Relevant bibliographies by topics / Enzyme-Substrate Interactions

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  1. Journal articles
  2. Dissertations / Theses
  3. Book chapters
  4. Conference papers

Academic literature on the topic 'Enzyme-Substrate Interactions'

Author: Grafiati
Published: 4 June 2021
Last updated: 15 February 2022

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Journal articles on the topic "Enzyme-Substrate Interactions"

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Kumar, Vinay, and K. K. Kannan. "Enzyme-Substrate Interactions." Journal of Molecular Biology 241, no. 2 (August 1994): 226–32. http://dx.doi.org/10.1006/jmbi.1994.1491.

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MILNER-WHITE, E. JAMES. "Displaying enzyme-substrate interactions." Biochemical Society Transactions 17, no. 4 (August 1, 1989): 681–82. http://dx.doi.org/10.1042/bst0170681.

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Lee, Ida, Barbara R. Evans, Lynette M. Lane, and Jonathan Woodward. "Substrate-enzyme interactions in cellulase systems." Bioresource Technology 58, no. 2 (November 1996): 163–69. http://dx.doi.org/10.1016/s0960-8524(96)00095-8.

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Guo, Qing, Yufan He, and H. Peter Lu. "Manipulating and probing enzymatic conformational fluctuations and enzyme–substrate interactions by single-molecule FRET-magnetic tweezers microscopy." Phys. Chem. Chem. Phys. 16, no. 26 (2014): 13052–58. http://dx.doi.org/10.1039/c4cp01454e.

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To investigate the critical role of the enzyme–substrate interactions in enzymatic reactions, the enzymatic conformation and enzyme–substrate interaction at a single-molecule level are manipulated by magnetic tweezers, and the impact of the manipulation on enzyme–substrate interactions are simultaneously probed by single-molecule FRET spectroscopy.
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Guerrier-Takada, C., N. Lumelsky, and S. Altman. "Specific interactions in RNA enzyme-substrate complexes." Science 246, no. 4937 (December 22, 1989): 1578–84. http://dx.doi.org/10.1126/science.2480641.

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Li, Huiying, and Thomas Poulos. "Crystallization of Cytochromes P450 and Substrate-Enzyme Interactions." Current Topics in Medicinal Chemistry 4, no. 16 (December 1, 2004): 1789–802. http://dx.doi.org/10.2174/1568026043387205.

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Freitag, Ruth. "Utilization of enzyme–substrate interactions in analytical chemistry." Journal of Chromatography B: Biomedical Sciences and Applications 722, no. 1-2 (February 1999): 279–301. http://dx.doi.org/10.1016/s0378-4347(98)00507-6.

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Bretz, Stacey Lowery, and Kimberly J. Linenberger. "Development of the enzyme-substrate interactions concept inventory." Biochemistry and Molecular Biology Education 40, no. 4 (June 18, 2012): 229–33. http://dx.doi.org/10.1002/bmb.20622.

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Peluso, Carolyn E., David Umulis, Young-Jun Kim, Michael B. O'Connor, and Mihaela Serpe. "Shaping BMP Morphogen Gradients through Enzyme-Substrate Interactions." Developmental Cell 21, no. 2 (August 2011): 375–83. http://dx.doi.org/10.1016/j.devcel.2011.06.025.

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Suzuki, T., Y. Zhang, T. Koyama, and K. Kurihara. "1P093 Specific Interactions between Enzyme-Substrate Complexes Depending on Substrate Chain Length." Seibutsu Butsuri 44, supplement (2004): S53. http://dx.doi.org/10.2142/biophys.44.s53_1.

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Dissertations / Theses on the topic "Enzyme-Substrate Interactions"

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Linenberger, Kimberly J. "Biochemistry Students' Understandings of Enzyme-Substrate Interactions as Investigated through Multiple Representations and the Enzyme-Substrate Interactions Concept Inventory." Miami University / OhioLINK, 2011. http://rave.ohiolink.edu/etdc/view?acc_num=miami1321309534.

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Qi, Xiaolin. "Enzyme-substrate interactions in PC1 #beta#-lactamase catalysis." Thesis, University of Newcastle Upon Tyne, 1991. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.315617.

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Ulrich, Magdalena Maria Wilhelmina. "Enzyme/substrate interactions of the vitamin K-dependent carboxylase." [Maastricht : Maastricht : Rijksuniversiteit Limburg] ; University Library, Maastricht University [Host], 1991. http://arno.unimaas.nl/show.cgi?fid=6207.

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Fransson, Linda. "Enzyme substrate solvent interactions : a case study on serine hydrolases." Doctoral thesis, KTH, Biokemi, 2008. http://urn.kb.se/resolve?urn=urn:nbn:se:kth:diva-4867.

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Reaction rates and selectivities were measured for transacylation of fatty acid esters in solvents catalysed by Candida antarctica lipase B and by cutinase from Humicola insolens. With these enzymes classical water-based enzymology can be expanded to many different solvents allowing large variations in interaction energies between the enzymes, the substrates and the surrounding. Further ,hydrolysis reactions catalysed by Bacillus subtilis esterase 2 were investigated. Thermodynamics analyses revealed that the enzyme contribution to reaction rate acceleration compared to acid catalysis was purely entropic. On the other hand, studies of differences in activation entropy and enthalpy between enantiomers and between homologous esters showed that high substrate specificity was favoured by enthalpic stabilisation. Solvent was found to have a profound effect on enzyme catalysis, affecting both reaction rate and selectivity. Differences in substrate solubility will impact enzyme specificity since substrate binding is an equilibrium between enzyme-bound substrate and substrate in free solution. In addition, solven tmolecules were found to act as enzyme inhibitors, showing both competitive and non-competitive behaviour. In several homologous data series enthalpy-entropy compensation relationships were encountered. A possible extrathermodynamic relationship between enthalpy and entropy can easily be lost under co-varying errors propagated from the experiments. From the data in this thesis, one instance was found of a real enthalpy-entropy compensation that could be distinguished from statistical errors, while other examples could not be verified.
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Pribowo, Amadeus Yeremia. "Enzyme-substrate interactions and their influence on enzyme recycling strategies as a way of reusing cellulases." Thesis, University of British Columbia, 2014. http://hdl.handle.net/2429/46481.

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Relatively high enzyme loadings are required for the bioconversion of lignocellulosic biomass, impeding the economical production of cellulosic sugars. The relative stability and robustness of these enzymes make enzyme recycling an attractive cost-reduction strategy. However, the efficiency of enzyme recycling has been limited by the complexity of enzyme-substrate interactions, which are influenced by enzyme, substrate, and physical factors. A lack of techniques to probe specific enzyme adsorption further limits our understanding of these interactions. Therefore, overcoming these challenges to better understand enzyme-substrate interactions is crucial if we are to improve the effectiveness of enzyme recycling strategies. Initial work compared various ways to assess enzyme adsorption during hydrolysis of steam pretreated corn stover (SPCS) using a complete commercial cellulase mixture. While the distribution of six individual enzymes could be followed, the initial approach used was laborious, highlighting the limitations of techniques used to quantify individual enzyme adsorption profiles. A quicker, more sensitive double antibody sandwich enzyme-linked immunosorbent assay (ELISA) was subsequently developed, to follow Cel7A, Cel6A, and Cel7B adsorption during hydrolysis, and shown to agree with earlier results. As enzyme, substrate, and physical factors were known to affect enzyme recycling performance, their influence on individual enzyme adsorption was evaluated. Although the lignin present in the SPCS did not appear to influence enzyme adsorption (although Cel6A adsorbed more readily to the lignin-containing SPCS), cellulose allomorphs and crystallinity did appear to influence enzyme adsorption. The addition of Auxiliary Activity (AA) family 9, an oxidative enzyme, increased desorption of Cel7A, likely by increasing the substrate’s negative charge. The AA9 itself remained primarily in the supernatant, which highlighted the importance of recovering enzymes from both the liquid and solid phases of the reaction. The influence of glucose and ethanol on enzyme adsorption was evaluated, and a reduction in enzyme adsorption was observed at high glucose but not ethanol concentrations. When the addition of fresh substrate was assessed as one way to recover enzymes, by combining enzyme recycling at low glucose concentrations with enzyme supplementation, good overall cellulose hydrolysis (~70%) over 5 rounds of enzyme recycle could be achieved with a 50% reduction in enzyme loading.
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Choudhury, Devapriya. "Functional implications of macromolecular recognition : assembly of adhesive pili and enzyme substrate interactions /." Uppsala : Swedish Univ. of Agricultural Sciences (Sveriges lantbruksuniv.), 2001. http://epsilon.slu.se/avh/2001/91-576-5820-X.pdf.

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Plach, Maximilian [Verfasser], and Reinhard [Akademischer Betreuer] Sterner. "Evolution of substrate specificity and protein-protein interactions in three enzyme superfamilies / Maximilian Plach ; Betreuer: Reinhard Sterner." Regensburg : Universitätsbibliothek Regensburg, 2017. http://d-nb.info/1131875826/34.

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Suh-Lailam, Brenda Bienka. "Development of Novel Methods and their Utilization in the Analysis of the Effect of the N-terminus of Human Protein Arginine Methyltransferase 1 Variant 1 on Enzymatic Activity, Protein-protein Interactions, and Substrate Specificity." DigitalCommons@USU, 2010. https://digitalcommons.usu.edu/etd/863.

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Protein arginine methyltransferases (PRMTs) are enzymes that catalyze the methylation of protein arginine residues, resulting in the formation of monomethylarginine, and/or asymmetric or symmetric dimethylarginines. Although understanding of the PRMTs has grown rapidly over the last few years, several challenges still remain in the PRMT field. Here, we describe the development of two techniques that will be very useful in investigating PRMT regulation, small molecule inhibition, oligomerization, protein-protein interaction, and substrate specificity, which will ultimately lead to the advancement of the PRMT field. Studies have shown that having an N-terminal tag can influence enzyme activity and substrate specificity. The first protocol tackles this problem by developing a way to obtain active untagged recombinant PRMT proteins. The second protocol describes a fast and efficient method for quantitative measurement of AdoMet-dependent methyltranseferase activity with protein substrates. In addition to being very sensitive, this method decreases the processing time for the analysis of PRMT activity to a few minutes compared to weeks by traditional methods, and generates 3000-fold less radioactive waste. We then used these methods to investigate the effect of truncating the NT of human PRMT1 variant 1 (hPRMT1-V1) on enzyme activity, protein-protein interactions, and substrate specificity. Our studies show that the NT of hPRMT1-V1 influences enzymatic activity and protein-protein interactions. In particular, methylation of a variety of protein substrates was more efficient when the first 10 amino acids of hPRMT1v1 were removed, suggesting an autoinhibitory role for this small section of the N-terminus. Likewise, as portions of the NT were removed, the altered hPRMT1v1 constructs were able to interact with more proteins. Overall, my studies suggest the the sequence and length of the NT of hPRMT1v1 is capable of enforcing specific protein interactions.
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Borel, Franck. "Contributions à l'étude des interactions entre les ARNtser et la séryl-ARNt synthétase d'Escherichia coli et de Saccharomyces cerevisiae." Université Joseph Fourier (Grenoble), 1994. http://www.theses.fr/1994GRE10142.

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Ce travail est consacre a l'etude des mecanismes responsables de la reconnaissance entre la seryl-arnt synthetase d'escherichia coli et son substrat macromoleculaire, l'arnt#s#e#r. La premiere etape de ces travaux a consiste a surexprimer l'arnt#s#e#r#2 et l'arnt#s#e#r ambre suppresseur d'e. Coli. Pour cela, les genes synthetiques des deux arnt#s#e#r ont ete construits par assemblage de sept oligonucleotides chevauchants. L'utilisation d'un plasmide dont le nombre de copies est regule par la temperature permet d'obtenir des cellules bacteriennes contenant un taux d'arnt#s#e#r vingt fois superieur a celui present dans des cellules depourvues de plasmide. Grace a cette surexpression, un protocole de purification comportant deux etapes chromatographiques a pu etre developpe. Les arnt purifies ont ensuite ete utilises pour determiner la contribution du domaine n-terminal de la seryl-arnt synthetase d'e. Coli, a l'efficacite et a la specificite de la reaction d'aminoacylation. Les resultats obtenus, montrent que le domaine n-terminal depourvu de role catalytique lors de la reaction d'activation de l'acide amine, est indispensable, d'une part, au maintien d'une efficacite de catalyse elevee lors du transfert de l'acide amine active sur l'arnt et, d'autre part, a la specificite de reconnaissance des arnt#s#e#r. Parallelement, la mesure des constantes de dissociation de l'arnt#s#e#r#2, revele que la fixation de deux molecules d'arnt sur la seryl-arnt synthetase est regit par un mecanisme de type cooperatif. La derniere partie de ce travail montre que la seryl-arnt synthetase de saccharomyces cerevisiae ne peut etre surexprimee chez e. Coli. Le contexte bacterien ne permettant pas a la seryl-arnt synthetase de s. Cerevisiae d'acquerir une structure tridimensionnelle homogene. Les etudes menees, en parallele sur la seryl-arnt synthetase de s. Cerevisiae surexprimee dans son contexte naturel, revelent une repartition de cette enzyme en deux populations distinctes, et une mobilite electrophoretique, sur sds-page, plus elevee que celle obtenue pour l'enzyme surexprimee chez e. Coli
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Pauthe, Emmanuel. "Approches cinétiques et moléculaires de la reconnaissance enzyme-substrat : application à l'étude de l'activité protéolytique de la thermolysine." Compiègne, 1998. http://www.theses.fr/1998COMP1139.

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L’accomplissement de tout acte protéolytique implique nécessairement la formation d'un complexe entre l'enzyme et son substrat. Par différentes approches cinétiques, spectroscopiques et moléculaires nous avons cherché à caractériser les phénomènes mis en jeu au cours de l'hydrolyse, par la thermolysine, de petits peptides en milieu biomimétiques. Cette étude a été conduite à l'interface entre la biochimie, la biophysique, la chimie et la physique. Dans un premier temps, nous nous sommes intéressés au comportement catalytique de la thermolysine sur des substrats modèles et en milieu modifié. Nous avons montré d'une part, que l'ajout d'additifs polyhydroxylés influence grandement l'activité de la thermolysine et d'autre part, affine les connaissances sur la spécificité et la sélectivité de cette enzyme (en particulier, mise en évidence de l'influence du résidu P'2 dans le mécanisme). Dans un deuxième temps, nous présentons des études structurales des peptides substrats en milieu modifié. Nous avons mis en évidence l'absence d'influence du micro-environnement contenant une forte proportion de glycérol sur la conformation des molécules de substrat et le rôle possible de leur structure tridimensionnelle quant à leur hydrolyse. Ces études ont été étendues à un autre modèle peptidique, de forme cyclique ou linéaire, et corrélées aux résultats cinétiques. Dans un troisième temps, par deux approches différentes, nous avons abordé l'étude des relations structure-fonction de la thermolysine. Expérimentalement, par des études cinétiques avec l'enzyme immobilisée et des déterminations de sa structure par spectroscopie laser Raman, nous montrons que l'enzyme est très peu sensible au micro-environnement. Théoriquement en analysant, par modélisation, l'interaction de la thermolysine avec un tripeptide substrat, nous avons mis en évidence des changements de conformation du substrat et/ou des mouvements du site actif enzymatique au cours de l'acte catalytique.
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Book chapters on the topic "Enzyme-Substrate Interactions"

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Page, M. I. "The specificity of enzyme—substrate interactions." In Accuracy in Molecular Processes, 37–66. Dordrecht: Springer Netherlands, 1986. http://dx.doi.org/10.1007/978-94-009-4097-0_3.

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Carloni, Paolo, and Frank Alber. "Density-Functional Theory Investigations of Enzyme-substrate Interactions." In 3D QSAR in Drug Design, 169–79. Dordrecht: Springer Netherlands, 2002. http://dx.doi.org/10.1007/0-306-46857-3_10.

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Chhabra, Aastha, and Vibha Rani. "Cell In Situ Zymography: Imaging Enzyme–Substrate Interactions." In Zymography, 133–43. New York, NY: Springer New York, 2017. http://dx.doi.org/10.1007/978-1-4939-7111-4_12.

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Kwiecień, Renata A., Andrzej Lewandowicz, and Piotr Paneth. "Substrate-Enzyme Interactions from Modeling and Isotope Effects." In Challenges and Advances in Computational Chemistry and Physics, 341–63. Dordrecht: Springer Netherlands, 2007. http://dx.doi.org/10.1007/1-4020-5372-x_7.

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Roberts, Gordon C. K. "NMR Studies of Enzyme-Substrate and Protein-Protein Interactions." In Dynamics and the Problem of Recognition in Biological Macromolecules, 37–47. Boston, MA: Springer US, 1996. http://dx.doi.org/10.1007/978-1-4615-5839-2_4.

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Kagamiyama, H., H. Hayashi, T. Yano, H. Mizuguchi, and S. Ishii. "Enzyme-substrate interactions modulating protonation and tautomerization states of the aldimines of pyridoxal enzymes." In Biochemistry of Vitamin B6 and PQQ, 43–47. Basel: Birkhäuser Basel, 1994. http://dx.doi.org/10.1007/978-3-0348-7393-2_7.

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Siezen, Roland J. "Modelling and Engineering of Enzyme/Substrate Interactions in Subtilisin-Like Enzymes of Unknown 3-Dimensional Structure." In Advances in Experimental Medicine and Biology, 63–73. Boston, MA: Springer US, 1996. http://dx.doi.org/10.1007/978-1-4613-0319-0_8.

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Hannon, Bruce, and Matthias Ruth. "Chance-Cleland Model for Enzyme-Substrate Interaction." In Dynamic Modeling, 80–82. Berlin, Heidelberg: Springer Berlin Heidelberg, 1994. http://dx.doi.org/10.1007/978-3-662-25989-4_9.

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Hannon, Bruce, and Matthias Ruth. "Chance-Cleland Model for Enzyme-Substrate Interaction." In Dynamic Modeling, 80–82. New York, NY: Springer US, 1994. http://dx.doi.org/10.1007/978-1-4684-0224-7_9.

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Hannon, Bruce, and Matthias Ruth. "Chance-Cleland Model for Enzyme-Substrate Interaction." In Dynamic Modeling, 129–32. New York, NY: Springer New York, 2001. http://dx.doi.org/10.1007/978-1-4613-0211-7_9.

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Conference papers on the topic "Enzyme-Substrate Interactions"

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Hester, Ronald E., and J. C. Austin. "Dehydrogenase enzyme/coenzyme/substrate interactions." In Moscow - DL tentative, edited by Sergei A. Akhmanov and Marina Y. Poroshina. SPIE, 1991. http://dx.doi.org/10.1117/12.57364.

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Fenton, J. W., J. I. Witting, and T. M. Miller. "THROMBIN KINETIC PARAMETERS WITH TRIPEPTIDE p-NITROANALIDES UNDER PHYSIOLOGICALLY RELEVANT CONDITIONS." In XIth International Congress on Thrombosis and Haemostasis. Schattauer GmbH, 1987. http://dx.doi.org/10.1055/s-0038-1643278.

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The Michaelis-Menten (Km ), catalytic (kcat ), and specificity (kcat/Km ) constants were determined for humSft α- and β-thrombins with the chromogenic substrate S-2238 (H-D-Phe-Pip-Arg-pNA), Chromozym-TH (Tos-Gly-Pro-Arg-pNA), and Spectrozyme-TH (H-D-HHT-Ala-Arg-pNA) in 0.15 M NaCl buffered with 10 mM HEPES ancTlO mM Tris-HCT at pH 7.4, 37°C. Spontaneous hydrolysis was insignificant for these substrates. Both S-2238 and Spectrozyme-TH exhibited limiting solubilities at ∼35 μM, while Chromozym-TH did not do so up to 50 μM. From initial estimates, Km's and k t's were refined by computer Gause-Newton iterations or nonlinearneast-square fits for the concentration of p-nitroanalide formed per second versus the initial substrate concentration. No major differences were found between a-thrombin (99% α, 91% esterolytically active enzyme, and > 3,500 kilo-U.S. clotting units/g with fibrinogen) and Yγ-thrombin (98% γ. 89% active enzyme, and < 10 kilo-units/g). For α- versus γ-thrombin and the three substrates, respectively, the Km 's were 6.75 ± 0.13 vs 7.62 ± 0.30, 18.4 ± 0.4 vs 23.0 ± 0.3, and 2.53 ± 0.02 vs 3.85 ± 0.51 μM; the kcat> s were 125 ± 1 vs 134 ± 2, 181 ± 2 vs 130 ± 1, and 35.5 ± 0.2 vs 52.4 ± 2.0 s−1 ; and the kcat/Km 's were 18.5 vs 17.6, 9.84 vs 5.65, and 10.1 vs 13.6 s−1 μm . These values closely approximate those of 7.2 ± 0.9 μM, 84 ± 4 s−1 , and 11.7 s−1 μM−1 determined by Higgins, Lewis, and Shafer (J. Biol. Chem. 258:9276-9282, 1983) for the Aα cleavage of human fibrinogen by human α-thrombin under physiologically relevant conditions. Thus, these chromogenic substrates have thrombin specificities similar to that of fibrinogen, although their amidolytic activities are independent of additional active-site regions required for fibrinogen clotting activity (α vs γ-thrombin). Fibrinogen interactions with such active-site regions might account for why the fibrinogen Aa site is an atypical thrombin susceptible bond and the high species variability of fibrinopeptides. (Supported in part by NIH grant HL-13160).
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Suttie, W. J., A. Cheung, and M. G. Wood. "ENZYMOLOGY OF THE VITAMIN K-DEPENDENT CARBOXYLASE: CURRENT STATUS." In XIth International Congress on Thrombosis and Haemostasis. Schattauer GmbH, 1987. http://dx.doi.org/10.1055/s-0038-1643991.

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The vitamin K-dependent microsomal carboxylase converts glutamyl residues in precursor proteins to γ-carboxyglutamyl (Gla) residues in completed proteins. The enzyme activity is present in significant activities in most non-skeletal tissues but has been studied most extensively in rat and bovine liver. Early studies of the enzyme utilized bound precursors of vitamin K-dependent clotting factors as substrates for the enzyme and demonstrated that the enzyme requires the reduced form of vitamin K (vitamin KH2), O2, and CO2. Subsequent investigations have taken advantage of the observation that the enzyme will carboxylate low-molecular-weight peptide substrates with Glu-Glu sequences. Utilizing a substrate such as Phe-Leu-Glu-Glu-Leu, it has been possible to demonstrate that γ-C-H release from the Glu residue of a substrate is independent of CO2 concentration. The formation of vitamin K 2,3-epoxide can also be demonstrated in a crude microsomal system, and it can be shown that the formation of this metabolite can be stimulated by the presence of a peptide substrate of the carboxylase. These observations have led to the general hypothesis that the mechanism of action of the enzyme involves interaction of vitamin KH2 with O2 to form an oxygenated intermediate that can interact with a substrate Glu residue to abstract a γ-hydrogen and in the process he converted to vitamin K epoxide (KO). The current evidence suggests that, either directly or indirectly, removal of the γ-C-H results in the formation of a carbanion at the γ-position of the Glu residue which can interact with CO2 to form Gla. The Glu residue intermediate which is formed can be demonstrated to partition between accepting a proton in the media to reform Glu, or interacting with CO2 to form Gla. Current data do not distinguish between the direct formation of a carbanion coupled to proton removal, or the participation of a reduced intermediate. Recent studies have demonstrated that the enzyme will carry out a partial reaction, the formation of vitamin K epoxide, at a decreased rate in the absence of a Glu site substrate. Epoxide formation under these conditions has the same for O2 as the carboxylation reaction and is inhibited in the same manner as the carboxylation reaction. In the presence of saturating concentrations of a Glu site substrate and C02, the ratio of KO formed, γ-C-H released, and C02 formed is 1:1:1. However, KO formation can be uncoupled from and proceeds at a higher rate than γ-C-H bond cleavage and Gla formation at low Glu site substrate concentrations. At saturating concentrations of CO2, Gla formation is equivalent to γ-C-H bond cleavage, and this unity is not altered by variations in vitamin KH2 or peptide substrate concentrations. Natural compounds with vitamin K activity are 2-Me-l,4-naphthoquinones with a polyprenyl side chain at the 3-position. Studies of vitamin K analogs have demonstrated that a 2-Me group is essential for activity but that the group at the 3-position can vary significantly. Modification of the aromatic ring of the naphthoquinone nucleus by methyl group substitution can result in alterations of either the rate of the carboxylation reaction or the apparent affinity of the enzyme for the vitamin. Studies of a large number of peptide substrates have failed to reveal any unique primary amino acid sequence which is a signal for carboxylation. However, current evidence from a number of sources suggests that a basic amino acid rich "propeptide" region of the intracellular form of the vitamin K-dependent proteins is an essential recognition site for the enzyme. This region of the precursor is lost in subsequent processing, and the manner in which it directs this posttranslational event is not yet clarified. Supported by NIH grant AM-14881.
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Bashkov, G., T. Kalishevekaya, and S. Strukova. "ALTERED INTERACTION OF THROMBIN AND ANTITHROMBIN III WITH THE VASCULAR WALL." In XIth International Congress on Thrombosis and Haemostasis. Schattauer GmbH, 1987. http://dx.doi.org/10.1055/s-0038-1643344.

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The role of the endothelial injury in the development of the thrombophylic state was studied in rats with nephrotic syndrome (NS,Heymann nephritis).There were a 6-fold increase of the soluble fibrin concentration and a 30% decrease of plasma antithrombin III (AT) activity in the NSIt was found that the plasma half-life of 125 J-labelled α-thrombin (10-7 M) is 3,0 ± 0,6 min in control animals and 4,0 ± 0,1 min in NS rats. At 20 min following the administration of bovine 125J-thrombin it was observed that in normal animals 84% of the radiolabelled enzyme was bound with vessel wall.while in NS rats the figure was only 63% (p< 0,05). The alteration of thrombin binding to the vascular wall lead to an increase in the amount of soluble fibrin-monomer and AT-proteinase complexes.AT-thrombin complexes and a proteolytically modified form of AT (Mr<68 kDa) were isolated from NS rats plasma by affinity chromatography on heparin-sepharose and chromatofocusing.At 3 min following injection of a 100-fold molar excess of bovine AT (1,7 .10-5 M) it was observed that 35% of thrombin reversibly bound to the endothelium could be detected in the circulation of normal rats. The same excess of AT induced only a 10% (p<0,001) release of 125J-thrombin to the blood stream in the NS rats through the formation of 125 J-thrombin complexes with Mr≥100 kDa.It is being proposed that injury of the vascular wall in the NS animals facilitated the interaction of the enzyme with the substrate (fibrinogen) and inhibitor (AT), and leads to ineffective inactivation of thrombin bound to the endothelium by AT.
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Vermeer, C., BA M. Soute, and MM W. Ulrich. "IN VITRO CARBOXYLATION OF EXOGENOUS PROTEIN SUBSTRATES BY VITAMIN K-DEPENDENT CARBOXYLASE." In XIth International Congress on Thrombosis and Haemostasis. Schattauer GmbH, 1987. http://dx.doi.org/10.1055/s-0038-1643994.

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In vivo treatment of experimental animals with vitamin K-antagonists induces the accumulation of non-carboxylated coagulation factor precursors in the liver, where they are tightly bound to vitamin K-dependent carboxylase. If hepatic carboxylase is isolated from warfarin-treated animals, it is obtained therefore almost exclusively in the form of an enzyme/substrate complex. If carboxylase is prepared from non-treated animals, on the other hand, the resulting enzyme is predominantly substrate-free. Small substrates like F L E E L or decarboxylated osteocalcinare carboxylated equally well by both types of carboxylase, but protein substrates(Mr > 30 000) are recognized exclusively by substrate-free carboxylase.Initial attempts to purify carboxylasewere performed with livers from warfarin-treated cows as a starting material. Antibodies against the normal blood coagulation factors crossreact with the hepatic precursor proteins so that the enzyme/substrate complexes could be specifically extracted from detergent-solubilized microsomes by the substrate/antibody interaction. This procedure resulted ina substantial purification of carboxylase, but because its endogenous substrate remained firmly bound, even after it had been carboxylated in vitro, the enzyme system was not suitable for the carboxylation of protein substrates.Therefore a second strategy was developed by which substrate-free carboxylase (from normal livers) was partly purified by sequential extraction of the microsomal membranes with detergents, followed by ammonium sulfate precipitation and size exclusion chromatography.This procedure resulted in a soluble carboxylase complex, still consisting of 7 proteins and phosphatidylcholine. Although further dissociation of the complex resulted in a complete loss of activity, it is not sure if all components play a role in the carboxylation reaction. Exogenous substrates which could be carboxylated by substrate-free carboxylase were: the penta-peptide F L E E L, descarboxyprothrombin from bovine plasma, thermally decarboxylated osteocalcin from bovine bone and non-car-boxy lated coagulaton factor precursors which had been produced by recombinant-DNA techniques in various laboratories. The . efficiency of CO^ incorporation was: 1 mole per 100 moles of F L E E L, 1 mole per 240 moles of descarboxy-prothrombin, 1 mole per mole of decarboxylated osteocalcin and 8 moles per mole of a recombinant factor IX precursor. We assume that the high efficiency with which the recombinant coagulation factor precursors were carboxylated is due to the presence of at least part of their leader sequence. The importance of the aminoacid chain preceding the first carboxylatable Glu residue is demonstrated by the fact that descarboxylated osteocalcin of bovine origin is carboxylated with a relatively high efficiency, whereas descarboxylated osteocalcin from monkey bone is not recognized atal.. Yet the only difference between the two substrates is found in their aminoacids 3 and 4, whereas the first carboxylatable Glu occurs at position 17. It seems, therefore, that the aminoacids 1-16 in bovine osteocalcin mimic to some extent part of the leader sequence in the coagulation factor precursors. Chemical or biochemical modification of decarboxylated osteocalcin might reveal which structural features contribute to its recognition by hepatic carboxylase.The optimal conditions for carboxylation include a high concentration of dithiols (e.g. DTT) and under these conditions disulfide bridges are reduced. Obviously this will lead to a complete destruction of the biological activity of various carboxylated products. Therefore we have searched for a more natural reducing system and it was found that the bacterial thioredoxin/thiore-doxin-reductase system in the presence of 40 uM NADFH was able to replace DTT in the reaction mixtures. Since a comparable system also occurs in calf liver it seems not unlikely that this is the physiological counterpart of the dithiols used in vitro.
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6

Heckel, A., and K. M. Hasselbach. "PREDICTION OF THE THREE-DIMENSIONAL STRUCTURE OF THE ENZYMATIC PART OF T-PA." In XIth International Congress on Thrombosis and Haemostasis. Schattauer GmbH, 1987. http://dx.doi.org/10.1055/s-0038-1644407.

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Up to now the three-dimensional structure of t-PA or parts of this enzyme is unknown. Using computer graphical methods the spatial structure of the enzymatic part of t-PA is predicted on the hypothesis, the three-dimensional backbone structure of t-PA being similar to that of other serine proteases. The t-PA model was built up in three steps:1) Alignment of the t-PA sequence with other serine proteases. Comparison of enzyme structures available from Brookhaven Protein Data Bank proved elastase as a basis for modeling.2) Exchange of amino acids of elastase differing from the t-PA sequence. The replacement of amino acids was performed such that backbone atoms overlapp completely and side chains superpose as far as possible.3) Modeling of insertions and deletions. To determine the spatial arrangement of insertions and deletions parts of related enzymes such as chymotrypsin or trypsin were used whenever possible. Otherwise additional amino acid sequences were folded to a B-turn at the surface of the proteine, where all insertions or deletions are located. Finally the side chain torsion angles of amino acids were optimised to prevent close contacts of neigh bouring atoms and to improve hydrogen bonds and salt bridges.The resulting model was used to explain binding of arginine 560 of plasminogen to the active site of t-PA. Arginine 560 interacts with Asp 189, Gly 19 3, Ser 19 5 and Ser 214 of t-PA (chymotrypsin numbering). Furthermore interaction of chromo-genic substrate S 2288 with the active site of t-PA was studied. The need for D-configuration of the hydrophobic amino acid at the N-terminus of this tripeptide derivative could be easily explained.
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7

Jordan, R. E., R. M. Nelson, and J. Kilpatrick. "KINETICS OF THE HEPARIN-DEPENDENT INACTIVATION OF ANTITHROMBIN BY NEUTROPHIL ELASTASE." In XIth International Congress on Thrombosis and Haemostasis. Schattauer GmbH, 1987. http://dx.doi.org/10.1055/s-0038-1643898.

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The rate of inhibition of coagulation enzymes by antithrombin III is greatly increased by an interaction between the inhibitor and a limited, anticoagulantly-active subfraction of heparin molecules. We have observed that this same heparin sub-fraction also greatly stimulates the rate of inactivation of antithrombin by neutrophil elastase. Inactivation rates were increased several hundred-fold by catalytic amounts of the anticoagulantly active heparin species and were unaffected by the inactive heparin fraction or other glycosaminoglycans. Addition of catalytic amounts of elastase (1:400) to a solution of antithrombin in the presence of saturating levels of anticoagulantly-active heparin caused an approximately 30% decrease in the ultraviolet fluorescence emission of the inhibitor. The rate of fluorescence loss corresponded exactly with the loss of inhibitory activity in a parallel incubation under the same conditions. The use of the fluorescence technique for kinetic measurement of inactivation rates indicated a Km of less than 1 uM for the heparin-antithrombin complex substrate and a turnover of several hundred per minute per enzyme molecule. Although the specificity of the heparin effect appears to be at the level of its interaction with antithrombin, an elastase-heparin interaction could also be detected in kinetic analyses. Chromatographic studies employing immobilized heparin confirmed that elastase itself binds tightly to the complex carbohydrate. These results suggest a subtle mechanism, of potential relevance to thrombosis associated with inflammatory conditions, in which both heparin and elastase act catalytically to direct the inactivation of antithrombin. Since anticoagulantly-active heparin species are present on endothelial cells, the above mechanism could markedly affect the balance between procoagulant and anticoagulant processes on the usually non-thrombogenic endothelial surface.
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8

Asakura, S., N. Yoshida, and M. Matsuda. "MONOCLONAL ANTIBODIES AGAINST THROHBIN-ANTITHROMBIN III COMPLEX: EPITOPE SPECIFICITY AND EFFECT ON THROMBIN-ANTITHROMBIN III INTERACTION." In XIth International Congress on Thrombosis and Haemostasis. Schattauer GmbH, 1987. http://dx.doi.org/10.1055/s-0038-1643673.

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Among monoclonal antibodies (MCA´s) raised against human thrombin (T)-antithrombin m (AT) complex (TAT), two MCA´s designated as JITAT-16 and 17 with high affinity, Kd = 4.6nMand 4.1 nfi, respectively, were selected and characterized for specificity and functions. Their respective immunoglobulin subclasses are IgGi and IgG2a, and epitopes were found to be different from each Dther as shown by crisscross inhibition experiments. Immuno-alotting of normal plasma and serum electrophoresed on non-SDS aolyacrylamide gel showed that these antibodies reacted with normal serum but not with plasma. This was verified by an anzyme-linked differential antibody immunosorbent assay using aither one of the MCA´s as the first antibody and the other MCA labeled with peroxidase as the second one. By immunoblotting after SDS-PAGE, we found that both antibodies reacted with TAT, nut not with its respective nascent constituent, AT or T. However, they reacted with reactive site-cleaved AT (or thrombin-nodified AT, ATM) and also a complex of AT with activated factor K (Xa-AT). These results indicate that both of these antibodies recognize enzyme-treated forms of AT, including AT molecules :omplexed with enzymes reversibly or irreversibly as well as ATM. Jpon incubation of T with AT in the presence of JITAT-16, T activity remained nearly unchanged and formation of irreversible rAT did not proceed as expected. Moreover, AT was preferentially :onverted to ATM. When JITAT-16 was added after completion of FAT formation, however, neither recovery of T activity nor generation of ATM was observed. These findings were not obtained vhen JITAT-17 had been substituted for JITAT-16. These data suggest that JITAT-16 may have converted AT from an inhibitor to a substrate for T after having recognized a possible intermediate reversible complex of AT with T. Undoubtedly, in the presence of a polyclonal antibody against AT, neither TAT formation nor ATM neneration was observed at all. The mechanism of the unique Function of JITAT-16 has not been fully clarified as yet, but this antibody seems to give us new information on the kinetic study of TAT formation and ATM generation when AT was allowed to react with enzymes.
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Mointire, V. L., A. J. Frangos, G. B. Rhee, G. S. Eskin, and R. E. Hall. "RHEOLOGY AND CELL ACTIVATION." In XIth International Congress on Thrombosis and Haemostasis. Schattauer GmbH, 1987. http://dx.doi.org/10.1055/s-0038-1643988.

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The subject of this work is to examine the hypothesis that some sublytic levels of mechanical perturbation of cells can stimulate cell metabolism. As a marker metabolite, we have chosen arachidonic acid. Principal metabolites for platelets include the cyclooxygenase product thromboxane A2(TXA2) and the lipoxygenase product 12-hydroperoxy-eicosatetraenoic acid (12-HPETE). Polymorphonuclear leukocytes (PMNLs) initally produce principally 5-HPETE, somtimes leading to the formation leukotrienes, though many other metabolites of arachidonic acid have been isolated from activated neutrophils. Human umbilical vein endothelial cells utilize arachidonic acid to produce mainly prostaglandin I2(PGI2). All of these metabolites are biologically active and modulate cell function - sometimes in quite contrasting ways. We will show that levels of sublytic mechanical stress exposure can stimulate arachidonic acid metabolism in all three of the cell types mentioned above. The biological implications of this stress/metabolism coupling may be quite far reaching.Human platelets, leukocytes and endothelial cells all appear to be sensitive to mechanical stress induced activation of arachidonic acid metabolism. Sheared PRP exhibited greatly increased synthesis of 12-HETE and surprisingly little thromboxane B2 production. This indicates that shear stress stimulation of platelets may produce quite different arachidonic acid metabolism than that seen with many direct chemical stimuli, such as thrombin or collagen.Our data demonstrate that a substance derived from shear induced platelet activation may activate the C-5 lipoxygenase of human PMNL under stress, leading to the production of LTB4. We hypothesize that this substance maybe 12-HPETE. LTB4 is known to be a very potent chemotactic factor and to induce PMNL aggregation and degranulation. Our studies provide further evidence that lipoxygenase products of one cell type can modulate production of lipoxygenase products in a second cell type, and that shear stress can initiate cell activation. This kind of coupling could have far reaching implications in terms of our understanding of cell/cell interaction in flowing systems, such as acute inflammation, artificial organ implantation and tumor metastasis.The data on PGI2 production by endothelial cells demonstrate that physiological levels of shear stress can dramatically increase arachidonic acid metabolism. Step increases in shear stress lead to a burst in production of PGI2 which decayed to a steady state value in several minutes. This longer term stimulation of prostacyclin production rate increased linearly with shear stress over the range of 0-24 dynes/cm2. In addition, pulsatile flow of physiological frequency and amplitude caused approximately 2.4 times the PGI2 production rate as steady flow with the same mean stress. Although only PGI2 was measured, it is likely that other arachidonic acid metabolites of endothelial cells are also affected by shear stress.The ability of cells to respond to external stimuli involves the transduction of a signal across the plasma membrane. One such external stimulus appears to be fluid shear stress. Steady shear flow induces cell rotation in suspended cells, leading to a periodic membrane loading, with the peak stress proportional to the bulk shear stress. On anchorage-dependent cells, such as endothelial cells, steady shear stress may act by amplifying the natural thermal or Brownian fluttering or rippling of the membrane. There are several possible mechanisms by which shear stress induced membrane perturbation could mimic a hormone/receptor interaction, leading to increased intracellular metabolism. Shear stress may induce increased phospholipase C activity, caused by translocation of the enzyme, increased substrate (arachidonic acid) pool availability to phospholipase C (particularly from that stored in phosphoinositols) due to shear-induced membrane movements or changes in membrane fluidity, direct activation of calcium - activated phospholipase A2 by increased membrane calcium ion permeability, or most probably by a combination of these mechanisms.
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