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Chen, Min. "Mechanistic insights into ATP hydrolysis by the ABC transporter TAP." [S.l.] : [s.n.], 2004. http://deposit.ddb.de/cgi-bin/dokserv?idn=972577971.
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Schwarzl, Sonja M. "Understanding the ATP hydrolysis mechanism in myosin using computer simulation techniques." [S.l. : s.n.], 2005. http://nbn-resolving.de/urn:nbn:de:bsz:16-opus-63890.
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Heidelberg, Univ., Diss., 2005.Aus: S.M. Schwarzl, Understanding the ATP hydrolysis mechanism in myosin using computer simulation techniques, Mensch und Buch Verlag Berlin 2006, ISBN 3-86664-044-7.
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Shaw, Sudipta. "Role of ATP Hydrolysis and Mechanism of Substrate Reduction in Nitrogenase." DigitalCommons@USU, 2017. https://digitalcommons.usu.edu/etd/5729.
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Nitrogenase consists of two metalloproteins, the MoFe protein and the Fe protein. The MoFe protein is an α2β2 heterotetramer and the Fe protein is an α2 homodimer. The catalytic cycle of nitrogenase involves binding of the Fe protein to each αβ catalytic half of the MoFe protein, electron transfer followed by ATP hydrolysis, Pi release and eventually dissociation of the two proteins. This cycle has to be repeated eight consecutive times to reduce one molecule of N2.
The two catalytic halves of the MoFe protein had been considered to be independent of each other. The research presented here showed that there is negative cooperativity associated between the two catalytic halves of the MoFe protein. The results suggested that only one half of the MoFe protein is operative during the first turnover of the enzyme.
In order to understand the substrate reduction mechanism of nitrogenase, the study focused on two important enzymes of the biogeochemical nitrogen cycle: nitrite (NO2 -) and nitrate (NO3 -). Two intermediates of NO2 - reduction were trapped by a remodeled nitrogenase (α-70Ala/α-195Gln MoFe protein) and characterized by advanced spectroscopic studies. These intermediates were found to be identical to the intermediates trapped during reduction of diazene (N2H2) and hydrazine (N2H4). The pathway for reduction NO2 - to ammonia (NH3) was also proposed.
NO3 - was established as a new substrate of nitrogenase. The advanced spectroscopic studies confirmed that the same two intermediates were trapped by the remodeled nitrogenase. Kinetic studies showed that two competing pathways lead to NO3 - reduction by nitrogenase, a primary 2 e- reduction pathway to form nitrite and a secondary 8 e- reduction pathway to form NH3. The pathways for reduction of NO3 - to NO2 - and NH3 were proposed.
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Balasubramanian, Krithika. "ATP hydrolysis in Rho: Identifying active site residues and their roles." Diss., Temple University Libraries, 2010. http://cdm16002.contentdm.oclc.org/cdm/ref/collection/p245801coll10/id/80319.
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BiochemistryPh.D.
Escherichia coli transcription termination factor Rho is a hexameric RNA/DNA helicase that terminates transcription using energy derived from the hydrolysis of ATP. The ATP binding sites of Rho are located at the interfaces of adjoining subunit Cterminal domains and have the Walker A and B motifs, characteristic of many ATPases (Skordalakes & Berger, 2003; Richardson 2002). Available Rho crystal structures capture the protein with its active site in an open configuration that must close to permit ATP hydrolysis. Because of this, the identities of active site residues predicted to mediate ATP hydrolysis are uncertain. To determine which amino acids activate water, stabilize transition state, sense the γ- phosphoryl group, and coordinate the magnesium ion of MgATP, we have carried out site-specific mutagenesis on candidate residues which are conserved across bacterial species, and characterized the relevant properties of the mutant proteins. The residues chosen were E211 as the water activator, R212 as the γ sensor, R366 as the arginine finger, and D265 as the residue that coordinates Mg2+. Each mutant protein was investigated for its ability to oligomerize as hexamers, assayed for ATPase activity, ATP and RNA binding, and pre-steady-state kinetics. The results show that the mutant proteins form hexamers similarly as to wild type Rho. The RhoE211 mutants display at least a 200-fold lower activity as ATPases, bind both ATP and RNA with similar affinities as the wild type protein, and display no burst in pre-steady-state kinetics. RhoR212A protein has 20-fold lower activity as an ATPase compared to wild type Rho, binds ATP with at least a 50-fold weaker affinity, and RNA with a 2-fold higher KD compared to wild type Rho. RhoR366A functions as an ATPase with 50-fold lower activity, binds RNA with similar affinity as wild type Rho and binds ATP with a 5- fold weaker affinity. RhoD265N displays 150-fold lower ATPase activity compared to the wild type enzyme, binds ATP with a 10-fold weaker affinity, and binds RNA with similar affinity as wild type Rho. Pre-steady-state kinetics studies indicate that the mutant proteins investigated show no burst kinetics, indicating a failure or a significantly slower rate of the hydrolysis (chemistry) step. It is possible that the rate-limiting step is the chemistry step in these mutant proteins, contrary to the wild type protein where the chemistry step is much faster (300/s). Together, the results obtained are consistent with the proposed roles for these residues: E211 is involved in activating a water molecule, R212 functions as the γ sensor, R366 functions as the arginine finger and D265 is involved in coordination of the Mg2+ ion. This study has elucidated the mechanism of ATP hydrolysis, by determining some of the key residues involved in the hydrolysis reaction. This study is only a part of the characterization of the active site residues. There might be other residues involved in one or all of the functions proposed. Utilizing the findings from this study, other experiments and models can be implemented to understand how Rho hydrolyzes ATP and utilizes the energy to move along the RNA molecule and functions as a helicase.
Temple University--Theses
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Oliveira, Ana Sofia Fernandes. "Molecular modelling of ABC transporters: from ATP hydrolysis to substrate transport." Doctoral thesis, Universidade Nova de Lisboa. Instituto de Tecnologia Química e Biológica, 2010. http://hdl.handle.net/10362/5793.
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Dissertação para a obtenção de grau de doutor em Bioquímica
pelo Instituto de Tecnologia Química e Biológica
da Universidade Nova de LisboaDespite the great advances that have been made in the past decades in the ABC transporters field, the molecular mechanisms involved in transport across membranes remains largely an enigma. To date, questions regarding the molecular mechanism of transport, nucleotide hydrolysis and inorganic phosphate exit from the binding sites are still unanswered. In this thesis the dynamic behavior of several ABC transporters during the ATP-hydrolytic cycle is investigated using molecular modeling methods. The content of this thesis is compiled in three main scientific publications [1-3], corresponding to sections 3, 4 and 5, respectively. Although these three works are performed in prokaryotic family ABC transporters, it is likely that eukaryotic ones use similar mechanisms for nucleotide hydrolysis, inter-domain communication and allocrite translocation.(...)
Esta tese teve o apoio financeiro da FCT e do FSE no âmbito do Quadro Comunitário de Apoio, BD nº SFRH/BD/21433/2005
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Kimura, Yasuhisa. "Analysis of ATP hydrolysis activities of ABC transporters involved in multidrug resistance and K[ATP] channel regulation." Kyoto University, 2005. http://hdl.handle.net/2433/59289.
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Kyoto University (京都大学)0048
新制・課程博士
博士(農学)
甲第11833号
農博第1523号
新制||農||918(附属図書館)
学位論文||H17||N4082(農学部図書室)
UT51-2005-K499
京都大学大学院農学研究科応用生命科学専攻
(主査)教授 植田 和光, 教授 植田 充美, 教授 矢﨑 一史
学位規則第4条第1項該当
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Hunter, Andrew W. "Coupling of ATP hydrolysis to microtubule depolymerization by mitotic centromere-associated kinesin /." Thesis, Connect to this title online; UW restricted, 2002. http://hdl.handle.net/1773/10549.
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Liu, Fei. "ATP Utilization by the DEAD-Box Protein DED1P." Case Western Reserve University School of Graduate Studies / OhioLINK, 2010. http://rave.ohiolink.edu/etdc/view?acc_num=case1259924176.
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Fung, Emma. "Dissecting the roles of ParA ATP binding and hydrolysis in P1 plasmid partition." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 2000. http://www.collectionscanada.ca/obj/s4/f2/dsk1/tape2/PQDD_0023/MQ50429.pdf.
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Angove, Hayley Clare. "Energy transduction by nitrogenase involving ATP hydrolysis coupled to proton and electron transfers." Thesis, University of Sussex, 1995. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.282081.
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Simpson, Brent W. "Genetic investigation of how an ATP hydrolysis cycle is coupled to lipopolysaccharide transport." The Ohio State University, 2018. http://rave.ohiolink.edu/etdc/view?acc_num=osu1523988371297363.
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Mishra, Ajay. "ATP hydrolysis and stable hinge dimerization is essential for cohesin's stable association with chromosomes." Thesis, University of Oxford, 2009. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.510187.
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Graham, Shirley. "Molecular and genetic analysis of the vha16 gene in Drosophila melanogaster." Thesis, University of Glasgow, 2000. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.341986.
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Xu, Jin. "Conformational Studies of Myosin and Actin with Calibrated Resonance Energy Transfer." Thesis, University of North Texas, 2000. https://digital.library.unt.edu/ark:/67531/metadc2438/.
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Resonance energy transfer was employed to study the conformational changes of actomyosin during ATP hydrolysis. To calibrate the technique, the parameters for resonance energy transfer were defined. With conformational searching algorithms to predict probe orientation, the distances measured by resonance energy transfer are highly consistent with the atomic models, which verified the accuracy and feasibility of resonance energy transfer for structural studies of proteins and oligonucleotides.
To study intramyosin distances, resonance energy transfer probes were attached to skeletal myosin's nucleotide site, subfragment-2, and regulatory light chain to examine nucleotide analog-induced structural transitions. The distances between the three positions were measured in the presence of different nucleotide analogs. No distance change was considered to be statistically significant. The measured distance between the regulatory light chain and nucleotide site was consistent with either the atomic model of skeletal myosin subfragment-1 or an average of the three models claimed for different ATP hydrolysis states, which suggested that the neck region was flexible in solution. To examine the participation of actin in the powerstroke process, resonance energy transfer between different sites on actin and myosin was measured in the presence of nucleotide analogs. The efficiencies of energy transfer between myosin catalytic domain and actin were consistent with the actoS1 docking model. However, the neck region was much closer to the actin filament than predicted by static atomic models. The efficiency of energy transfer between Cys 374 and the regulatory light chain was much greater in the presence of ADP-AlF4, ADP-BeFx, and ADP-vanadate than in the presence of ADP or no nucleotide. These data detect profound differences in the conformations of the weakly and strongly attached crossbridges which appear to result from a conformational selection that occurs during the weak binding of the myosin head to actin.
The resonance energy transfer data exclude a number of versions of the swinging lever arm model, and indicate that actin participation is indispensable for conformational changes leading to force generation. The conformational selection during weak binding at the actomyosin interface may precock the myosin head for the ensuing powerstroke.
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Davey, Megan Jeannette. "The P1 plasmid partition protein ParA, roles for ATP binding and hydrolysis in plasmid partition." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 1997. http://www.collectionscanada.ca/obj/s4/f2/dsk2/tape16/PQDD_0006/NQ27904.pdf.
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Bhat, Anayat [Verfasser]. "Live Cell Fluorescence Imaging of Nucleotide Dynamics : ATP Hydrolysis and DNA Damage Response / Anayat Bhat." Konstanz : KOPS Universität Konstanz, 2021. http://d-nb.info/1229351094/34.
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Vineyard, Diana. "MECHANISTIC CHARACTERIZATION OF THE ATP HYDROLYSIS ACTIVITY OF ESCHERICHIA COLI LON PROTEASE USING KINETIC TECHNIQUES." Case Western Reserve University School of Graduate Studies / OhioLINK, 2007. http://rave.ohiolink.edu/etdc/view?acc_num=case1164049966.
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Mikita, Natalie. "DEVELOP SPECTROSCOPIC APPROACHES TO STUDY NON-PROTEOSOMAL ATP-DEPENDENT PROTEOLYSIS." Case Western Reserve University School of Graduate Studies / OhioLINK, 2014. http://rave.ohiolink.edu/etdc/view?acc_num=case1401814273.
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Niedermayer, Thomas. "On the depolymerization of actin filaments." Phd thesis, Universität Potsdam, 2012. http://opus.kobv.de/ubp/volltexte/2013/6360/.
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Actin is one of the most abundant and highly conserved proteins in eukaryotic cells. The globular protein assembles into long filaments, which form a variety of different networks within the cytoskeleton. The dynamic reorganization of these networks - which is pivotal for cell motility, cell adhesion, and cell division - is based on cycles of polymerization (assembly) and depolymerization (disassembly) of actin filaments. Actin binds ATP and within the filament, actin-bound ATP is hydrolyzed into ADP on a time scale of a few minutes. As ADP-actin dissociates faster from the filament ends than ATP-actin, the filament becomes less stable as it grows older. Recent single filament experiments, where abrupt dynamical changes during filament depolymerization have been observed, suggest the opposite behavior, however, namely that the actin filaments become increasingly stable with time. Several mechanisms for this stabilization have been proposed, ranging from structural transitions of the whole filament to surface attachment of the filament ends.
The key issue of this thesis is to elucidate the unexpected interruptions of depolymerization by a combination of experimental and theoretical studies. In new depolymerization experiments on single filaments, we confirm that filaments cease to shrink in an abrupt manner and determine the time from the initiation of depolymerization until the occurrence of the first interruption. This duration differs from filament to filament and represents a stochastic variable. We consider various hypothetical mechanisms that may cause the observed interruptions. These mechanisms cannot be distinguished directly, but they give rise to distinct distributions of the time until the first interruption, which we compute by modeling the underlying stochastic processes. A comparison with the measured distribution reveals that the sudden truncation of the shrinkage process neither arises from blocking of the ends nor from a collective transition of the whole filament. Instead, we predict a local transition process occurring at random sites within the filament.
The combination of additional experimental findings and our theoretical approach confirms the notion of a local transition mechanism and identifies the transition as the photo-induced formation of an actin dimer within the filaments. Unlabeled actin filaments do not exhibit pauses, which implies that, in vivo, older filaments become destabilized by ATP hydrolysis.
This destabilization can be identified with an acceleration of the depolymerization prior to the interruption. In the final part of this thesis, we theoretically analyze this acceleration to infer the mechanism of ATP hydrolysis. We show that the rate of ATP hydrolysis is constant within the filament, corresponding to a random as opposed to a vectorial hydrolysis mechanism.Aktin ist eines der am häufigsten vorkommenden und am stärksten konservierten Proteine in eukaryotischen Zellen. Dieses globuläre Protein bildet lange Filamente, die zu einer großen Vielfalt von Netzwerken innerhalb des Zellskeletts führen. Die dynamische Reorganisation dieser Netzwerke, die entscheidend für Zellbewegung, Zelladhäsion, und Zellteilung ist, basiert auf der Polymerisation (dem Aufbau) und der Depolymerisation (dem Abbau) von Aktinfilamenten. Aktin bindet ATP, welches innerhalb des Filaments auf einer Zeitskala von einigen Minuten in ADP hydrolysiert wird. Da ADP-Aktin schneller vom Filamentende dissoziiert als ATP-Aktin, sollte ein Filament mit der Zeit instabiler werden. Neuere Experimente, in denen abrupte dynamische Änderungen während der Filamentdepolymerisation beobachtet wurden, deuten jedoch auf ein gegenteiliges Verhalten hin: Die Aktinfilamente werden mit der Zeit zunehmend stabiler. Mehrere Mechanismen für diese Stabilisierung wurden bereits vorgeschlagen, von strukturellen Übergängen des gesamten Filaments bis zu Wechselwirkungen der Filamentenden mit dem experimentellen Aufbau. Das zentrale Thema der vorliegenden Dissertation ist die Aufklärung der unerwarteten Unterbrechungen der Depolymerisation. Dies geschieht durch eine Kombination von experimentellen und theoretischen Untersuchungen. Mit Hilfe neuer Depolymerisationexperimente mit einzelnen Filamenten bestätigen wir zunächst, dass die Filamente plötzlich aufhören zu schrumpfen und bestimmen die Zeit, die von der Einleitung der Depolymerisation bis zum Auftreten der ersten Unterbrechung vergeht. Diese Zeit unterscheidet sich von Filament zu Filament und stellt eine stochastische Größe dar. Wir untersuchen daraufhin verschiedene hypothetische Mechanismen, welche die beobachteten Unterbrechungen verursachen könnten. Die Mechanismen können experimentell nicht direkt unterschieden werden, haben jedoch verschiedene Verteilungen für die Zeit bis zur ersten Unterbrechung zur Folge. Wir berechnen die jeweiligen Verteilungen, indem wir die zugrundeliegenden stochastischen Prozesse modellieren. Ein Vergleich mit der gemessenen Verteilung zeigt, dass der plötzliche Abbruch des Depolymerisationsprozesses weder auf eine Blockade der Enden, noch auf einen kollektiven strukturellen Übergang des gesamten Filaments zurückzuführen ist. An Stelle dessen postulieren wir einen lokalen Übergangsprozess, der an zufälligen Stellen innerhalb des Filaments auftritt. Die Kombination von weiteren experimentellen Ergebnissen und unserem theoretischen Ansatz bestätigt die Vorstellung eines lokalen Übergangsmechanismus und identifiziert den Übergang als die photo-induzierte Bildung eines Aktindimers innerhalb des Filaments. Nicht fluoreszenzmarkierte Aktinfilamente zeigen keine Unterbrechungen, woraus folgt, dass ältere Filamente in vivo durch die ATP-Hydrolyse destabilisiert werden. Die Destabilisierung zeigt sich durch die Beschleunigung der Depolymerisation vor der Unterbrechung. Im letzten Teil der vorliegenden Arbeit untersuchen wir diese Beschleunigung mit theoretischen Methoden, um auf den Mechanismus der ATP-Hydrolyse zu schließen. Wir zeigen, dass die Hydrolyserate von ATP innerhalb des Filaments konstant ist, was dem sogenannten zufälligen Hydrolysemechanismus entspricht und im Gegensatz zum sogenannten vektoriellen Mechanismus steht.
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Ayed, Saud. "Insights into the Role of Conformational Change, Membrane Interactions and ATP Hydrolysis in the Min Protein Regulators of Bacterial Cell Division." Thesis, Université d'Ottawa / University of Ottawa, 2018. http://hdl.handle.net/10393/37981.
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Ayed, Saud. "Investigation of the Effect of Changes in Lipid Bilayer Properties on the Activity of the Bacterial Cell Division Regulator Protein MinD." Thèse, Université d'Ottawa / University of Ottawa, 2012. http://hdl.handle.net/10393/23258.
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Bacterial cell division requires formation of the cytokinetic cell division septum at the mid-cell position, a process that is determined by three Min proteins; MinC, MinD and MinE. Regulation of cell division by Min proteins occurs via a multi-step process involving interactions between various Min proteins, as well as the membrane. In this cycle, ATP-bound MinD binds to the membrane surface where it can recruit MinC to inhibit formation of the cell division septum. MinE binding to this complex displaces MinC and stimulates ATP hydrolysis, leading to the dissociation of MinD from the membrane. These interactions give rise to a dynamic pattern of Min protein localization that appears to involve a polymeric state that is designed to create a zone that is permissive to cell division at the mid-point of the cell. The interaction between MinD and the membrane is a critical aspect of this cycle, yet the role of the lipid bilayer in MinD activation, localization and polymerization is not well understood. To probe the role of membrane charge and fluidity on MinD activation and polymerization, we developed a kinetic assay of MinE-stimulated MinD ATPase activity. We found that membrane charge is essential for MinD activation and that differences in membrane fluidity give rise to changes in its activity. Moreover, a burst phase was also observed during the first few minutes of reaction, but only on the most fluid anionic lipid tested. To help determine if the observed membrane-dependent changes in MinD activity are linked to any changes in MinD polymer structure, we have begun to develop a method to identify surface exposed regions of MinD through a combination of covalent labeling and mass spectrometry. Optimization of various steps for the assay has been done, and the assay can be applied to the future characterization of MinD polymer structure. Results from this assay, in combination with those from the kinetic measurements described here, will help to improve understanding about how membrane properties modulate MinD ATPase activity, and how this can influence the Min protein oscillation that is required to ensure normal bacterial cell division.
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Burress, Helen. "Modulation of cholera toxin structure and function by host proteins." Doctoral diss., University of Central Florida, 2014. http://digital.library.ucf.edu/cdm/ref/collection/ETD/id/6251.
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Cholera toxin (CT) moves from the cell surface to the endoplasmic reticulum (ER) where the catalytic CTA1 subunit separates from the holotoxin and unfolds due to its intrinsic thermal instability. Unfolded CTA1 then moves through an ER translocon pore to reach its cytosolic target. Due to the instability of CTA1, it must be actively refolded in the cytosol to achieve the proper conformation for modification of its G protein target. The cytosolic heat shock protein Hsp90 is involved with the ER-to-cytosol translocation of CTA1, yet the mechanistic role of Hsp90 in CTA1 translocation remains unknown. Potential post-translocation roles for Hsp90 in modulating the activity of cytosolic CTA1 are also unknown. Here, we show by isotope-edited Fourier transform infrared (FTIR) spectroscopy that Hsp90 induces a gain-of-structure in disordered CTA1 at physiological temperature. Only the ATP-bound form of Hsp90 interacts with disordered CTA1, and its refolding of CTA1 is dependent upon ATP hydrolysis. In vitro reconstitution of the CTA1 translocation event likewise required ATP hydrolysis by Hsp90. Surface plasmon resonance (SPR) experiments found that Hsp90 does not release CTA1, even after ATP hydrolysis and the return of CTA1 to a folded conformation. The interaction with Hsp90 allowed disordered CTA1 to attain an active state and did not prevent further stimulation of toxin activity by ADP-ribosylation factor 6, a host cofactor for CTA1. This activity is consistent with its role as a chaperone that refolds endogenous cytosolic proteins as part of a foldosome complex consisting of Hsp90, Hop, Hsp40, p23, and Hsc70. A role for Hsc70 in CT intoxication has not yet been established. Here, biophysical, biochemical, and cell-based assays demonstrate Hsp90 and Hsc70 play overlapping roles in the processing of CTA1. Using SPR we determined that Hsp90 and Hsc70 could bind independently to CTA1 at distinct locations with high affinity, even in the absence of the Hop linker. Studies using isotope-edited FTIR spectroscopy found that, like Hsp90, Hsc70 induces a gain-of-structure in unfolded CTA1. The interaction between CTA1 and Hsc70 is essential for intoxication, as an RNAi-induced loss of the Hsc70 protein generates a toxin-resistant phenotype. Further analysis using isotope-edited FTIR spectroscopy demonstrated that the addition of both Hsc70 and Hsp90 to unfolded CTA1 produced a gain-of-structure above that of the individual chaperones. Our data suggest that CTA1 translocation involves a ratchet mechanism which couples the Hsp90-mediated refolding of CTA1 with extraction from the ER. The subsequent binding of Hsc70 further refolds CTA1 in a manner not previously observed in foldosome complex formation. The interaction of CTA1 with these chaperones is essential to intoxication and this work elucidates details of the intoxication process not previously known.Ph.D.
Doctorate
Molecular Biology and Microbiology
Medicine
Biomedical Sciences
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Helle, Steve. "Biosurfactants & cellulose hydrolysis." Thesis, McGill University, 1992. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=61308.
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The action of many antimicrobial agents is dependent on their ability to interact with biological membranes. A group of polypeptide antibiotics was found to have surface activite properties. One of them, gramicidinS, produced a minimum in the surface tension curve, which was attributed to instabilities in the intra-molecular hydrogen bonds. Biosurfactants were found to have a great effect on the two phase hydrolysis of cellulose by cellulase. Seven times as much sugar was produced by the hydrolysis of Sigmacell 100 when the biosurfactant sophorolipid was present. The surfactant affects the adsorption of cellulase onto cellulose, and prevents the cellulase from binding irreversibly to the cellulose and becoming inactive.
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Swatkoski, Stephen James. "Integration of ASP-specific microwave-accelerated acid hydrolysis into proteomic analyses." College Park, Md. : University of Maryland, 2007. http://hdl.handle.net/1903/7252.
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Thesis (Ph. D.)--University of Maryland, College Park, 2007.Thesis research directed by: Chemistry. Title from t.p. of PDF. Includes bibliographical references. Published by UMI Dissertation Services, Ann Arbor, Mich. Also available in paper.
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Moore, Andrew. "A unified ligand effect in Co(III) complexes : decarboxylation, phosphate diester hydrolysis and methyl acetate hydrolysis." Thesis, McGill University, 1990. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=39231.
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The effect of ligand structure on the reactivity of a diverse group of aqua-hydroxytetraazacobalt(III) complexes towards the hydrolysis of phosphate diesters and carboxylic esters has been investigated. It has been shown that the magnitude of the N-Co-N bond angle that lies opposite to the aqua-hydroxy ligands is influential in determining which complexes are best able to stabilize a strained four-membered ring, and this has been related to the relative rates at which these complexes promote reactions involving four-membered ring formation. The great range of rates of acid catalyzed decarboxylation of the carbonato derivatives of these complexes has been explained in terms of the same ligand-effect, and a correlation between the rate of this reaction and Co(III) aqua-hydroxy promoted phosphate diester hydrolysis and carboxylic ester hydrolysis has been established.The relationship between reactivity of a complex towards phosphate diester hydrolysis and mode of binding of inorganic phosphate has been elucidated.
The true catalysis of methyl acetate hydrolysis by the two complexes ((13aneN$ sb4)$Co(OH)(OH$ sb2)$) $ sp{2+}$ and ((cyclen)Co(OH)(OH$ sb2)$) $ sp{2+}$ has been demonstrated, it has been shown that complexes which are capable of this kind of catalysis can bind acetate as a chelate.
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Mrejen, Karen. "Copper complex catalyzed hydrolysis of amides." Thesis, McGill University, 1991. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=60516.
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Diaqua Cu(II) complexes are effective catalysts in promoting the hydrolysis of activated and unactivated amides.The complex (Cu(2,2$ sp prime$-dipyridylamine)(OH$ sb2$)$ sb2$) $ sp{+2}$ efficiently catalyzes the hydrolysis of the acyl-activated amides trifluoro-N-methyl-p-nitroacetanilide (MNTA), p-nitrotrifluoroacetanilide (NTA), and p-methoxytrifluoroacetanilide (MTA).
A cooperative effect between N-methylmorpholine buffer and (Cu(2,2$ sp prime$-dipyridylamine)(OH$ sb2$)$ sb2$) $ sp{+2}$ is observed in the hydrolysis of p-methoxytrifluoroacetanilide.
(Cu(2,2$ sp prime$-dipyridylamine)(OH$ sb2$)$ sb2$) $ sp{+2}$ hydrolyzes the unactivated amides with poor leaving groups formamide (FA) and N-methylformamide (MFA). In contrast, the monoaqua complex (Cu(2,2$ sp prime$:6$ sp prime$,2$ sp{ prime prime}$-terpyridine)(OH$ sb2$)) $ sp{+2}$ is not active. A detailed mechanism of the copper complex catalyzed hydrolysis reactions is proposed to explain the structural requirements of an amide-cleaving catalyst.
A copper complex is shown to be an effective metalloprotein model. A potential hapten capable of generating catalytic metalloantibodies with peptidase activity has been proposed. The role of the metal ion in carboxypeptidase A is compared to that of the metal ion in (Cu(2,2$ sp prime$-dipyridylamine)(OH$ sb2$)$ sb2$) $ sp{+2}$.
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Mahey, Rajesh. "Calcium transport and ATP hydrolytic activities in guinea-pig pancreatic acinar plasma membranes." Thesis, University of British Columbia, 1991. http://hdl.handle.net/2429/31044.
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The aim of the present investigation was to determine whether a plasma membrane high affinity Ca²+-ATPase plays an integral role in the maintenance of cytoplasmic free Ca²+ in pancreatic acinar cells. To achieve this, the Ca²+-transport and Ca²+-ATPase activities were characterized and their properties compared. Plasma membranes from guinea-pig pancreatic acini were shown to contain an ATP-dependent high affinity Ca²+-pump and a high affinity Ca²+-dependent ATPase activity. In addition, a low affinity ATPase activity was also observed. The high affinity Ca²+-ATPase activity as well as the Ca²+-transport were found to be dependent on Mg²+, whereas the low affinity ATPase activity appeared to be inhibited by Mg²+. The high affinity ATPase activity was 7-fold greater in magnitude than the Ca²+-transport. Whereas the Ca²+-transport was very specific for ATP as a substrate, the high affinity Ca²+-ATPase showed little specificity for various nucleotide triphosphates. These data would suggest that the Ca²+-transport and the high affinity Ca²+-dependent ATPase in guinea-pig pancreatic acinar plasma membranes may be two distinct activities
To further investigate whether the two activities were related, we investigated how the Ca²+-transport and Ca²+-ATPase activities were regulated by intracellular mediators. Regulation of the two activities by calmodulin, cyclic AMP-dependent protein kinase, Protein kinase C and inositol phosphates was investigated. Calmodulin failed to stimulate either activity. In addition, calmodulin antagonists, trifluoperazine and compound 48/80 produced a concentration-dependent inhibition of Ca²+-transport. These data suggested the presence of endogenous calmodulin. Both antagonists failed to influence the Ca²+-dependent ATPase activity. Experiments using boiled extracts from guinea-pig pancreatic acinar plasma membranes and erythrocyte plasma membranes Ca²+-ATPase confirmed the presence of endogenous calmodulin.
The catalytic subunit of cyclic AMP-dependent protein kinase stimulated Ca²+ transport, suggesting that cyclic AMP may have a role in the regulation of Ca²+-pump-mediated Ca²+ efflux from pancreatic acini. Ca²+-dependent ATPase activity, on the other hand, was not affected by the catalytic subunit. HA 1004, a specific inhibitor of cAMP-dependent protein kinase, failed to inhibit the Ca²+-transport and Ca²+-dependent ATPase activities. Since, this inhibitor was also ineffective at inhibiting the catalytic-subunit-stimulated Ca²+ transport, it may be concluded that HA 1004 is ineffective in blocking the actions of cAMP-dependent protein kinase in pancreatic acinar plasma membranes.
In our studies, purified protein kinase C, the phorbol ester TPA and the diacylglycerol derivative, SA-DG, failed to stimulate the Ca²+-uptake activity. However, these agents produced stimulation of the Ca²+-dependent ATPase activity in the presence of phosphatidylserine. CGP 41 251, a potent and selective inhibitor of protein kinase C, did not inhibit the Ca²+-transport or Ca²+-dependent ATPase activities. These observations suggest that protein kinase C may not be involved in the regulation of the plasma membrane Ca²+-pump in guinea-pig pancreatic acinar cells. These results also point to another difference between Ca²+-transport and the Ca²+-ATPase activities in guinea-pig pancreatic acinar plasma membranes.
Neither inositol trisphosphate nor inositol tetrakisphosphate produced a statistically significant effect on Ca²+-uptake, suggesting that IP₃- and/or IP₄-mediated Ca²+ releasing pathways may not operate in the isolated guinea-pig pancreatic acinar plasma membrane vesicles.
In summary, the results presented here provide evidence to suggest that the high affinity Ca²+-ATPase is not the biochemical expression of plasma membrane Ca²+-transport in panreatic acini. Our results imply a role for calmodulin and cAMP-dependent protein kinase, but not protein kinase C, in the regulation of Ca²+ efflux from pancreatic acinar cells.Medicine, Faculty of
Anesthesiology, Pharmacology and Therapeutics, Department of
Graduate
28
Zhou, Rong. "Topoisomerase II and drug resistance in leukemic cells /." Stockholm, 2001. http://diss.kib.ki.se/2001/91-628-4738-4/.
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Hagi-Pavli, Eleni. "Synthesis of transition state analogues designed to generate antibodies that catalyse the hydrolysis of cocaine." Thesis, Queen Mary, University of London, 1996. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.338895.
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Wahnon, Daphne C. "A dinuclear approach to phosphate diester hydrolysis." Thesis, McGill University, 1995. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=39888.
Full textAbstract:
The catalysis of the transesterification reaction of 2-hydroxypropyl p-nitrophenyl phosphate HPNP by a series of copper(II) complexes is investigated. Dichloro- ((bis(benzimidazol-2-ylmethyl)amine)copper(II)$ rm rbrack{ cdot}CH sb3OH (Cu(N3))$ and chloro- ((bis(benzimidazol-2ylmethyl)hydroxyethylamine)copper(II)$ rm rbrack chloride{ cdot}3(H sb2O)$ (Cu(N3OH)) are the most reactive mononuclear catalysts for promoting HPNP cleavage. A second order dependence on catalyst and a second order hydroxide dependence is observed for the transesterification reaction of HPNP promoted by Cu(N3). A mechanism is proposed in which a dimerized complex provides double Lewis acid activation followed by nucleophilic attack of the internal alkoxide of HPNP.A dinuclear copper(II) complex dichloro- ((N,N,N$ sp prime$,N$ sp prime$-tetrakis(2-benzimidazolyl)-2-hydroxy-$1, 3$-di-aminopropane)dicopper(II)) chloride LCu$ sb2$ is also shown to be extremely reactive in promoting the cleavage of HPNP. LCu$ sb2$ (2 mM) cleaves HPNP with an observed psuedo first order rate constant of $ rm 3.9 times 10 sp{-3} s sp{-1}$ at pH 7 and 25$ sp circ$C. The half-life for this reaction is 3 mins. LCu$ sb2$ is as effective as Cu(N3) and Cu(N3OH) for promoting the transesterification reaction of HPNP. A mechanism is proposed which involves double Lewis acid activation and facilitation of nucleophilic attack by the internal alkoxide of HPNP. A value for the rate enhancement expected by a double Lewis acid mechanism $6 times 10 sp6$ in the cleavage reaction of phosphate diesters is reported. The novel compound LCu$ sb2$DMP is synthesized and characterized. The structure of LCu$ sb2$DMP provides supporting evidence for the mechanism proposed.
Two novel and structurally interesting dinuclear cobalt(III) complexes, $ mu$-dimethylphosphato-di-$ mu$-hydroxy-bis ((1,4,7-triazacylononane)cobalt(III)) triperchlorate tacn$ sb2$Co$ sb2$(OH)$ sb2$DMP and $ mu$-(methyl-p-nitro-phenylphosphato)-di-$ mu $-hydroxy-bis ((1,4,7-triazacylononane)cobalt(III)) triperchlorate tacn$ sb2$Co$ sb2$(OH)$ sb2$MPNP are synthesized and characterized. The second order rate constant for the hydroxide caltalyzed hydrolysis of the doubly coordinated methyl (p-nitrophenyl) phosphate MPNP is $ rm 1.1 times 10 sp6 M sp{-1}s sp{-1}$ at 45$ sp circ$C. The proposed mechanism involves double Lewis activation of the phosphate diester and nucleophilic attack by a bridging oxy nucleophile. The breakdown of the species formed by this nucleophilic attack involves Co-O bond cleavage. Oxygen labeling experiments, pH-rate data and the identified reaction products are consistent with the proposed mechanism. A rate acceleration of $6 times 10 sp{11}$ fold is observed for the hydrolysis of the P-O bond in tacn$ sb2$Co$ sb2$OH$ sb2$MPNP over the background hydrolysis rate of MPNP.
31
Richter, Klaus. "Die ATP-Hydrolyse des molekularen Chaperons Hsp90 und ihre Regulation durch Co-Chaperone." [S.l. : s.n.], 2003. http://deposit.ddb.de/cgi-bin/dokserv?idn=970225741.
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32
Houde, R. L. "Canola phytate : enzymatic hydrolysis and nitrogen-phytate relationships." Thesis, McGill University, 1988. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=63918.
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33
Ona, Ikuba John. "Enzyme hydrolysis of cassava peels for ethanol production." Thesis, University of Strathclyde, 2013. http://oleg.lib.strath.ac.uk:80/R/?func=dbin-jump-full&object_id=20827.
Full textAbstract:
The enzyme hydrolysis of cassava peels for ethanol production provides an interesting research opportunity to convert starch rich lignocellulose waste into renewable fuel production. The research involved the pretreatment of cassava peels with steam explosion and hot water pretreatment processes as well as combining both amylolytic and cellulolytic enzymes to produce simple sugars. This research compared different enzyme treatment strategies; a separate hydrolysis that involved the treatment of the peels with either cellulolytic enzymes or amylolytic enzymes, a consecutive hydrolysis process which is a follow up of the separate hydrolysis in which sugars were washed from the initial enzyme treatment (amylase or cellulase treatment) and the cassava peels resuspended for further enzyme treatment was also investigated. Another treatment strategy employed in this study was the simultaneous hydrolysis by amylases and cellulases of the cassava peels. The hydrolysis rate and yield were c ompared for each process. Minor changes that incorporated steam explosion pretreatment and hot water pretreatment were also studied. A separate hydrolysis of milled cassava peels treated by amylolytic and cellulolytic enzymes yielded a maximum reducing sugar of 0.41g (as glucose) per gram of peels and 0.31g per gram of peels respectively. Also steam exploded cassava peels treated by amylolytic and cellulolytic enzymes yielded maximum reducing sugars of 0.24g per gram of peels and 0.37g per gram of peels respectively. Results also showed that a consecutive treatment that incorporates an initial hydrolysis by cellulolytic enzymes followed by a subsequent treatment by amylolytic treatment yielded reducing sugars of 0.64g per gram of milled cassava peels. A reverse treatment where the cellulolytic enzymes were used to first treat the peels before a second treatment by amylolytic enzymes yielded 0.61g reducing sugar per gram of milled cassava peels. A simultaneous hydrolysis by both cellulolytic and amylolytic enzymes produced a maximum reducing sugar of 0.58g per gram of milled cassava peels. A modification that incorporates hot water pretreatment, simultaneous and consecutive treatment was carried out. The milled cassava peels treated with hot water at 1000C and amylase enzymes for 2 hours were further subjected to a simultaneous saccharification by cellulases and glucoamylase enzymes yielded a reducing sugar of 0.62g per gram of peels. Fermentation experiments were also carried out with Kluyveromyces marxianus at 400C and results showed a maximum ethanol yield of 0.12g ethanol per g of cassava peels for a separate hydrolysis and fermentation process and 0.18g ethanol per g of cassava peels for the simultaneous saccharification and fermentation process. It was concluded that cassava peels presents a very good source of sugars for bioethanol production.
34
Banaszczyk, Mariusz G. "Artificial hydrolytic enzymes." Thesis, McGill University, 1989. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=74289.
Full textAbstract:
The efficiencies of many rigidly held cis-aquahydroxotetraazacobalt(III) complexes in promoting the hydrolysis of phosphate esters (BDNPP, BNPP, NPP) have been compared. The phosphate diester bond in ((trpn)Co(OH)(BNPP)) $ sp{+}$ is hydrolyzed at about the same rate as BNPP bound to a real enzyme from Enterobacter aerogenes and about 10$ sp{10}$ times more rapidly than free BNPP. The dramatic increase in the activity of the Co(III) complex with change in the tetraamine ligand structure can be explained in terms of a detailed mechanism of the reaction.Co(III) complexes, ((tren)Co(OH$ sb2)$(OH)) $ sp{2+}$ and ((trpn)Co(OH$ sb2)$(OH)) $ sp{2+}$ have been shown to be highly efficient in promoting the hydrolysis of phosphate di- and monoesters with poor leaving groups. ((trpn)Co(OH$ sb2)$(OH)) $ sp{2+}$ promoted hydrolysis of phosphate monoesters leads to a novel binuclear phosphato complex which is a model for the active site of Purple Acid Phosphatases.
For the first time it has been shown that hydrolysis of carboxylic esters catalyzed by ((trpn)Co(OH$ sb2)$(OH)) $ sp{2+}$ is truly catalytic. The hydrolytic activity of ((trpn)Co(OH$ sb2)$(OH)) $ sp{2+}$ has been linked to the fact that this complex chelates carboxylic acids as shown by $ sp{13}$C NMR spectroscopy and the crystal structure of ((trpn)Co($ eta sp2$-OCC(CH$ sb3) sb3) rbrack sp{2+}$. This complex is the first example of a cobalt(III) complex in which carboxylic acid is chelated.
35
Burton, Simon Alexander Quentric. "Ureolytic nitrification at low pH." Thesis, University of Aberdeen, 1993. http://digitool.abdn.ac.uk/R?func=search-advanced-go&find_code1=WSN&request1=AAIU052828.
Full textAbstract:
Laboratory studies of ureolytic nitrification were carried out to determine whether the ability of ammonia oxidisers to hydrolyse urea could explain their persistence and activity in acid soils. Ammonia oxidising bacteria were isolated from a number of acid soils, using previously described and novel techniques, and isolates tested for their ability to hydrolyse urea. None of the 17 isolated strains were found to be ureolytic, nor were they active below pH 7, indicating the persistence of neutrophilic ammonia oxidisers in acidic soils. The failure to isolate ureolytic and acidophilic strains suggested either their absence in these soils or inadequacies with the isolation procedure. Ten strains of ammonia oxidisers, previously isolated by other workers, were also tested for ureolytic activity and two were found to be ureolytic, Nitrosospira sp. (NPAV) and Nitrosospira sp. The growth of Nitrosospira sp. (NPAV) in liquid batch culture was studied in buffered and unbuffered media revealing that, in the presence of urea, growth and activity could be maintained in media with a pH value of 4-7 whereas growth on ammonium sulphate only occurred at or above pH 7. This suggested that ureolytic strains were capable of growth and activity in acidic conditions if urea was present, providing an explanation for the nitrification in acid soils. The oxidation of urea to nitrite by cultures was incomplete and ammonium accumulated. Growth appeared to inhibited at pH 8 in some media suggesting inhibition of growth by urea in these conditions. The growth and activity of Nitrosospira sp. (NPAV) was studied in continuous flow columns at low pH. Activity could be initiated in continuous flow columns by medium containing urea at pH 4 whereas ammonia was only oxidised at or above pH 6 when medium containing ammonium sulphate was supplied. When effluent nitrite production was constant and a steady state had been established, urea was completely hydrolysed by Nitrosospira sp., causing an increase in the pH, indicating the formation of NH3.
36
Sallis, P. J. "The characterization of hydrolytic enzyme activities in a freshwater sediment." Thesis, University of Kent, 1985. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.333017.
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37
Nayeri, Gita. "Enzymatic hydrolysis of shrimp for recovery of taste active compounds." Thesis, McGill University, 1992. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=61080.
Full textAbstract:
The muscle of shellfish, in particular shrimp, is characterized by high amounts of free amino acids especially glycine as well as proline, serine and alanine which all contribute to the overall pleasant and desirable flavour perceived. The two major proteolytic enzymes, chymotrypsin and trypsin, were used at different ratios (E/S, 0.0-0.3%), temperatures (25$ sp circ$C-45$ sp circ$C) and time of hydrolysis (1-3h) for frozen and fresh shrimp. These conditions were optimized in order to generate a product with desirable sensory and chemical characteristics. The thermal stability of chymotrypsin and trypsin were investigated, to determine the suitability of heat to stop the hydrolysis reactions at desired conditions. The results for the frozen shrimp showed that chymotrypsin was found to be inactivated after incubation for 1 min at 80$ sp circ$C while trypsin was found to be relatively heat stable. The commercial soybean trypsin inhibitor was used to inactivate trypsin. The use of both enzymes accelerate the rate of hydrolysis to some extent. Higher free amino acids yield for glycine, serine, and proline were obtained for chymotrypsin-treated hydrolysates. (Abstract shortened by UMI.)
38
Bielmann, Regula. "Specific receptor recognition and cell wall hydrolysis by bacteriophage structural proteins /." Zürich : ETH, 2009. http://e-collection.ethbib.ethz.ch/show?type=diss&nr=18255.
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Dueck, Corinne L. "Hydrodynamics of a fluidized bed reactor for urea hydrolysis by microencapsulated urease." Thesis, McGill University, 1985. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=65922.
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40
Williams, Daniel M. "Phosphate ester hydrolysis promoted by lanthanide (III) and transition metal complexes." Thesis, McGill University, 1999. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=35551.
Full textAbstract:
Phosphate ester linkages are omnipresent in nature and are found in many biologically important molecules such as DNA, RNA and ATP. Many enzymes that hydrolyze phosphate esters are activated by metal ions. Since the structure of biological enzymes can be quite complicated, it is often useful to work with simpler artificial metalloenzymes to help elucidate the mechanisms by which phosphate ester hydrolysis is activated by metals.In principle, simple dinuclear metal complexes could hydrolyze phosphate esters by double Lewis acid activation or by a combination of single Lewis acid activation and metal-hydroxide activation. These two mechanisms are kinetically indistinguishable. In this study, two different phosphate substrates are used to distinguish these mechanisms.
Lanthanide (iii) salts have proven extraordinarily effective in accelerating the rate of phosphate ester hydrolysis by several orders of magnitude. Consequently, there has been much effort in recent years to develop a ligand that would bind lanthanides so as to further improve their ability to hydrolyze phosphates. These efforts have met with limited success. Reported here is a dinuclear lanthanide (iii) complex which hydrolyzes BNPP (bis p-nitrophenyl phosphate) with unprecedented reactivity at pH 7.0 and 25°C.
A mononuclear copper (ii) 6,6'-diamino-2,2 '-bipyridyl complex is synthesized which gives one of the fastest rate accelerations reported for hydrolyzing 2',3 '-cAMP. A hydrogen bonding mechanism is proposed for the acceleration of the rate of hydrolysis of the cyclic phosphate.
41
Rusendi, Dadi. "Enzymatic hydrolysis of potato processing waste for the production of biopolymers." Thesis, McGill University, 1994. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=55528.
Full textAbstract:
Biopolymers are polymers produced by certain microorganisms, that are readily degradable in the environment. These biodegradable plastics have the potential to be used as substitutes for conventional petroleum based plastic provided that the production costs can be greatly reduced. The high cost of biopolymer production is due to the cost of substrate which mainly is glucose.The enzymatic hydrolysis of potato processing wastes was to produce glucose as a least expensive feedstock substrate for the production of biopolymers of polyhydroxybutirate (PHB) from the bacterium Alcaligenes eutrophus was studied. The enzymatic hydrolysis experiments were carried out using $ alpha$-amylase liquefaction enzymes from Aspergillus oryzae and barley-malt, and amyloglucosidase saccharification enzyme from Rhizopus.
The results indicated that the production of glucose from potato starch waste to be used as a substrate to produce biopolymers was both technically and economically feasible. A 10 to 90 ratio of barley-malt to potato starch waste gave the highest conversion of starch to glucose of 194.30 gL$ sp{-1}$ (96.56%), and the lowest liquefaction enzyme cost ($0.054) to hydrolyze one kg of potato starch waste. { it A. eutrophus /} produced PHB of 5.0 gL$ sp-1$ (76.9 % of biomass) using the glucose substrate generated from the potato starch waste.
42
Ma, Yating. "Deactivation of soybean agglutinin by enzyme hydrolysis and identification of active peptides from soy proteins." [Ames, Iowa : Iowa State University], 2010. http://gateway.proquest.com/openurl?url_ver=Z39.88-2004&rft_val_fmt=info:ofi/fmt:kev:mtx:dissertation&res_dat=xri:pqdiss&rft_dat=xri:pqdiss:1476322.
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Jeyaragavan, Tharmalingam. "Effect of genetic variants on hydrolysis of -casein by chymosin and pepsin." Thesis, McGill University, 2001. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=31245.
Full textAbstract:
Several studies have demonstrated that certain genetic variants of beta-casein are closely related to milk production, milk composition and technological properties of milk such as coagulation properties during cheese making, calcium precipitation and water binding properties. The objective of current study is to investigate the effect of the genetic variants on hydrolysis of beta-casein by chymosin and pepsin. On the basis of a preliminary analysis, a total of 50 milk samples which provided representatives of different available genetic variants of beta-casein were collected from different herds in Quebec. Casein was prepared from milk samples by the acid precipitation and the genetic variants of beta-casein were identified by both alkaline and acid urea-PAGE. An anion exchange chromatography was employed for the separation of beta-casein from whole casein. An initial hydrolysis of beta-casein of different phenotypes by chymosin and pepsin were achieved under the optimized hydrolytic conditions. Hydrolysates were periodically removed from the reaction mixture and they were analyzed by both RP-HPLC and SDS-PAGE in order to study the hydrolytic pattern of each beta-casein variant with the increasing hydrolytic time. (Abstract shortened by UMI.)
44
Challinor, John M. "Thermally assisted hydrolysis and derivatisation techniques for the characterisation of organic materials." Curtin University of Technology, School of Applied Chemistry, 1998. http://espace.library.curtin.edu.au:80/R/?func=dbin-jump-full&object_id=10601.
Full textAbstract:
This thesis describes the development of a novel method for the rapid identification of complex organic materials, including macromolecules, that involves a high temperature simultaneous hydrolysis and derivatisation reaction. In this procedure, aqueous quaternary alkylammonium hydroxides are made to react with a wide range of complex molecular species, including synthetic and natural polymers, under high temperature flash heating conditions. The hydrolysis products are converted to derivatives, such as alkyl esters or alkyl ethers. The reaction forms the basis for a modified pyrolysis gas chromatography (Py-GC) identification technique. Although the process is primarily intended for the rapid identification of polymers which are susceptible to hydrolysis, it is also valuable for characterisation of a variety of hydrolysable lower molecular weight species, such as polymer additives, triglycerides and natural waxes.The reaction takes place when an intimate mixture of an aqueous quaternary alkylammonium hydroxide solution is flash heated with the analyte in a conventional pyrolysis unit, and "on-line" GC-MS is used to separate and identify the reaction products. Analytes included synthetic polyester resins and phenolic polymers, natural products such as lipids and wood extractives, and natural polymers including lignocellulose, proteins and kerogen.Reaction variables, such as temperature, pH analyte particle size, substrate, and the derivatising reagent were studied, in order to find the optimum conditions for the reaction. While the reaction occurs at temperatures as low as 358 degrees celsius, a 770 degrees celsius reaction temperature was adopted to allow direct comparison with Py-GC data. A high pH of the derivatising reagent was found to be necessary to achieve an efficient hydrolysis of the macromolecule. Small particle size gives better conversion to derivatised ++products. The nature of the heating substrate did not appear to influence the reaction. Tetraalkylammonium hydroxides (TAAH) were found to be the most effective derivatising reagents for the reaction. Tetramethylammonium hydroxide (TMAH) was the most useful derivatising reagent, since the methyl derivatives of the hydrolysed products were conveniently chromatographed and usually had well known mass spectra. Other TA-AHs were useful for (i) producing higher molecular weight alkyl derivatives of low molecular weight side chains in some polymers, e.g., acetate groups in polyvinyl acetate, (ii) the purpose of determining sites of pre-existing methylation in natural products such as lignocellulose, or (iii) cases where methylation products could be confused with existing pyrolysis products.The reaction mechanism is believed to involve hydrolysis of the organic material, formation of the tetra-alkylammonium salt, and thermal degradation of the quaternary ammonium salt to alkylated derivatives. Some evidence is presented to support this mechanism, which is considered to be ionic in character, rather than a free radical reaction.A detailed study of the reaction of alkyd resins indicated that polyhydric alcohols, polybasic acids, degree of cure, oil length, and rosin acid and epoxy modification could be determined. The reaction of rosin modified phenolic resins (tert-butyl phenol formaldehyde and para-nonyl phenol formaldehyde), gave rosin acid methyl esters and easily identifiable products from the synthetic components.Fatty acid methyl esters could be obtained directly from lipids, such as vegetable oils, without time consuming preparative steps. The problems of base catalysed isomerisation of the double bonds in polyunsaturated fatty acids were overcome by reducing the amount of base used for the reaction. The reaction facilitated the identification of fatty acids in ++
woolwax, the triglycerides in cosmetic products, and lipids in trace quantities of human fingerprint deposits.A more reliable representation of the chemical structure of lignocellulose in softwood and hardwood species was obtained by the reaction, as compared to conventional PyGC which underestimates the aromatic carboxylic acid moieties. Gymnosperm or angiosperm origin was indicated by the presence of solely guaiacyl, or both guaiacyl and syringyl derived groups, respectively. Other extraneous extractable material was identified simultaneously, including aliphatic and aromatic acids, which would not normally be detected by conventional Py-GC.An alternative method involved extracting the wood with TMAH, followed by pyrolysis of the extract, to give less complex but more specific GC profiles. The TMAH extraction procedure also indicated some characteristic biomarker species as well as guaiacyl and syringyl derived compounds. The pyrolysis of tetraethylammonium hydroxide (TEAH) extracts revealed the sites of pre-existing methylation in the Eucalyptus marginata species.The thermally assisted hydrolysis and alkylation method which has been developed is usually superior to the conventional Py-GC procedure for those polymers which are prone to hydrolysis, since it results in products which are more readily related to the polymer structure. For example, concerted hydrolysis and alkylation of polyester resins results in alkyl carboxylate esters and the alkyl ethers, whereas in conventional Py-GC the products are alkenes and carboxylic acids. Carboxylic acids are more difficult to chromatograph by GC, and aromatic carboxylic acids in particular are susceptible to decarboxylation under the pyrolysis conditions.The reaction procedure has provided an alternative approach to the characterisation of submicrogram quantities of a range of synthetic polymers, natural products and ++
natural polymers, which has not previously been possible without lengthy chemical degradation procedures. Although it has not displaced the conventional Py-GC technique, it has given a new dimension to the characterisation of organic materials, providing a powerful tool for forensic science investigations and the analysis of complex materials.
45
Awafo, Victor Ankang. "Biosynthesis of cellulase-system from Trichoderma reseei [i.e. reesei] characteristics." Thesis, McGill University, 1997. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=41972.
Full textAbstract:
There are generally four factors recognized as delimiting in the study of lignocelluloses for fuel ethanol production, viz., the source of the cellulase-system and its quality characteristics for cellulose hydrolysis, the substrate and pretreatment method, the process for cellulase production and bioreactor design, and the ability of yeast to ferment mixed hexose and pentose sugars. Wheat straw (WS) and T. reesei mutants were used in the study to evaluate the production of cellulase-systems. Hydrolysis of cellulose revealed the superiority of mild NaOH pretreatment over steam explosion for cellulase production with T. reesei MCG 80 and QMY-1. Response surface models were capable of predicting that NaOH could be used for the pretreatment of WS at 4% (w/w) without urea in the fermentation medium to yield optimum filter paper activity (FPA) of 9.9 IU/mL (247 IU/g WS) and beta-glucosidase activity ($ beta$GA) of 6.4 IU/mL (159 IU/g WS) under solid-state fermentation (SSF) conditions. Multiple regression analysis with multiple coefficients of correlation, R, between 0.957 and 0.99 from the experimental data showed close agreement between the cellulase activities (FPA and $ beta$GA) from the experiments and predicted values.The superiority of SSF over liquid-state fermentation (LSF) in the production of cellulase-systems was also established, and a prototype pan-bioreactor showed good potential for upgrading cellulase production under SSF conditions. The economics of fuel ethanol production was considered in the optimization model that sought to establish threshold cellulase loadings needed to achieve maximum cellulose hydrolysis for fermentation. High substrate concentrations of up to 7.5% were hydrolyzed with cellulase loadings of 24-30 IU/g and fermented by Pichia stipitis to achieve 90-100% conversion into ethanol.
Crude unextracted cellulase yielded over 90% hydrolysis of delignified wheat straw and proved to be better than extracted cellulase and commercial cellulases for the hydrolysis of pure cellulose and pretreated wheat straw. Studies were also conducted to demonstrate the importance of the ratio of $ beta$GA- to FPA in cellulose hydrolysis which showed that ratios closer to one (1), produced more sugars and lowered the cellobiose content in the hydrolysates. It was also shown that the source of the cellulase is important in eliminating the accumulation of cellobiose during hydrolysis as was demonstrated with cellulase from mixed cultures of T. reesei and Aspergillus phoenicis. Higher $ beta$GA from the latter were implicated since A. phoenicis is a good $ beta$-glucosidase producer.
Delignified wheat straw at 5% concentration when subjected to separate hydrolysis and fermentation and simultaneous hydrolysis and fermentation resulted in similar volumetric productivities (g/L/h) of ethanol.
46
Bayingana, Claude. "The prevalence of members of the "red complex" in pregnant women as revealed by PCR and BANA hydrolysis." Thesis, University of the Western Cape, 2005. http://etd.uwc.ac.za/index.php?module=etd&.
Full textAbstract:
Increased levels of oestrogen and progesterone during pregnancy may lead to periodontal disease. The anaerobic Gram-negative bacteria called red complex (Porphyromonas gingivalis, Tannerella forsythensis and Treponema denticola) are frequently associated with periodontal disease. Periodontopathogens produce toxins and enzymes which can enter the bloodstream and cross the placenta to harm the foetus. The response of the mother&rsquos immune system to infection by these periodontopathogens, brings about the release of inflammatory mediators which may trigger preterm labour or result in low birth-weight infants. The purpose of this study was to examine the prevalence of red complex, using BANA and PCR in subginginval plaque samples from pregnant women. Subgingival plaque samples were obtained from pregnant women between the ages of 17 to 45 years attending a Mitchells Plain ante-natal clinic. Plaque samples were analyzed by the enzymatic BANA-test for detection of the presence of red complex and DNA was extracted and analyzed using 16 rDNA-Polymerase Chain Reaction (PCR).
Seventy-nine percent of pregnant women showed gingival index scores of &ge
1 of which 74.24% harboured by at least one of the members of the red complex. P.gingivalis was the most prevalent of the three members of the red complex. Findings of this study confirmed a need for dental preventive measures in pregnant women and microbial monitoring of suspected periodontopathogenes. This could be achieved by joint cooperation between Maternity Obstetric Units (MOU), Dentistry and oral microbiology departments. The results of this study revealed that although PCR is more sensitive than BANA in detecting members of the red complex, BANA showed a better association with the indices used to diagnose periodontal disease.
47
Kim, Jung-Hee. "Developing artificial proteases and nucleases : catalytic hydrolysis of unactivated amides, nitriles and phosphates." Thesis, McGill University, 1992. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=39317.
Full textAbstract:
Cis-diaqua cobalt complex ((L)Co(III)(OH$ sb2) sb2 rbrack sp{3+}$ promoted hydrolyses of phosphate esters, nitriles and carboxy amides are examined (L represents 1,4,7,10-tetraazacyclododecane (cyclen) and its N-methylated derivatives). ((Cyclen)Co(OH$ sb2) sb2 rbrack sp{3+}$ is more active than any other catalysts reported to date for hydrolyzing dimethyl phosphate, acetonitrile and formyl morpholine.((Cyclen)Co(OH$ sb2) sb2 rbrack sp{3+}$ efficiently hydrolyzes dimethyl phosphate under mild conditions (k = 3.7 $ times$ 10$ sp{-5}$ M$ sp{-1}$ sec$ sp{-1}$ at pD 6.3, 60$ sp circ$C). This represents the first hydrolysis of dimethyl phosphate (P-O bond cleavage) at neutral pH. Mechanism for the cobalt complex promoted hydrolysis of dimethyl phosphate and its implication on the role of metal ions in ribozymes is discussed.
((Cyclen)Co(OH$ sb2) sb2 rbrack sp{3+}$ catalyzes the hydration of nitriles to amides. Acetonitrile coordinated to the cobalt complex is hydrated 10$ sp9$ times more rapidly than the uncoordinated acetonitrile at pH 7 and 40$ sp circ$C. Catalytic turnover for the hydration reaction is demonstrated for the first time with the Co(III) complex. Chelated benzamide, a key intermediate in the catalytic hydration of benzonitrile, is isolated and its crystal structure determined. Detailed kinetics and mechanistic analyses of the cobalt complex catalyzed hydration of acetonitrile including the equilibrium constant for complexation of acetonitrile to the cobalt complex (K = 0.6 M$ sp{-1})$ are reported. Synthetic utility of the catalyst including acrylamide production is discussed.
((Cyclen)Co(OH$ sb2) sb2 rbrack sp{3+}$ efficiently hydrolyzes formyl morpholine under mild conditions (k = 7.97 $ times$ 10$ sp{-5}$ M$ sp{-1}$ sec$ sp{-1}$ at pD 6, 60$ sp circ$C). The equilibrium constant for complexation of formyl morpholine to the cobalt complex is 0.4 M$ sp{-1}.$ The equilibrium constant for complexation of amides to metal complexes had not been previously measured. The efficiency and mechanism of the cobalt complex for hydrolyzing amides are compared to those of carboxypeptidase A.
48
Mirshokraie, S. Ahmad (Seyed Ahmad). "Reactions of a-substituted non-phenolic lignin model compounds under alkaline hydrolysis conditions." Thesis, McGill University, 1988. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=75783.
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Side-chain ether groups in lignin model compounds of the general structure 1-(3$ sp prime$,4$ sp prime$-dimethoxyphenyl)-1,2-diaryloxyethane exhibited hydrolytic cleavage (the extent depending on the substituents on the 1-aryloxy groups) in aqueous 2N NaOH at 150$ sp circ$C (i.e. under wood pulping conditions). The level of hydrolysis increased in phenylpropane models (i.e. with $ gamma$-CH$ sb2$OH). Thus the commonly accepted generalization that 1,2-diethers of 3$ sp prime$,4$ sp prime$-dimethyoxyphenylpropane are stable in hot alkali is valid only for 1-alkoxy compounds. The newly observed hydrolytic cleavage is influenced by a combination of steric effects, electronic effects and solubility parameters. These observations now make it possible to consider alkaline delignification in the light of gel-degradation theory.A series of compounds of the general structure 1-(3$ sp prime$-4$ sp prime$,dimethoxyphenyl)-1-aryl-2-guaiacyloxyethane, where the 1-aryl group bore a hydroxy substituent, also exhibited alkaline hydrolysis, at 150$ sp circ$C, at the $ beta$-carbon. The extent was greater when the 1-aryl group bore an $o$-OH than when it bore a $p$-OH. Thus, the reaction was assisted by the nucleophilic attack of the $o$-phenoxide anion on the $ beta$-carbon.
A relatively high release of guaiacol occurred on treatment of 1-(3$ sp prime$,4$ sp prime$-dimethoxyphenyl)-1-thio-2-guaiacyloxyethane with 2N NaOH at 150$ sp circ$C, and the yield of guaiacol was increased when the terminal sidechain carbon bore a $-$CH$ sb2$OH group. Analogous $ alpha$-thioaryl and $ alpha$-thioalkyl compounds also exhibited greater ether cleavage than their oxy counterparts. The hydrolysis of the $ beta$-guaiacyl group was also enhanced by the presence of $ alpha$-seleno-containing groups.
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Piatkowski, Nicolas. "Zn-nanoparticle in-situ hydrolysis for hydrogen production in a high quench rate reactor." Zürich : Departement Maschinenbau und Verfahrenstechnik, Eidgenössische Technische Hochschule, 2007. http://e-collection.ethbib.ethz.ch/show?type=dipl&nr=312.
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Hediger, Thomas. "Die enzymatische Hydrolyse der Lactose mit Hohlfaserreaktoren /." [S.l.] : [s.n.], 1985. http://e-collection.ethbib.ethz.ch/show?type=diss&nr=7933.
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