x 10(-8) M. Comparison with the native inhibitor indicates that oxidation decreases kass. by a factor of 18.8 and increases kdiss. by a factor of 6.4, and therefore leads to a 120-fold increase in Ki. Yet, the oxidized inhibitor may still act as a potent elastase inhibitor in the upper respiratory tract where its concentration is 500-fold higher than Ki, i.e. where the elastase inhibition is pseudo-irreversible. Experiments in vitro with fibrous human lung elastin, the most important natural substrate of elastase, support this view: 1.35 microM elastase is fully inhibited by 5-6 microM oxidized inhibitor whether the enzyme-inhibitor complex is formed in the presence or absence of elastin and whether elastase is pre-adsorbed on elastin or not.
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Brown, Nicholas G., Dar-Chone Chow, and Timothy Palzkill. "BLIP-II Is a Highly Potent Inhibitor of Klebsiella pneumoniae Carbapenemase (KPC-2)." Antimicrobial Agents and Chemotherapy 57, no. 7 (April 15, 2013): 3398–401. http://dx.doi.org/10.1128/aac.00215-13.
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ABSTRACTβ-Lactamase inhibitory protein II (BLIP-II) is a potent inhibitor of class A β-lactamases. KPC-2 is a class A β-lactamase that is capable of hydrolyzing carbapenems and has become a widespread source of resistance to these drugs for Gram-negative bacteria. Determination of association and dissociation rate constants for binding between BLIP-II and KPC-2 reveals a very tight interaction with a calculated (koff/kon) equilibrium dissociation constant of 76 fM (76 × 10−15M).
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LOHSE, Anders, Tore HARDLEI, Astrid JENSEN, Igor W. PLESNER, and Mikael BOLS. "Investigation of the slow inhibition of almond β-glucosidase and yeast isomaltase by 1-azasugar inhibitors: evidence for the ‘direct binding’ model." Biochemical Journal 349, no. 1 (June 26, 2000): 211–15. http://dx.doi.org/10.1042/bj3490211.
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(-)-1-Azafagomine [(3R,4R,5R)-4,5-dihydroxy-3-hydroxymethylhexahydropyridazine; inhibitor 1] is a potent glycosidase inhibitor designed to mimic the transition state of a substrate undergoing glycoside cleavage. The inhibition of glycosidases by inhbitor 1 and analogues has been found to be a relatively slow process. This ‘slow inhibition’ process was investigated in the inhibition of almond β-glucosidase and yeast isomaltase by inhibitor 1 and analogues. Progress-curve experiments established that the time-dependent inhibition of both enzymes by inhibitor 1 was a consequence of relatively slow dissociation and association of the inhibitor from and to the enzyme, and not a result of slow interchanges between protein conformations. A number of hydrazine-containing analogues of inhibitor 1 also inhibited β-glucosidase and isomaltase slowly, while the amine isofagomine [(3R,4R,5R)-3,4-dihydroxy-5-hydroxymethylpiperidine; inhibitor 5] only inhibited β-glucosidase slowly. Inhibitor 1 and related inhibitors were found to leave almond β-glucosidase with almost identical rate constants, so that the difference in Ki values depended almost entirely on changes in the binding rate constant, kon. The same trend was observed for the inhibition of yeast isomaltase by inhibitor 1 and a related inhibitor. The values of the rate constants were obtained at 25 °C and at pH 6.8.
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Lindahl, P., E. Raub-Segall, S. T. Olson, and I. Björk. "Papain labelled with fluorescent thiol-specific reagents as a probe for characterization of interactions between cysteine proteinases and their protein inhibitors by competitive titrations." Biochemical Journal 276, no. 2 (June 1, 1991): 387–94. http://dx.doi.org/10.1042/bj2760387.
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Papain was labelled by attachment of the fluorescent groups 2-(4′-acetamidoanilino)naphthalene-6-sulphonic acid (AANS) or N-(acetylaminoethyl)-8-naphthylamine-1-sulphonic acid (AEDANS) to the active-site cysteine residue, with the aim of using the labelled papains as probes in competitive titrations of unlabelled cysteine proteinases with their inhibitors. The interaction between the labelled papains and cystatins was accompanied by an increase in fluorescence emission of up to 38-fold for AANS-papain and approximately 3.5-fold for AEDANS-papain. Fluorescence titrations gave dissociation equilibrium constants of 3.1 and 0.6 microM for the binding of chicken cystatin and recombinant human cystatin C respectively to AANS-papain and of 11.9 microM for the binding of chicken cystatin to AEDANS-papain. The kinetics of interaction of chicken cystatin with AANS-papain showed an unusual biphasic dependence of the observed pseudo-first-order rate constant on inhibitor concentration, consistent with the reaction occurring via both pathways of a general two-step binding mechanism. AANS-papain was selected as the most suitable probe for competitive titrations of unlabelled active or inactivated cysteine proteinases with inhibitors. This technique, which provides stoichiometries and dissociation constants for the interaction between unlabelled enzyme and inhibitor, allows monitoring of the interactions by a large fluorescent signal in a wavelength region where the interacting proteins do not contribute to the observed fluorescence. Such competitive titrations of active papain or actinidin with chicken cystatin or recombinant human cystatin C all gave inhibitor/enzyme stoichiometries of close to 1.0. A dissociation constant of 1.8 microM for the reaction of chicken cystatin with a papain derivative, S-[N-(3-carboxypropyl)succinimidyl]-papain, was also determined by the same technique. These results show the usefulness of the fluorescent papains for the characterization of interactions between cysteine-proteinase inhibitors and their target enzymes.
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Lindhout, T., G. Willems, R. Blezer, and H. C. Hemker. "Kinetics of the inhibition of human factor Xa by full-length and truncated recombinant tissue factor pathway inhibitor." Biochemical Journal 297, no. 1 (January 1, 1994): 131–36. http://dx.doi.org/10.1042/bj2970131.
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The inhibition equilibrium and kinetics of association and dissociation of the binding of three types of recombinant tissue factor pathway inhibitor (TFPI), namely full-length TFPI, C-terminal-truncated TFPI, and TFPI without the third Kunitz domain (TFPI1-161), to factor Xa have been measured. Formation and dissociation of the complexes were monitored by continuous measurement of the changes in the rate of hydrolysis of a peptidyl-p-nitroanilide substrate. Progress curves of product formation were fitted to a set of equations describing a one-step bimolecular inhibitory reaction in the presence of a competing substrate. For full-length TFPI the rate constants of association (kon) and dissociation (koff) were (5.1 +/- 0.7) x 10(6) M-1.s-1 and (2.6 +/- 0.9) x 10(-4)s-1 respectively. Thus, although the inhibition constant (50 pM) is far below the plasma concentration (2.5 nM) of TFPI, the half-time for transition to equilibrium in plasma is rather long (66s). The truncated forms of TFPI differ in that they have a 4-fold lower kon value but a similar dissociation rate constant. Therefore the inhibition constant, Ki, is 4-fold higher (0.2 nM) and the half-time to achieve equilibrium is prolonged to 250 s. The kon values of full-length and C-terminal-truncated TFPI, but not that of TFPI1-161, were found to decrease with increasing ionic strength.
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Ahmad, Suhail, R. K. Bhatnagar, and T. A. Venkitasubramanian. "Ornithine transcarbamylase from Mycobacterium smegmatis ATCC 14468: purification, properties, and reaction mechanism." Biochemistry and Cell Biology 64, no. 12 (December 1, 1986): 1349–55. http://dx.doi.org/10.1139/o86-177.
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Ornithine transcarbamylase (EC 2.1.3.3) has been purified 980-fold from Mycobacterium smegmatis and has a molecular weight of 116 000. Initial velocity determinations indicated that the reaction proceeds via a sequential kinetic mechanism. The limiting Michaelis constants for carbamyl phosphate (KmA) and ornithine (KmB) and the dissociation constant for carbamyl phospate (Kia) were found to be 0.20, 0.25, and 0.07 mM, respectively. Ornithine at higher concentrations acted as an uncompetitive inhibitor when carbamyl phosphate was the variable substrate. Phosphate was a competitive inhibitor with carbamyl phosphate as variable substrate and showed noncompetitive or mixed type inhibition when ornithine was the variable substrate. Norvaline acted as a competitive inhibitor with ornithine as variable substrate and as an uncompetitive inhibitor when carbamyl phophate was the variable substrate. Such inhibitory patterns are characteristic of reactions that proceed via sequential ordered mechanisms. Although the enzyme activity was strongly inhibited by arginine, several arginine analogs had no effect on the enzyme activity. The results suggest that, even though the enzyme from M. smegmatis is unique in the sense that it is feedback inhibited by arginine, the reaction mechanism is similar to the ornithine transcarbamylase isolated from other microorganisms.
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Jin, Byung-Ju, Jay R. Thiagarajah, and A. S. Verkman. "Convective washout reduces the antidiarrheal efficacy of enterocyte surface–targeted antisecretory drugs." Journal of General Physiology 141, no. 2 (January 28, 2013): 261–72. http://dx.doi.org/10.1085/jgp.201210885.
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Secretory diarrheas such as cholera are a major cause of morbidity and mortality in developing countries. We previously introduced the concept of antisecretory therapy for diarrhea using chloride channel inhibitors targeting the cystic fibrosis transmembrane conductance regulator channel pore on the extracellular surface of enterocytes. However, a concern with this strategy is that rapid fluid secretion could cause convective drug washout that would limit the efficacy of extracellularly targeted inhibitors. Here, we developed a convection–diffusion model of washout in an anatomically accurate three-dimensional model of human intestine comprising cylindrical crypts and villi secreting fluid into a central lumen. Input parameters included initial lumen flow and inhibitor concentration, inhibitor dissociation constant (Kd), crypt/villus secretion, and inhibitor diffusion. We modeled both membrane-impermeant and permeable inhibitors. The model predicted greatly reduced inhibitor efficacy for high crypt fluid secretion as occurs in cholera. We conclude that the antisecretory efficacy of an orally administered membrane-impermeant, surface-targeted inhibitor requires both (a) high inhibitor affinity (low nanomolar Kd) to obtain sufficiently high luminal inhibitor concentration (>100-fold Kd), and (b) sustained high luminal inhibitor concentration or slow inhibitor dissociation compared with oral administration frequency. Efficacy of a surface-targeted permeable inhibitor delivered from the blood requires high inhibitor permeability and blood concentration (relative to Kd).
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Bernal, A. López, S. Buckley, C. M. P. Rees, and J. M. Marshall. "Meclofenamate inhibits prostaglandin E binding and adenylyl cyclase activation in human myometrium." Journal of Endocrinology 129, no. 3 (June 1991): 439–45. http://dx.doi.org/10.1677/joe.0.1290439.
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ABSTRACT The effect of sodium meclofenamate on the binding of [3H]prostaglandin E2 ([3H]PGE2) to membranes from human myometrium was investigated. Meclofenamate inhibited the binding of [3H]PGE2 to high-affinity (dissociation constant 1·5 nmol/l) sites in a reversible dose-dependent manner (inhibition constant 11 μmol/l). The mechanism of inhibition was mainly competitive, but at high doses of meclofenamate (≥ 100 μmol/l) there was loss of PGE receptor sites. Of several PG synthesis inhibitors tested, only meclofenamate and, to a lesser extent, mefenamic acid had a significant inhibitory effect. PGE2 stimulated cyclic AMP generation in slices of human myometrium and this was inhibited by meclofenamate in a dose-dependent manner (50% inhibition occurred at 9 μmol/l). Again, this effect was specific for meclofenamate and fitted a competitive mechanism at doses in the range 1–10 μmol/l and a non-competitive mechanism at higher doses. The data show that meclofenamate, in addition to its traditional role as a PG synthesis inhibitor, affects directly PGE receptor binding and activation. Journal of Endocrinology (1991) 129, 439–445
Dissertations / Theses on the topic "Dissociation constant of inhibitor"
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Mann, Maretta Clare, and n/a. "Sialylmimetics as Potential Inhibitors fo Vibrio Cholerae Sialidase." Griffith University. Institute for Glycomics, 2004. http://www4.gu.edu.au:8080/adt-root/public/adt-QGU20061006.083947.
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Cholera is an epidemic infectious diarrhoeal disease that for centuries has proven its frightening ability to cause rapid and widespread loss of human life. All symptoms associated with cholera are a result of rapid dehydration due to infection by pathogenic strains of the bacterium Vibrio cholerae. The damaging effects associated with cholera are mainly attributed to the toxin, which is secreted by the bacterium and infects cells lining the gastrointestinal tract. A sialidase, also secreted by the bacterium, is believed to facilitate toxin uptake by the gastrointestinal epithelium. V. cholerae sialidase is therefore a potential target for therapeutic intervention. A survey of the literature reveals that sialidases from different species share common features with respect to their structure, substrate specificity and catalytic mechanism. The unsaturated sialic acid, Neu5Ac2en, inhibits most exosialidases with a dissociation constant of inhibitor of -10-4 to-10-6 M and has frequently been used as a template in the design of more potent sialidase inhibitors. In the case of V. cholerae sialidase, there have been no inhibitors reported to date that are significantly more potent than Neu5Ac2en itself The present research aimed to develop a range of mimics of Neu5Ac2en, which contain various substituents to replace the C-6 glycerol side chain, as potential inhibitors of V cholerae sialidase. The x-ray crystal structure of V cholerae sialidase was used to explore potential interactions between active site residues and C-6 modified Neu5Ac2en mimetics of known inhibitory potency. Opportunities for interactions within the glycerol side chain pocket in the active site of V cholerae sialidase are discussed. A novel synthetic strategy was developed for the synthesis of a series of glucuronidebased Neu5Ac2en mimetics starting from readily available GIcNAc. This approach was employed for the preparation of Neu5Ac2en mimetics that contained an ether or thioether substituent as replacement of the glycerol side chain of Neu5Ac2en. Progress was also made towards the synthesis of a series of C-6 acylamino Neu5Ac2en mimetics. Analysis by 1H NMR spectroscopy showed that the acylamino derivatives adopted a half-chair conformation that was similar to the conformation of Neu5Ac2en but different to the conformation adopted by the ether and thioether derivatives prepared. The inhibitory activity of the C-6 ether and thioether Neu5Ac2en mimetics prepared was evaluated in vitro using an enzyme assay. It was found that most of the derivatives inhibited V. cholerae sialidase with a K1 of approximately 1O-4 M. The derivatives containing a hydrophobic side chain were found to be slightly more potent compared to derivatives with more hydrophilic side chains. A more detailed study of binding interactions between the C-6 thioether Neu5Ac2en mimetics and V cholerae sialdiase was carried out using STD 1H NMR spectroscopy and computational molecular modelling.
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Mann, Maretta Clare. "Sialylmimetics as Potential Inhibitors fo Vibrio Cholerae Sialidase." Thesis, Griffith University, 2004. http://hdl.handle.net/10072/367187.
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Cholera is an epidemic infectious diarrhoeal disease that for centuries has proven its frightening ability to cause rapid and widespread loss of human life. All symptoms associated with cholera are a result of rapid dehydration due to infection by pathogenic strains of the bacterium Vibrio cholerae. The damaging effects associated with cholera are mainly attributed to the toxin, which is secreted by the bacterium and infects cells lining the gastrointestinal tract. A sialidase, also secreted by the bacterium, is believed to facilitate toxin uptake by the gastrointestinal epithelium. V. cholerae sialidase is therefore a potential target for therapeutic intervention. A survey of the literature reveals that sialidases from different species share common features with respect to their structure, substrate specificity and catalytic mechanism. The unsaturated sialic acid, Neu5Ac2en, inhibits most exosialidases with a dissociation constant of inhibitor of -10-4 to-10-6 M and has frequently been used as a template in the design of more potent sialidase inhibitors. In the case of V. cholerae sialidase, there have been no inhibitors reported to date that are significantly more potent than Neu5Ac2en itself The present research aimed to develop a range of mimics of Neu5Ac2en, which contain various substituents to replace the C-6 glycerol side chain, as potential inhibitors of V cholerae sialidase. The x-ray crystal structure of V cholerae sialidase was used to explore potential interactions between active site residues and C-6 modified Neu5Ac2en mimetics of known inhibitory potency. Opportunities for interactions within the glycerol side chain pocket in the active site of V cholerae sialidase are discussed. A novel synthetic strategy was developed for the synthesis of a series of glucuronidebased Neu5Ac2en mimetics starting from readily available GIcNAc. This approach was employed for the preparation of Neu5Ac2en mimetics that contained an ether or thioether substituent as replacement of the glycerol side chain of Neu5Ac2en. Progress was also made towards the synthesis of a series of C-6 acylamino Neu5Ac2en mimetics. Analysis by 1H NMR spectroscopy showed that the acylamino derivatives adopted a half-chair conformation that was similar to the conformation of Neu5Ac2en but different to the conformation adopted by the ether and thioether derivatives prepared. The inhibitory activity of the C-6 ether and thioether Neu5Ac2en mimetics prepared was evaluated in vitro using an enzyme assay. It was found that most of the derivatives inhibited V. cholerae sialidase with a K1 of approximately 1O-4 M. The derivatives containing a hydrophobic side chain were found to be slightly more potent compared to derivatives with more hydrophilic side chains. A more detailed study of binding interactions between the C-6 thioether Neu5Ac2en mimetics and V cholerae sialdiase was carried out using STD 1H NMR spectroscopy and computational molecular modelling.Thesis (PhD Doctorate)
Doctor of Philosophy (PhD)
School of Biomolecular and Physical Sciences
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Sooväli, Lilli. "Spectrophotometric measurements and their uncertainty in chemical analysis and dissociation constant measurements /." Online version, 2006. http://dspace.utlib.ee/dspace/bitstream/10062/627/5/soovalililli.pdf.
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Ronneburg, Henrike [Verfasser], Jürgen [Akademischer Betreuer] Dittmer, and Kurt [Akademischer Betreuer] Engeland. "Prognostische Relevanz von Rho-GDP dissociation inhibitor Proteinen beim Mammakarzinom / Henrike Ronneburg. Betreuer: Jürgen Dittmer ; Kurt Engeland." Halle, Saale : Universitäts- und Landesbibliothek Sachsen-Anhalt, 2009. http://d-nb.info/1024895807/34.
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Evangelista, Jaqueline Pesciutti. "Selenoproteínas: Seril-tRNA Sintetase e as selenoproteínas do Trypanosoma brucei." Universidade de São Paulo, 2014. http://www.teses.usp.br/teses/disponiveis/76/76132/tde-13112014-171709/.
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O aminoácido selenocisteína (Sec) representa a principal forma biológica de selênio sendo requerida uma complexa maquinaria molecular para sua síntese e incorporação co-traducional em selenoproteínas. A Seril-tRNA sintetase (SerRS) inicia essa via, aminoacilando o Ser-tRNASec (SelC) com uma serina e também aminoacila os tRNAsSer. Sendo assim, um dos focos deste trabalho foi estudar a interação da SerRS de Trypanosoma brucei (T. brucei) com os tRNAsSer e o SelC utilizando a técnica de anisotropia de fluorescência para determinar suas constantes de dissociação. Em Kinetoplastidae, além da via de síntese de selenocisteína, há três selenoproteínas: SelT, SelK e SelTryp. No entanto, pouco se sabe a respeito das mesmas, sendo o estudo destas selenoproteínas o outro foco deste trabalho. Os fragmentos de DNA que codificam estas selenoproteínas foram subclonados em vetor de expressão pET 28a e 29a para posterior uso em células de Escherichia coli (E. coli). Para as proteínas SelK e SelTryp os ensaios de expressão apresentaram resultados insuficientes para dar continuidade aos experimentos planejados, pois o rendimento foi baixo e a purificação não foi possível. Já com a proteína SelT, devido à grande dificuldade encontrada para tornà-la solúvel, descobriu-se, no decorrer do trabalho, que tratava-se de uma proteína de membrana, ocasionando mudanças de alguns objetivos previamente propostos e consequentemente busca por novas estratégias. Conseguiu-se expressá-la na de forma solúvel e purificá-la por cromatografias. Ensaios realizados no SEC-MALLS mostraram uma estabilidade do complexo proteína-detergente. Com a TbSerRS é possível concluir que a organização de especificidade de ligação da enzima com seus ligantes se dá crescentemente: SelC>tRNASer7>tRNASer3a>tRNASer3b. E com as selenoproteínas do T. brucei faz-se necessários novas contruções para SelK e SelTryp e dar continuidade aos experimentos com a SelT tentando cristalizá-la, já que prototolo para a obtenção do complexo proteína-detergente está montado e estabilizado.Selenocysteine (Sec) amino acid is the major biological form of selenium and requires a complex molecular machinery for its synthesis and co-translational incorporation into selenoproteins. The Seryl-tRNA synthetase (SerRS) starts this biosynthesis and matches the tRNASec (SELC) with a serine and the tRNAsSer, therefore the focus of this study is on SerRS of Trypanosoma brucei (T. brucei) and tRNAsSer and SELC interactions, with fluorescence anisotropy techinic to determinat dissociation constants. Three selenoproteins, namely SelT, SelK and SelTryp, besides the route of selenocysteine synthesis there be in Kinetoplastidae. DNA fragments that coding for these selenoproteins were subcloned in 28a and 29a to use into Escherichia coli (E. coli) cells. For Selk and SelTryp proteins, the expression protocol did not show an unsatisfactory result to continue the experiments. Many difficulties were encountered in studies with Selt protein, mainly in attempts to make it soluble. Our analyses revealed SelT was a membrane protein, therefore it could cause changes in some objectives and search for new strategies. It could be expressed and purified in cromatographis. SEC-MALLS assays showed a stability of the protein detergent complex. With TbSerRS is possible to conclude that the organization of binding specificity of the enzyme with its ligands occurs increasingly: SelC>tRNASer7>tRNASer3a>tRNASer3b. And selenoproteins in T. brucei, it is necessary for new constructions to SelK and SelTryp to continue the experiments trying to crystallizes SelT, since prototolo for obtaining the protein-detergent complex is assembled and stabilized.
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Karbanová, Kateřina. "Disociační chování přírodních biokoloidů." Master's thesis, Vysoké učení technické v Brně. Fakulta chemická, 2017. http://www.nusl.cz/ntk/nusl-316163.
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This diploma thesis is focused on the study of dissociation behaviour of natural biocolloids, namely humic acids and fulvic acids. Humic and fulvic acids are natural, heterogeneous, high molecular weight substances which behave as weakly acidic polyelectrolytes and they have complex not exactly described structure. They are formed by biochemical transformations of organic residues (mainly plants). They are part of the soil, water, peat, sediments and coal. Solubility of humic acids is affected by pH value. The higher the pH value is the higher the solubility is. Fulvic acids are soluble in whole range of pH values. The aim of this diploma thesis is to determine the dissociation constant for the five kinds of humic acids and four kinds of fulvic acids, which have been isolated from various natural sources. These samples were purchased from IHSS. Dissociation constants were determined by the conductometric method and a combination of measurment pH and the content of acidic functional groups in Na2SO4. UV-VIS spectrophotometry method was used to characterize the quality of humic acids and fulvic acids.
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Viktorinová, Jana. "Využití chronopotenciometrické titrace v huminovém výzkumu." Master's thesis, Vysoké učení technické v Brně. Fakulta chemická, 2013. http://www.nusl.cz/ntk/nusl-216933.
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Humic acids are natural substances belonging to the group of humic substances. They arise mainly decomposition of plant residues. They are contained in soils, peat, sediments, young coal, water and even in the air. Humic acids are only partially soluble in water with increasing pH increases their solubility. Diploma thesis focuses on the use of chronopotentiometric titration of humic research. This method is mainly used for the determination of trace concentrations of analytes. This work is focused on the determination of acidity by potentiometric titration and the determination of dissociation constants using chronopotentiometry with measurement of pH of prepared samples.
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Chaudhury, Chaity. "Identification and biochemical characterization of a novel receptor:ligand interaction between FcRn and albumin." The Ohio State University, 2005. http://rave.ohiolink.edu/etdc/view?acc_num=osu1110211148.
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Smallman, Matthew John. "Spatial regulation of Rho GTPase signalling during root hair development in Arabidopsis thaliana is reliant upon the guanine nucleotide dissociation inhibitor SCN1." Thesis, University of Bristol, 2008. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.496221.
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The scn1-1 mutants of Arabidopsis are characterised by spatially deregulated sites of root hair growth. This studydemonstrates that this phenotype is the result of the loss of regulation of members of the plant specific sub-family of Rho small GTPases by the Guanine nucleotide Dissociation Inhibitor (GDI), encoded by SCN1. The small GTPases R0P2, R0P4 and R0P5 of Arabidopsis are members of the I subset of type I ROPs that terminate with a conserved CXXL box and undergo 3 prenylation. These small GTPases are expressed in trichoblasts, and localize in patters suggestive of specific roles throughout root hair development. Loss-of function ROP2, R0P4 or ROP5 mutants display distinct root hair phenotypes lending support to the hypothesis that : R0P2, R0P4 and ROP5 control the establishment of the site of root hair initiation and subsequent tip-growth. Our findings also reveal SCNl/GDIl and R0P2 co-localize in a similiar pattern in developing root hairs in planta, but SCNl/GDIl is able to associate directly with ROP2, ROP4 and ROP5 in vitro.This suggests root hair morphogenesis relies on the negative regulation of ROP activity by SCNl/GDI 1 and is further supported by morphological phenotypes of scnl-l plants which can be rescued by the over expression of CFP:GD11 fusion placed under the control of the root hair specific PRP3 (Proline Rich Protein3) promoter. These observations imply that ROPs can be selectively sequestered away from the from plasma membrane of elongating root hairs during tip growth thereby promoting growth along a planar axis. Differences in the observed binding affinity of R0P2 in comparison to R0P4 and ROPS for SCNl/GDIl provides evidence that the CXXL box may play a critical role in ROP regulation, and that R0P2 and R0P4 are differentially prenylated.
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Li, Muchen. "Determination of dissociation constant of DNA/DNA hybridization by three different surface techniques : comparison of surface plasmon resonance, fluorescent microarray and evanescent field fluorescence." Thesis, Lyon, 2018. http://www.theses.fr/2018LYSEC028/document.
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Les biocapteurs sont des outils de détection et d'analyse puissants qui ont été largement utilisés dans les domaines de la santé, de la recherche biomédicale et de l’environnement. Cependant, différents biocapteurs utilisent différents transducteurs qui varient par la nature des substrats utilisés et par la chimie de surface. Tous ces paramètres peuvent avoir un effet sur les réactions biomoléculaires aux interfaces et conduire à des variations de la mesure de la constante de dissociation Kd. Dans ce contexte, ce travail de thèse visait à comparer trois techniques différentes : biopuce avec une détection par fluorescence, biocapteur à fluorescence par champ évanescent et biocapteur par résonance de plasmon de surface (SPR). Ces trois techniques ont été comparées pour la détermination de la constant de dissociation de l'hybridation de l'ADN. Pour la biopuce à fluorescence classique, le substrat est une lame de verre et la mesure est effectuée à la fin de l'expérience. Dans le cas du biocapteur à fluorescence à champ évanescent, le polystyrène est le substrat et une détection en temps réel est réalisée. La SPR est réalisée sur un film d'or mince. C'est une technique en temps réel et sans marquage. Les deux techniques basées sur la fluorescence nécessitent de marquer les cibles avec un fluorophore avant la mesure. Un facteur important déterminant la performance de l'analyse est la chimie de surface du capteur. Ici, nous avons optimisé la chimie de la surface de l'or pour le greffage d'ADN modifié thiol. Nous avons étudié deux méthodes de nettoyage: la solution de piranha et le plasma d'oxygène, dans le but d'obtenir une surface d'or propre sans oxydation de l'or. Ensuite, nous avons optimisé les paramètres lors de la mesure SPR comme par exemple la structure interfaciale du capteur, la force ionique .... Enfin, ces trois techniques ont été utilisées pour mesurer la constante de formation du duplex ADN/ADN. Les résultats ont montré que les Kd sont du même ordre de grandeur pour les trois techniques. De plus, pour les trois techniques, une augmentation de la densité de sonde de surface a entraîné une baisse d’affinité telle que mesuréeBiosensors are powerful detection and analysis tools that have been widely applied in pharmaceuticals, healthcare, biomedical research, and environmental monitoring. However different biosensors use different transducers and therefore different substrates and surface chemistries. All of these parameters may have an effect on the biomolecular reactions at the interface and lead to a deviation in dissociation constant Kd measurements. In this context, this PhD work aimed at comparing three different techniques: fluorescent microarray, evanescent field fluorescence biosensor and surface plasmon resonance (SPR) biosensor, to determine DNA hybridization Kd. For the classical fluorescence microarray, the substrate is a glass slide and the detection is performed at the end of the experiment. In the case of evanescent field fluorescence biosensor, polystyrene is the substrate and it permits a real-time detection. SPR is performed on thin gold film. It is a real-time and a label-free technique. The two fluorescent based techniques require to label the targets with fluorescent dyes prior to the measurements. One important factor determining the performance of the analysis is the surface chemistry of the sensor chip. Herein, we have optimized gold surface chemistry for thiol modified DNA grafting. We studied two cleaning methods: piranha solution and oxygen plasma, aiming at obtaining a clean gold surface without oxidation of the gold. Then, we optimized SPR assay parameters such as interfacial structure of sensor chip, ionic strength... After, these three techniques were used to measure the DNA hybridization Kd. The results showed that the Kds measured are similar for the three techniques. In addition, increasing surface probe density resulted in an increase of Kd of DNA hybridization
Books on the topic "Dissociation constant of inhibitor"
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Raney, Donald C., and M. L. Gillette. Determining the Dissociation Constant of a Weak Acid Using Ph Measurements. Chemical Education Resources, 1989.
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Gillette, Marcia L., and H. Anthony Neidig. Evaluating the Dissociation Constant of a Weak Acid Using Microscale Techniques. Chemical Education Resources, 1991.
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Carter, K. N. Determining the Formula and Estimating the Dissociation Constant of a Complex Ion. Chemical Education Resources, 1994.
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Siino, Joseph S. Replacement of conserved threonines by alanine residues: Effect on the dissociation constant of recombinant human HMG-I. 1992.
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Stafford, Dan. Determiming the Equivalant Mass and Dissociation Constant of an Unknown Weak Acid by Titrimetry Modular Laboratory Program in Chemistry. Chemical Education Resources, 1990.
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Gillette, M. L., and Richard C. Bell. Determiming the Equivalant Mass and Dissociation Constant of an Unknown Weak Acid by Titrimetry Modular Laboratory Program in Chemistry. Chemical Education Resources, 1990.
Find full textBook chapters on the topic "Dissociation constant of inhibitor"
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Gooch, Jan W. "Dissociation Constant." In Encyclopedic Dictionary of Polymers, 237. New York, NY: Springer New York, 2011. http://dx.doi.org/10.1007/978-1-4419-6247-8_3861.
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Gooch, Jan W. "Dissociation Constant." In Encyclopedic Dictionary of Polymers, 888. New York, NY: Springer New York, 2011. http://dx.doi.org/10.1007/978-1-4419-6247-8_13585.
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Ellenbroek, Bart, Alfonso Abizaid, Shimon Amir, Martina de Zwaan, Sarah Parylak, Pietro Cottone, Eric P. Zorrilla, et al. "Equilibrium Dissociation Constant." In Encyclopedia of Psychopharmacology, 489–90. Berlin, Heidelberg: Springer Berlin Heidelberg, 2010. http://dx.doi.org/10.1007/978-3-540-68706-1_796.
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Speer, Tod W., Christin A. Knowlton, Michelle Kolton Mackay, Charlie Ma, Lu Wang, Larry C. Daugherty, Brandon J. Fisher, et al. "Dissociation Constant (Kd)." In Encyclopedia of Radiation Oncology, 157–58. Berlin, Heidelberg: Springer Berlin Heidelberg, 2013. http://dx.doi.org/10.1007/978-3-540-85516-3_675.
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Nahler, Gerhard. "acid dissociation constant." In Dictionary of Pharmaceutical Medicine, 2. Vienna: Springer Vienna, 2009. http://dx.doi.org/10.1007/978-3-211-89836-9_13.
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Tallarida, Ronald J., and Rodney B. Murray. "Dissociation Constant I: Agonists." In Manual of Pharmacologic Calculations, 44–47. New York, NY: Springer New York, 1987. http://dx.doi.org/10.1007/978-1-4612-4974-0_13.
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Tallarida, Ronald J., and Rodney B. Murray. "Dissociation Constant II: Partial Agonists." In Manual of Pharmacologic Calculations, 47–50. New York, NY: Springer New York, 1987. http://dx.doi.org/10.1007/978-1-4612-4974-0_14.
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Tallarida, Ronald J., and Rodney B. Murray. "Dissociation Constant III: Perturbation Methods (Rate Constants in the Drug-Receptor Reaction)." In Manual of Pharmacologic Calculations, 50–53. New York, NY: Springer New York, 1987. http://dx.doi.org/10.1007/978-1-4612-4974-0_15.
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Yan, Dong, Tetsumei Urano, Yumiko Takada, and Akikazu Takada. "The Dissociation of α2-Plasmin-Inhibitor-Plasmin Complex to Active Plasmin by SDS Treatment." In Current Aspects of Blood Coagulation, Fibrinolysis, and Platelets, 140–43. Tokyo: Springer Japan, 1993. http://dx.doi.org/10.1007/978-4-431-68323-0_24.
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Vogel, Marc, and Beatrix Suess. "Label-Free Determination of the Dissociation Constant of Small Molecule-Aptamer Interaction by Isothermal Titration Calorimetry." In Methods in Molecular Biology, 113–25. New York, NY: Springer New York, 2016. http://dx.doi.org/10.1007/978-1-4939-3197-2_9.
Full textConference papers on the topic "Dissociation constant of inhibitor"
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Visser, A., I. M. A. Verhamme, D. G. Meuleman, and G. W. K. van Dedem. "AN INDIRECT KINETIC METHOD FOR ESTIMATING THE AFFINITY BETWEEN HEPARIN AND HEPARIN COFACTOR II." In XIth International Congress on Thrombosis and Haemostasis. Schattauer GmbH, 1987. http://dx.doi.org/10.1055/s-0038-1644352.
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The heparin-dependent inactivation of alpha-thrombin by heparin cofactor II was studied in buffer media with a pH ranging from 6 to 9 and an ionic strength from 0.05 M to 0.80 M. We used a heparin fraction with a mean Mr of 16,000 .The log dose response curves (logarithm of the 2nd order inactivation constant vs. the logarithm of heparin concentration) display a sigmoidal behavior. The lower and upper limiting plateau and the steepness of the ascending limb are characteristic for every pH and ionic strength. Similar log dose response curves can be observed for the heparin-mediated inactivation of factor Xa and plasmin by ATIII,indicating that enhancement of the inhibition only depends on heparin-inhibitor binding despite the presence of heparin-enzyme complexes.This type of inactivation mechanism is clearly different from the one observed for the thrombin- and factor IXa - ATIII interactions which are characterized by a maximum in the log dose response curves.Therefore we can make the assumption that heparin-inhibitor binding is of major importance in the heparin-mediated thrombin-HCII interaction.Our experimental data were fit to a model which describes the dependence of the observed inactivation constant upon the concentration of heparin-HCII complex.This complex concentration is a function of the initial heparin and HCII concentrations and the dissociation constant K0 of the heparin-HCII complex.The model allows the estimation of K0 in various buffer media.A decrease of pK0 with increasing buffer concentration can be observed.The upper limiting plateau value for kobs which is often referred to as the intrinsic activity of heparin also decreases with increasing ionic strength.At pH 7 and 8 and an ionic strength of 0.4 M we-found K0 values of 1.00E-07 M.At pH 6 and an ionic strength of 0.8 M Kq eguals 4.00E-04 M indicating a markedly decreased affinity of heparin for HCII.Through a detailed analysis of the pH profile for K0 we might gain insight in the nature of the binding sites for heparin on the inhibitor.
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Sahari Moghaddam, Farzan, Maziyar Mahmoodi, Marziyeh Zare, Fatemeh Goodarzi, Majid Abdi, and Lesley James. "Natural Gas Hydrate Equilibria in Brine Including the Effect of Inhibitors on Hydrate Formation." In SPE Canadian Energy Technology Conference. SPE, 2022. http://dx.doi.org/10.2118/208890-ms.
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Abstract Preventing hydrate formation is critical to safely and economically manage subsea tiebacks. Thermodynamic Hydrate Inhibitors (THI) and Low Dosage Hydrate Inhibitors (LDHI) help manage hydrate formation. Here, we use a novel isothermal approach using a PVT cell to experimentally find the hydrate equilibrium point of natural gas and brine. In addition, a constant temperature and pressure condition is used to compare hydrate formation with and without hydrate inhibitors. First, to better understand the novel isothermal technique, natural gas-brine equilibrium experiments were conducted. Secondly, a constant pressure and temperature approach is used to investigate Kinetic Hydrate Inhibitors (KHIs) and low dosage methanol performance on hydrate formation. The formation and dissociation points are detected through a sudden drop or peak in the pressure profile, respectively, and by visual observation. To evaluate inhibitor performance, the experiments were conducted at challenging temperatures between -0.5°C to 3°C, applicable to the environment offshore Newfoundland and Labrador. Two commercial KHIs and one THI were tested. Both KHIs showed good performance up to certain level of subcooling according to their concentration. However, KHI-B performed better at inhibiting hydrates compared to KHI-A despite its lower concentrations compared to KHI-A. The induction time for 1 wt% KHI-A under 10°C subcooling at a temperature of 0.75°C was 311 min. The induction time for 1 wt% KHI-B under 12°C subcooling at a temperature of 2.66°C was 184 min. Yet, in the case of KHI B, with half the concentration (0.5 wt%), no hydrate formed at temperature of 1.21°C and 10°C subcooling. Low dosage methanol (a common THI) was also assessed. Although the induction time under 10.36°C subcooling and constant temperature of −0.43°C was only 47 min, no hydrate formed within 22 hours at −0.12°C under 7.5°C subcooling. This work uses a novel experimental isothermal approach by PVT cell to investigate hydrate equilibrium and the effectiveness of different inhibitors. Hence, a better understanding of natural gas hydrate equilibrium in brine is developed. Based on significant costs associated with injecting high quantities of THI (e.g., methanol) to prevent hydrate formation, this work also compares the performance of KHIs and low dosage THI (methanol).
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Church, F. C., R. E. Treanor, and H. C. Whinna. "ACTIVATION OF HEPARIN COFACTOR II BY PHOSVITIN, A PHOSPHOGLYCO-PROTEIN, AND OTHER PHOSPHATE-CONTAINING POLYANIONS." In XIth International Congress on Thrombosis and Haemostasis. Schattauer GmbH, 1987. http://dx.doi.org/10.1055/s-0038-1643867.
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We are characterizing the specificity of the polyanion-binding domain of the heparin/dermatan sulfate-dependent plasma protease inhibitor, heparin cofactor II (HCII). Various phosphate-containing polyanions accelerate the HCII-catalyzed inhibition of thrombin (T). Phosvitin, a phosphoprotein, enhances the HCII/T reaction at 25°C and pH 8.0 with the apparent second-order rate constant value (K2) increasing from 5 × 104 M−1 min−1 (in the absence of phosvitin) to 8 x 10' M”1 min as phosvitin increased from 0.05 to 30 ug/ml and then decreases as phosvitin is increased above 30 ug/ml. Apparent dissociation constant values for phosvitin-HCII and phosvitin-T are 450 nM and 10 nM, respectively. Polynucleotides accelerate the HCII/T reaction and have the following specificity (concentrations examined from 1-200 ug/ml): poly(guanylic acid) >> poly(adeny-lic acid, guanylic acid) > poly(inosinic acid) > poly(guanylic acid, uridylic acid) > poly(uridylic acid) = poly(adenylic acid) > poly(cytidylic acid). Polyphosphate anions (phosphate chain length, n, ranging from 5-100) enhance the HCII/T reaction. When compared at an equimolar phosphate concentration (1 mM), the rate was saturated at n = 50 with a maximum V.2 of about 5 × 107 M−1 min−1. Ca2+ (or Mg2+)-phosvitin/polyphosphate anion complexes and salmon protamine-polynucleotide complexes have lost the ability to enhance the HCII/T reaction. Phospho-pyridoxylated-HCII (lysine modified), with greatly reduced heparin cofactor activity, has lost its accelerating effect with phosvitin, polynucleotides and the polyphosphate anions. None of the above mentioned polyphosphate-containing compounds are effective at accelerating either the HCII-catalyzed inhibition of chymotrypsin or the antithrombin Ill-catalyzed T reaction. Our results suggest that (i) HCII is activated by the multiple negative charges of phosphate polyanions but they alone are not sufficient; (ii) the effective phosphate polyanions must also possess a specific conformation for maximum activity; and (iii) the phosphoserine-containing protein, phosvitin, can serve as a "template" for HCII/T.
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Lian, E. C. Y., and F. A. Siddigui. "BINDING OF 37-DKa PLATELET AGGLUTINATING PROTEIN TO HUMAN PLATELETS." In XIth International Congress on Thrombosis and Haemostasis. Schattauer GmbH, 1987. http://dx.doi.org/10.1055/s-0038-1643976.
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We have previously reported the purification of a 37-KDa platelet agglutinating protein (PAP p37) from the plasma of a patient with thrombotic thrombocytopenic purpura. using 125I-labeled p37, the properties of its binding to platelets were studied. The binding of p37 to washed human platelets from 4 normal subjects and two TTP patients after recovery was specific, concentration dependent and saturable. The Scatchard analysis revealed that the binding sites for p37 was about 100,000 per platelet with a dissociation constant of 48 × 10−9 M. The binding of p37 to erythrocytes was very little and non-specific. Stimulation of platelets by thrombin or ADP did not have any effect on the binding of p37 to platelets. The monoclonal antibodies to platelet GP lb (6D1) and GP Ilb-llla (10E5)(A gift of Dr. Barry coller) did not inhibit the binding of p37 to platelets. Fibrinogen (1 mg/ml) and FVIII/vWF (250 ug/ml) reduced the binding slightly. The polyclonal antibodies to p37 as well as concanavalin-A inhibited the binding of p37 to platelets through their direct interaction with p37. Other lectins such as phytohemagglutinin, potato lectin and helix pomatia lectin did not have any effect. At 40 mM, sialic acid, α-D-(+)-glucose, D-(+)-mannose and D-fructose caused 91%,44%,79%, and 63% inhibition of p37 binding respectively. D-(+)-galactose did not interfere with the binding. It is concluded that p37 binds to platelets on the sites other than GP lb and Gp IIb-IIIa and its binding to platelets is inhibited by certain sugars.
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Contant, G., J. L. Martinoli, and C. Thirion. "INFLUENCE OF PLASMINOGEN ACTIVATOR INHIBITOR (PAI) ON THE TISSUE PLASMINOGEN ACTIVATOR (tPA) ASSAY IN EUGLOBULIN FRACTIONS AND IN PLASMA." In XIth International Congress on Thrombosis and Haemostasis. Schattauer GmbH, 1987. http://dx.doi.org/10.1055/s-0038-1644861.
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Current liquid-phase methods for thedetermination of tPA in plasma involve inactivation of both tPA and plasmin inhibitors by either plasma acidificationor euglobulin fractionation. In a firststep, our liquid-phase assay was performed in euglobulin fractions (EF) and the inactivation of PAI was monitored.In a second step, we studied the influenceof PAI onthe tPA assay in plasma.When EF were used (1:10 dilution, pH 5.9), the reference curve was done with tPA (0 to 1 IU/ml) diluted in buffer.The diluted samples (tPA or EF) were incubated with fibrinogen fragments as Stimulator (0.5 mg/ml), an excess of plasminogen (0.15 pM/ml) and chromogenic substrate CBS 30.41 (3.75 pM/ml).The tPA level of EF obtained after venous stasis was found tobe2.7 IU/ml, whatever the dilution was (1:4 to 1:64). For EF obtained at rest, a paradoxical increase in tPA activity was found from 1:4 to 1:16 until a plateau was reached (usually ar dilution 1:16 to 1:32).This is due to the partial dissociation of tPA/PAI complexes which increases with the dilution as demonstrated by adding tPA to highly diluted (1:64) EF : noinhibition occurred. To avoid the influence of PAI and to measure the total tPA activity, a sufficient dilution was needed.When the tPA assay was performed in plasma, ∝ 2 antiplasmin (∝ 2AP) was shown to be mainly responsiblefor a gradual loss of tPA and for inhibition of plasmin generation (using anti ∝2AP IgG). To maintain the concentration of ∝2AP constant in samples, they were 1:10 diluted in a tPA/PAI depleted plasma (plasmin inhibitors were brought in excess by the depleted plasma). The reference curve was done with tPA (0 to2 IU/ml) diluted in the depleted plasma instead of buffer. TPA activities were shown in plasma obtained after venous stasis, when low PAI values (0 to 2.5 IU/ml) were associated. No tPA activity was found in plasma obtained at rest, associated with normal PAI values. It can be concluded that a tPA activity is displayed only when PAI is saturated by released tPA.A free tPA activity can be detected in plasma, while in the EF, the tPA measured is dissociated from tPA/PAI complexes.
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Bakhit, C., D. Lewis, R. Billings, and B. Malfroy. "CELLULAR CATABOLISM OF RECOMBINANT TISSUE-TYPE PLASMINOGEN ACTIVATOR: IDENTIFICATION AND CHARACTERIZATION OF A NOVEL HIGH AFFINITY UPTAKE SYSTEM ON RAT HEPATOCYTES." In XIth International Congress on Thrombosis and Haemostasis. Schattauer GmbH, 1987. http://dx.doi.org/10.1055/s-0038-1644400.
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The uptake, internalization and intracellular degradation of 125I-labeled rt-PA (125I-rt-PA) by isolated rat hepatocytes was investigated. Incubation at 37°C resulted in internalization of 125I-rt-PA, followed by the appearance of labeled trichloroacetic acid-soluble (TCA) material in the inclubation media due to degradation of rt-PA. Degradation of rt-PA was inhibited by the presence of NH4Cl (10mM) or chloroquine (ImM) (lysosoma tropic agents) in the incubation media. This suggests that rt-PA degradation occurs intracellularly, perhaps within the lysosomes. 125I-rt-PA was taken up by rat hepatocytes through a specific, high affinity mechanism. Scatchard analysis of the data indicated that 106 molecules of rt-PA were taken up per cell/hour and the calculated dissociation constant was lOnM. Uptake of 125I-rt-PA was not inhibited by glycopeptides isolated from rt-PA nor by several other glycoproteins known to be cleared by identified hepatic receptors. These results suggest that the uptake of rt-PA by rat hepatocytes involves a receptor specific for t-PA and is not mediated by a carbohydrate specific receptor.
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Preissner, K. T., E. Anders, and G. Müller-Berghaus. "INTERACTION OF S PROTEIN/VITRONECTIN WITH CULTURED ENDOTHELIAL CELLS: PROMOTION OF ATTACHMENT AND SPECIFIC BINDING." In XIth International Congress on Thrombosis and Haemostasis. Schattauer GmbH, 1987. http://dx.doi.org/10.1055/s-0038-1643635.
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The interaction of the complement inhibitor S protein, which is identical to the serum spreading factor, vitronectin, with cultured human endothelial cells of macro- and microvas- cular origin was investigated. Purified S protein, coated for 2 h on polystyrene petri dishes, induced concentration- and time-dependent attachment and spreading of human umbilical vein endothelial cells (HUVEC) as well as human omental tissqe microvasular endothelial cells (HOTMEC) at 37°C. With 3 × 105 cells/ml (final concentration) more than 50% of the cells attached within 2 h incubation at 0.3 - 3 μg/ml S protein. The effect of S protein was specific, since only monospecific antibodies against S protein prevented attachment of cells, while antibodies against fibronectin, fibrinogen or von Wille-brand factor were uneffective. The pentapeptide Gly-Arg-Gly-Asp-Ser, which contains the cell-attachment site of these adhesive proteins including S protein, inhibited the activity of S protein to promote attachment of endothelial cells in a concentration-dependent fashion; at 200 μM peptide, less than 10% of the cells became attached. Direct binding of S protein to HUVEC and HOTMEC was studied with cells in suspension at a concentration of 1 × 106 cells/ml in the presence of 1% (w/v) human serum albumin and 1 mM CaCl2 and was maximal after 120 min. Both cell types bound S protein in a concentration-dependent fashion with an estimated dissociation constant KD=0.2pM. More than 80% of bound radiolabelled S protein was displaced by unlabelled S protein, whereas binding was reduced to about 50% by the addition in excess of either fibronectin, fibrinogen, von Willebrand factor or the pentapeptide. These findings provide evidence for the specific association of S protein with endothelial cells, ultimately leading to attachment and spreading of cells. Although the promotion of attachment was highly specific for S protein, other adhesive proteins than S protein, also known to associate with endothelial cells, may in part compete with direct S protein binding.
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Higashiyama, S., I. Ohkubo, H. Ishiguro, and M. Sasaki. "A NEW FUNCTION OF HUMAN KININOGENS: THE AMINO-TERMINAL REGION OF DOMAIN 1 INVOLVES AN EF HAND-LIKE STRACTURE FOR METAL BINDING." In XIth International Congress on Thrombosis and Haemostasis. Schattauer GmbH, 1987. http://dx.doi.org/10.1055/s-0038-1642851.
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Two types of kininogens in mammalian plasma, high molecular weight (HMW) and low molecular weight (LMW) kininogens, are the precursors of kinin. Especially, HMW kininogen circulates in the plasma as a complex with prekallikrein and factor XI, and functions as a cofactor in the initial phase reactions of intrinsic blood coagulation cascade. Recently, it has been found that the kininogens have inhibitory activity toward cysteine proteinases. The heavy chain portion, which is identical for HMW and LMW kininogens, is composed of three domains, domain 1, 2 and 3. Each the domain 2 and 3 has a reactive site as a cysteine proteinase inhibitor. However, physiological function of domain 1 remains still unknown. By using the antibody recognizing the interaction between HMW kininogen and Ca2+ (anti-HMW kininogen-Ca2+ antibody) as a probe, we newly found the Ca2+ binding site in the domain 1.Anti-HMW kininogen-Ca2+ antibody was isolated from anti-HMW kininogen antiserum as an antibody which bound to a HMW kininogen-Sepharose column equilibrated with 40 mM Tris-HCl buffer, pH 7.5, containing 1.0 M NaCl and 1 mM CaCl2, and was eluted with 3 mM EDTA. Resulting from the characterization by ELISA, this antibody specifically recognized the CB-1 region (CNBr-cleavage fragment 1: 1-160 amino acid sequence) of the heavy chain of kininogen molecules in the presence of Ca2+ or Mg2+. Furthermore, circular dichroism (CD) experiments showed that the conformational changes of HMW kininogen and heavy chain were induced by the addition of metal ions such as Ca2+ or Mg2+, and that this change was due to the conformational change of the CB-1 region. The dissociation constant (Kd) for heavy chain measured by Ca2+ titration analysis by CD at 214 nm was found to be 0.33 ± 0.09 mM. The number of Ca2+ binding sites of heavy chain calculated from Hill plot was 1.15 ± 0.04. The EF handlike structure found in the amino-terminal portion of the heavy chain of kininogen molecules strongly supported the above data. This indicates a possibility that kininogens play an important role as a Ca2+ binding protein.
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LOFTUS, J. C., E. F. Plow, A. L. Frelinger III, M. A. Smith, S. D’ouza, and M. H. Ginsberg. "LOCALIZATION AND CHEMICAL SYNTHESIS OF A DIVALENT CATION REGULATED EPITOPE IN PLATELET MEMBRANE GPIIb." In XIth International Congress on Thrombosis and Haemostasis. Schattauer GmbH, 1987. http://dx.doi.org/10.1055/s-0038-1643959.
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Platelet membrane glycoprotein (GP)IIb-IIIa is a component of a common adhesive protein receptor for fibrinogen, fibronectin, and von Willebrand factor. A monoclonal antibody, PMI-1, defines a divalent cation dependent regulation of the surface orientation of the heavy chain of GPIIb. Exposure of the PMI-1 epitope inversely correlates with the capacity of platelets to bind fibrinogen and aggregate. We have now localized and chemically synthesized this epitope. A 1.1 Kb cDNA clone which directs the synthesis of a fusion protein which bears the PMI-1 epitope was isolated from a lambda gt 11 expression library constructed from mRNA from the human erythroleukemia (HEL) cell line. The position of the N-terminal sequence of the light chain of GPIIb in the deduced amino acid sequence of the clone defined the orientation of the light and heavy chains of GPIIb. Analysis of the amino acid sequence corresponding to the heavy chain of GPIIb identified a single region with a high likelihood of containing a continuous epitope. A synthetic 17 residue peptide, corresponding to the predicted antigenic site, inhibited the binding of PMI-1 to platelets. Two uM peptide was required to inhibit binding 50% in the presence of 1 uM PMI-1, indicating an approximate dissociation constant of 1.5 uM for the peptide-antibody complex. This figure should be compared to a Kd of 0.95 uM (JCI 78:1103, 1986) for PMI-1 binding to GPIIb. A second peptide, corresponding to the region immediately adjacent to the predicted antigenic site, failed to inhibit PMI-1 binding. Neither peptide inhibited the binding of two other monoclonal anti GPIIb-IIIa’s to platelets. The peptides had similar effects on PMI-1 interaction with purified GPIIb-IIIa in detergent solution. These data localize the PMI-1 epitope to a 17 amino acid region located near the carboxyl terminal of the heavy chain of GPIIb. Thus, they chemically define a region of GPIIb whose surface expression reflects the competence of GPIIb-IIIa as a component of a platelet receptor for adhesive proteins.
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Weizhen Liang, Sanjay B Shah, John J Classen, and Ratna Sharma-Shivappa. "Dissociation Constant of Ammonium and Henry's Law Constant of Ammonia in Broiler Litter." In 2011 Louisville, Kentucky, August 7 - August 10, 2011. St. Joseph, MI: American Society of Agricultural and Biological Engineers, 2011. http://dx.doi.org/10.13031/2013.37373.
Full textReports on the topic "Dissociation constant of inhibitor"
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Shomer, Ilan, Ruth E. Stark, Victor Gaba, and James D. Batteas. Understanding the hardening syndrome of potato (Solanum tuberosum L.) tuber tissue to eliminate textural defects in fresh and fresh-peeled/cut products. United States Department of Agriculture, November 2002. http://dx.doi.org/10.32747/2002.7587238.bard.
Full textAbstract:
The project sought to understand factors and mechanisms involved in the hardening of potato tubers. This syndrome inhibits heat softening due to intercellular adhesion (ICA) strengthening, compromising the marketing of industrially processed potatoes, particularly fresh peeled-cut or frozen tubers. However, ICA strengthening occurs under conditions which are inconsistent with the current ideas that relate it to Ca-pectate following pectin methyl esterase (PME) activity or to formation of rhamnogalacturonan (RG)-II-borate. First, it was necessary to induce strengthening of the middle lamellar complex (MLX) and the ICA as a stress response in some plant parenchyma. As normally this syndrome does not occur uniformly enough to study it, we devised an efficient model in which ICA-strengthening is induced consistently under simulated stress by short-chain, linear, mono-carboxylic acid molecules (OAM), at 65 oC [appendix 1 (Shomer&Kaaber, 2006)]. This rapid strengthening was insufficient for allowing the involved agents assembly to be identifiable; but it enabled us to develop an efficient in vitro system on potato tuber parenchyma slices at 25 ºC for 7 days, whereas unified stress was reliably simulated by OAMs in all the tissue cells. Such consistent ICA-strengthening in vitro was found to be induced according to the unique physicochemical features of each OAM as related to its lipophilicity (Ko/w), pKa, protonated proportion, and carbon chain length by the following parameters: OAM dissociation constant (Kdiss), adsorption affinity constant (KA), number of adsorbed OAMs required for ICA response (cooperativity factor) and the water-induced ICA (ICAwater). Notably, ICA-strengthening is accompanied by cell sap leakage, reflecting cell membrane rupture. In vitro, stress simulation by OAMs at pH<pKa facilitated the consistent assembly of ICAstrengthening agents, which we were able to characterize for the first time at the molecular level within purified insoluble cell wall of ICA-strengthened tissue. (a) With solid-state NMR, we established the chemical structure and covalent binding to cell walls of suberin-like agents associated exclusively with ICA strengthening [appendix 3 (Yu et al., 2006)]; (b) Using proteomics, 8 isoforms of cell wall-bound patatin (a soluble vacuolar 42-kDa protein) were identified exclusively in ICA-strengthened tissue; (c) With light/electron microscopy, ultrastructural characterization, histochemistry and immunolabeling, we co-localized patatin and pectin in the primary cell wall and prominently in the MLX; (d) determination of cell wall composition (pectin, neutral sugars, Ca-pectate) yielded similar results in both controls and ICA-strengthened tissue, implicating factors other than PME activity, Ca2+ or borate ions; (e) X-ray powder diffraction experiments revealed that the cellulose crystallinity in the cell wall is masked by pectin and neutral sugars (mainly galactan), whereas heat or enzymatic pectin degradation exposed the crystalline cellulose structure. Thus, we found that exclusively in ICA-strengthened tissue, heat-resistant pectin is evident in the presence of patatin and suberinlike agents, where the cellulose crystallinity was more hidden than in fresh control tissue. Conclusions: Stress response ICA-strengthening is simulated consistently by OAMs at pH< pKa, although PME and formation of Ca-pectate and RG-II-borate are inhibited. By contrast, at pH>pKa and particularly at pH 7, ICA-strengthening is mostly inhibited, although PME activity and formation of Ca-pectate or RG-II-borate are known to be facilitated. We found that upon stress, vacuolar patatin is released with cell sap leakage, allowing the patatin to associate with the pectin in both the primary cell wall and the MLX. The stress response also includes formation of covalently bound suberin-like polyesters within the insoluble cell wall. The experiments validated the hypotheses, thus led to a novel picture of the structural and molecular alterations responsible for the textural behavior of potato tuber. These findings represent a breakthrough towards understanding of the hardening syndrome, laying the groundwork for potato-handling strategies that assure textural quality of industrially processed particularly in fresh peeled cut tubers, ready-to-prepare and frozen preserved products.