Dissertations / Theses on the topic 'Enzymatic regulations'

ENZYMATIC REGULATION OF OPIOID ANTINOCICEPTION AND TOLERANCE By Lynn C. Hull, Ph.D. A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy at Virginia Commonwealth University. Virginia Commonwealth University, 2009 Director: William L. Dewey, Ph.D. Department of Pharmacology and Toxicology The involvement of kinases in opioid actions has long been established. The acute actions of opioids, through the Gi/Go G-proteins, cause the inhibition of adenylyl cyclase and therefore a decrease in protein kinase A (PKA) activation. Additionally, acute opioid administration may cause the G-protein to activate the phospholipase C (PLC)-mediated cascade leading to the activation of protein kinase C (PKC). The phosphorylation of the MOR which can lead to both desensitization by uncoupling of the G-protein coupled receptors (GPCRs) from the G-proteins and to internalization by recruitment of β-arrestins has long been identified as a key process in tolerance. Phosphorylation by PKA and PKC leads primarily to uncoupling of the receptor from the G-proteins. Phosphorylation of the receptor by G-protein coupled receptor kinase (GRK) leads to the recruitment of β-arrestins and internalization of the receptor. Many in vitro studies have come to the conclusion that GRK induced internalization plays a more central role in the tolerance to high efficacy opioids and a lesser role in low- and moderate-efficacy opioid tolerance. In fact it has been hypothesized that morphine, a moderate-efficacy opioid, causes no internalization at all, while the desensitization of the receptor via phosphorylation by PKA and PKC plays a more central role in low- and moderate-efficacy opioid tolerance. We sought to test these in vitro findings in an in vivo model of opioid tolerance. Animals were made tolerant to one of a number of opioids of varying efficacy (low-efficacy meperidine, moderate-efficacy morphine and fentanyl, and high-efficacy [D-Ala2, N-MePhe4, Gly-ol]-enkephalin (DAMGO)) over an 8 hour period and then were administered one of the kinases’ inhibitors. Tolerance reversal was determined by challenging these mice with the same opioids to which they were tolerant. Calcium is known to play an important role in the acute antinociceptive actions of opioids as well as in opioid tolerance. Therefore it is important to determine how opioids are affecting the regulation of intracellular calcium. Our laboratory has previously shown that Calcium Induced Calcium Release (CICR), the ryanodine receptor and intracellular microsomal Ca2+ pools all play a role in opioids’ actions. It is also well known that mammalian ADP-ribosyl cyclase, CD38’s, product cADPR acts on the ryanodine receptor to cause Ca2+ release into the intracellular space. We chemically and genetically altered CD38 and then tested the acute effect of morphine as well as what effect these treatments had on morphine tolerance to determine what role if any, that CD38 may play in the acute actions of morphine antinociception as well as in morphine tolerance. Together, studies focusing on the role of an ADP-ribosyl cyclase, CD38, and 3 separate kinases, PKA, PKC and GRK, in opioids’ actions were performed in order to better understand the roles of these enzymes’ pathways in the actions of opioid-induced antinociception and subsequent development of tolerance. It is hoped that the results herein add useful knowledge to the general understanding of this drug class, and will one day be of use in the development of future analgesics and in the clinical treatment of pain and reduction in tolerance.

Pulmonary surfactant is a critical surface-active substance consisting of dipalmitoylphosphatidylcholine (DPPtdCho) and key apoproteins that are produced and secreted into the airspace from alveolar type II epithelial cells. Deficiency of the surfactant leads to severe lung atelectasis, ventilatory impairment, and gas-exchange abnormalities. The generation of DPPtdCho in cells occurs via two integral routes: the de novo and remodeling pathways. The interplay between these pathways has not been investigated. Overexpression of the remodeling enzyme, acyl-CoA:lysophosphatidylcholine acyltransferase (LPCAT1), in epithelia decreases de novo PtdCho synthesis without significantly altering cellular phospholipid mass; this occurs through increased degradation of cholinephosphotransferase (CPT1), the terminal enzyme of the de novo pathway. CPT1 is degraded by multi-ubuiquitination and trafficking via the lysosomal pathway. When expressed in lung epithelia, CPT1 mutants harboring arginine substitutions at multiple carboxyl-terminal lysine residues exhibited proteolytic resistance to effects of LPCAT1 overexpression. Cellular expression of these CPT1 mutants also restores de novo PtdCho synthesis to levels normally observed in lung epithelia. Further studies demonstrate that the SCF (Skip-Cullen-F-box) ubiquitin E3 ligase component, β-TrCP, was sufficient to degrade CPT1. Similar to CPT1, LPCAT1 levels are also regulated at the level of protein stability. However, LPCAT1 is a polyubiquitinated enzyme processed within the proteasome. Similar to CPT1, β-TrCP is the putative E3 ubiquitin ligase subunit responsible for LPCAT1 ubiquitination. β-TrCP appears to dock and ubiquitinate LPCAT1 within its amino-terminus. Collectively, these observations indicate the presence of cross-talk between the phospholipid remodeling and de novo pathways; this involves tight regulation by site-specific ubiquitination of indispensable regulatory enzymes catalyzed by SCF ubiquitin E3 ligase members that mechanistically provide homeostatic control of cellular phospholipid content.

Pulmonary surfactant is a critical surface-active substance consisting of dipalmitoylphosphatidylcholine (DPPtdCho) and key apoproteins that are produced and secreted into the airspace from alveolar type II epithelial cells. Surfactant deficiency leads to severe lung atelectasis, ventilatory impairment, and gas-exchange abnormalities. These are features of the acute lung injury syndrome, characterized by a strong pro-inflammatory component where cytokines or bacteria infections greatly impair surfactant DPPtdCho biosynthesis. The key enzyme needed to produce surfactant DPPtdCho is a rate-limiting enzyme CTP: phosphocholine cytidylyltransferase (CCTalpha). Calmodulin (CaM), rather than disruption of an NH2-terminal PEST sequence, stabilizes CCTalpha from actions of the proteinase, calpain. Mapping and site-directed mutagenesis of CCTalpha uncovered a motif (LQERVDKVK) harboring a vital recognition site, Q243, whereby CaM directly binds to the enzyme. Mutagenesis of CCTalpha Q243 not only resulted in loss of CaM binding, but also led to complete calpain resistance in vitro and in vivo. These data suggest that CaM, by antagonizing calpain, serves as a novel binding partner for CCTalpha that stabilizes the enzyme under pro-inflammatory stress. We further show that CCTalpha does not undergo polyubiquitination and proteasomal degradation. Rather, the enzyme is monoubiquitinated at a molecular site (K57) juxtaposed near its NLS resulting in disruption of its interaction with importin, nuclear exclusion, and subsequent degradation within the lysosome. Importantly, by using CCTalpha-ubiquitin hybrid constructs that vary in the intermolecular distance between ubiquitin and the NLS, we show that CCTalpha monoubiquitination masks its NLS resulting in cytoplasmic retention. These results unravel a unique molecular mechanism whereby monoubiquitination governs the trafficking of a critical regulatory enzyme in vivo. Last, we identify FBXL2 as a novel F-box E3 ubiquitin ligase that targets CCTalpha for degradation. Interestingly, FBXL2 also interacts with CaM, and CaM directly disrupts CCTalpha and FBXL2 interaction. This study demonstrates in the first time that adenoviral gene transfer of CaM attenuates the deleterious effects of P. aeruginosa infection by improving several parameters of pulmonary mechanics in animal models of sepsis-induced acute pulmonary injury. Collectively, these studies reveal a novel regulatory mechanism for phosphatidylcholine synthesis that may provide important clues to understanding the pathobiology of acute lung injury.

Human pyruvate kinase performs the last step in glucose glycolysis in all cells and organisms and can be a key regulator of glycolytic flux. Pyruvate produced by PYK is transported into the mitochondria to fuel the TCA cycle, which enables the production of ATP; the main energy source of the cell. Human PYK contains four isoforms: M1 (found in muscle, heart and brain), M2 (in foetal cells and tumours), L (liver), and R (red blood cells) PYK. M2PYK plays a crucial role in tumour cell proliferation; by down-regulating metabolic flux, upstream metabolites can be used for protein and DNA synthesis. Reprogramming the metabolism of fast proliferating cells is called the 'Warburg effect'. The biological relevance of the different isoform activities is also discussed. For example RPYK in red blood cells is exposed to slowly altering metabolite concentrations, especially after intestinal absorption in plasma and RBCs uptake some of the metabolites. This thesis describes biochemical and biophysical studies of human M1PYK, M2PYK, LPYK, and RPYK. PYK is allosterically regulated by a range of metabolites. A comparative enzyme kinetics study of the four isoforms was performed to examine the mechanisms of activation and inhibition of these small molecule regulators, including all 20 amino acids and the thyroid hormone T3. The redox state of the environment was also found to be an important regulator of PYK activity. All four PYK isoforms were successfully expressed and purified. Interestingly, only M2PYK and RPYK were strongly regulated by amino acids and metabolites. We also found that the redox state regulates the activity of all four PYK isoforms as well as the sensitivity of M2PYK in response to natural regulators. These studies also confirmed the dissociation of tetrameric PYK into inactive monomers as an important mechanism of regulation, particularly for M2PYK activity. Nuclear magnetic resonance (NMR) and Small-angle X-ray scattering (SAXS) studies were performed to investigate the conformational behaviour of PYK isoforms in solution and to compare the effects of ligand binding. NMR data of all four isoforms reveal a conserved binding mechanism between isoforms and specific amino acids. SAXS data of all four isoforms demonstrate that ligands affect tetramerisation of PYK isoforms.

Doctor of Philosophy
Department of Anatomy and Physiology
Timothy I. Musch
Nitric oxide (NO) is synthesized via distinct NO synthase (NOS) enzymes and constitutes an essential cardiovascular signaling molecule. Whereas important vasomotor contributions of endothelial NOS (eNOS) have been well-described, the specific vasomotor contributions of nNOS-derived NO in healthy subjects during exercise are unknown. The purpose of this dissertation is to test the global hypothesis that nNOS-derived NO is a critical regulator of exercising skeletal muscle vascular control. Specifically, we utilized the selective nNOS inhibitor S-methyl-L-thiocitrulline (SMTC) to investigate the effects of nNOS-derived NO on skeletal muscle vascular function within established rodent models of exercise performance. The first investigation (Chapter 2) identifies that nNOS inhibition with SMTC increases mean arterial pressure (MAP) and reduces rat hindlimb skeletal muscle blood flow at rest whereas there are no effects during low-speed (20 m/min) treadmill running. In Chapter 3 it is reported that nNOS inhibition with SMTC reduces blood flow during high-speed treadmill running (>50 m/min) with the greatest relative effects found in highly glycolytic fast-twitch muscles and muscle parts. Chapter 4 demonstrates that nNOS-derived NO modulates contracting skeletal muscle blood flow (increases), O2 consumption (VO2, increases), and force production (decreases) in the rat spinotrapezius muscle and thus impacts the microvascular O2 delivery-VO2 ratio (which sets the microvascular partial pressure of O2, PO2mv, and represents the pressure head that drives capillary-myocyte O2 diffusion). In Chapter 5 we report that systemic administration of the selective nNOS inhibitor SMTC does not impact lumbar sympathetic nerve discharge. This reveals that the SMTC-induced peripheral vascular effects described herein reflect peripheral nNOS-derived NO signaling as opposed to centrally-derived regulation. In conclusion, nNOS-derived NO exerts exercise-intensity and muscle fiber-type selective peripheral vascular effects during whole-body locomotor exercise. In addition, nNOS-derived NO modulates skeletal muscle contractile and metabolic function and, therefore, impacts the skeletal muscle PO2mv. These data identify novel integrated roles for nNOS-derived NO within healthy skeletal muscle and have important implications for populations associated with reduced NO bioavailability and/or impaired nNOS structure and/or function specifically (e.g., muscular dystrophy, chronic heart failure, advanced age, etc.).

Enzyme-catalyzed reactions are important to regulate steroidogenesis and nuclear receptor activation. The present investigation examines the role of steroid metabolism catalyzed by CYP7B1 for regulation of hormone receptor activation and the effects of vitamin D on enzymatic regulation of steroidogenesis. The study reports data indicating that CYP7B1 can regulate estrogenic signaling by converting estrogens into inactive or less active metabolites. Similar results were obtained for CYP7B1-mediated metabolism of some androgen receptor ligands, indicating that CYP7B1 can be involved also in the regulation of androgenic signaling. CYP7B1 substrates and metabolites were found to exert androgenic effects in a cell line-specific manner. Furthermore, cell line differences were observed in the expression pattern for androgen receptor comodulators. This thesis reports that 1α,25-dihydroxyvitamin D3 alters the gene expression and enzyme activity of CYP21A2 and CYP17A1 leading to suppressed production of aldosterone, dehydroepiandrosterone and androstenedione in adrenocortical cells. These are novel findings on vitamin D action. A mechanism is reported for the vitamin D-mediated regulation of the CYP21A2 gene. Data indicate that vitamin D receptor interacting repressor (VDIR) and Williams syndrome transcription factor (WSTF) are key comodulators in this novel vitamin D receptor (VDR)-mediated mechanism. Furthermore, the results indicate that altered expression levels of VDIR and WSTF can shift the suppressing effect of vitamin D to a stimulatory effect. Also, epigenetic components were found to be involved in the effects of vitamin D on CYP21A2 transcriptional rate. In addition, a functional vitamin D response element was identified in the CYP21A2 promoter. This study also reports that 1α,25-dihydroxyvitamin D3 affects sex hormone production in a tissue-specific way. Gene expression and enzyme activity of aromatase were found to be downregulated in cells derived from breast, but not in cells derived from prostate and adrenal cortex. The production of estradiol and dihydrotestosterone was altered in a tissue-selective manner following vitamin D treatment. These findings are of importance for the discussion on vitamin D as a potential anti-breast cancer agent.

Dans ce travail, nous avons établi un modèle dynamique d’équations différentielles de la glycolyse de Bacillus subtilis, comprenant à la fois les régulations transcriptionnelles et enzymatiques. La validation complète d’un modèle aussi complexe n’est pas faisable à l’heure actuelle. De ce fait, nous avons développé une stratégie de validation, guidée par le modèle, pour diminuer le nombre d’expériences et pour se focaliser uniquement sur les points clés de la régulation. L’analyse du modèle à l’équilibre a donc fait ressortir : les propriétés structurelles fortes de la glycolyse, les enzymes clés impliqués et les régulations enzymatiques indispensables. Notre objectif était donc de valider les régulations enzymatiques prédites et de démontrer les propriétés structurelles du modèle d’un point de vue biologique en les perturbant. Premièrement, le modèle prédit un certain nombre de régulations enzymatiques critiques que nous avons vérifié expérimentalement. Notre approche, guidée par le modèle mathématique, nous a permis de faire des découvertes inattendues. Le modèle prédit que la glucose-6-phosphate déshydrogénase et la phosphofructokinase sont inactivées par le phosphoenolpyruvate (PEP). Nous avons purifié les enzymes de B. subtilis et nous avons pu démontrer pour les deux enzymes une inhibition non compétitive par le PEP. Le modèle prédit également que la pyruvate kinase est activée par le ribose-5-phosphate (R5P). De manière inattendue, les essais enzymatiques avec la pyruvate kinase purifiée de B. subtilis et taguée en N-terminus n’a montré aucune activation par le R5P ou par aucun des activateurs connus de la pyruvate kinase chez d’autres espèces. En revanche, la pyruvate kinase purifiée de B.subtilis, mais taguée en C-terminus est bien activée par le R5P, démontrant ainsi l’implication de la partie N-terminale de la protéine dans la stabilité de l’enzyme. Enfin, le modèle a également démontré que la pyruvate kinase et la phosphofructokinase sont fortement corrélées afin de maintenir la robustesse de la glycolyse. Il est donc intéressant de constater que les gènes codant pour ces deux enzymes forment un opéron (pfk-pyk). Afin de perturber cette régulation, nous avons découplé les gènes de cet opéron, chacun sous contrôle d’un promoteur inductible. Les résultats montrent que la robustesse de la glycolyse de B. subtilis est très forte, et qu’elle est très difficile à perturber. Pour conclure, ce travail a permis de valider le modèle mathématique établi en amont, tout en démontrant les difficultés et les atouts d’une collaboration entre mathématiques et biologie, pour expliquer les réseaux biologiques aussi complexes
In this study, we established a dynamical differential equation model of glycolysis in Bacillus subtilis that couples enzymatic and transcriptional regulation. Full experimental validation of such a complex model is currently not feasible. Thus, we built a model-driven validation strategy to decrease the number of experiments and focus only on several key points of the regulation. Model analysis at steady-state pointed out: strong structural properties of glycolysis, key enzymes involved and enzymatic regulations that seem indispensable. Our objective was to validate the predicted enzymatic regulation, demonstrate the structural properties from a biological perspective and perturb them in order to validate the model. First, the model predicted critical enzymatic regulations that we verified experimentally. This in silico-driven approach led us to some unexpected discoveries. Glucose-6-phosphate dehydrogenase and phosphofructokinase were both predicted by the model to be inactivated by phosphoenolpyruvate (PEP). We purified the enzymes from B. subtilis and were able to demonstrate uncompetitive inhibition by PEP for both of them. Moreover, pyruvate kinase, catalyzing the last step of glycolysis, was predicted to be activated by ribose-5-phosphate (R5P). Enzymatic assays with N-terminally tagged B. subtilis pyruvate kinase showed no activation by R5P, or any known activator of pyruvate kinases from other species. By contrast, enzymatic assays with C-terminally tagged B. subtilis pyruvate kinase showed the predicted R5P activation, suggesting the implication of the N-terminus in B. subtilis pyruvate kinase stability. Finally, the model analysis showed that pyruvate kinase and phosphofructokinase need to be strongly correlated to maintain the robustness of glycolysis. This notion is supported by the fact that genes coding for these enzymes constitute an operon (pfk-pyk). In order to perturb the robust regulation of glycolysis, we constructed B. subtilis with the genes pfk and pyk uncoupled, each under control of a separate inducible promoter. The results show high-robustness of B. subtilis glycolysis that was difficult to perturb. In the end, the mathematical model has been validated. This work has demonstrated the shortcomings and the advantages of working at the interface between mathematics and biology, which is necessary for full understanding of high-complexity biological networks

Biological systems, their physical structure and their functions, are built, maintained, and controlled by the activity of enzymes. Understanding how enzymes contribute to the regulation of various pathways and processes allows us to gain a deeper understanding of the entirety of the biological system. As changes in enzyme activity are often essential for the pathogenesis of multiple and varied diseases, identifying these changes represents a crucial step to both understanding the disease and preventing its progression within the individual. Enzymes’ functional output can be controlled by numerous different mechanisms, including control of transcription and translation, subcellular localisation, co-factor interactions, or chemical modification to specific amino acids. Activity-based protein profiling allows the potential for activity of target enzymes to be measured, thereby gaining a more accurate representation of the functional state of the biological system. In this work, profiling differential enzyme activity allows the discovery of previously unknown links between metabolic regulatory enzymes and infection by the hepatitis C virus (HCV). The novel probe wortmannin-yne is described and is shown to be able to report on the activity multiple kinases, including MAPK1, whose activity is dysregulated during HCV replication. Novel probes designed to target a smaller selection of kinases, phosphatidylinositol kinases, are reported and are shown to be capable of measuring HCV-induced changes to not only kinase activity but also regulatory protein-protein interactions with the phosphoinositide kinases. Lastly, the role of microRNA-27b in the HCV-induced dysregulation of lipid metabolic enzymes is examined. Three novel targets of microRNA-27b are identified, and their dysregulation is shown to have an effect on the life cycle of HCV. Altogether, this work has developed new tools for the study of metabolic enzymes and identified new avenues of investigation into the dysregulation of lipid metabolism.

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C4 plants have been classified into three groups based on the enzymes used to decarboxylate C4 acids in the bundle sheath: NADP-ME, NAD-ME and PEPCK subtypes. Numerous molecular, biochemical and physiological evidences indicate that C4 plants could exhibit a certain degree of flexibility in the use of the three established decarboxylation mechanisms, depending on environmental factors. In this context, the objective of this work was to evaluate the modulation of the pathways of decarboxylation of the C4 photosynthesis in sugarcane (NADP-ME specie) under water deficit. The experiment was realized with two genotypes - RB92579 (tolerant to water deficit) and SP80-3280 (susceptible to water deficit) submitted to four treatments: control (normal conditions of water supply), moderate stress (-1,5 to -1,8 MPa), severe stress (below -2,0 MPa) and recovery (48 hours after rehydration). Leaf water potential, leaf gas exchange and biomass production were measured for the physiological characterization of the plants. Changes in the transcriptional responses of genes encoding C4-cycle enzymes (NADP-ME, NAD-ME, PEPCK, AspAT, AlaAT, PEPC, NADP-MDH and PPDK) and the activities of the enzymes NADP-ME, NAD-ME, PEPCK, AspAT and AlaAT were analyzed by RT-qPCR and spectrophotometry, respectively. Under water deficit conditions the genotypes SP80-3280 was more sensitive to drought stress in terms of increased loss of above ground biomass and lower leaf water potential. It also showed less capacity of photosynthetic recovery following rehydration compared to the tolerant genotypes RB92579. The analysis of transcriptionals and of the activities of enzymes involved in the C4 photosynthetic pathway showed that sugarcane uses the PEPCK pathway as a decarboxylation mechanism in addition to the NADP-ME, which was more evident under water deficit conditions for both the drought tolerant and susceptible genotypes. Finally, the results here obtained, together with the existing information, do not support the established classification of sugarcane (Saccharum spp.) as a classical NADP-ME C4 subtype, but instead should be considered as a NADP-ME + PEPCK species.
As plantas C4 são classificadas em três grupos de acordo com as enzimas utilizadas para descarboxilação de ácidos C4 na bainha dos feixes vasculares: subtipos NADP-ME, NAD-ME e PEPCK. Inúmeras evidências moleculares, bioquímicas e fisiológicas indicam que plantas C4 podem exibir certo nível de flexibilidade na utilização dos três mecanismos de descarboxilação, dependendo de fatores ambientais. Neste contexto, o objetivo deste trabalho foi avaliar a modulação das vias de descarboxilação da fotossíntese C4 em cana-de-açúcar (espécie NADP-ME) sob déficit hídrico. O experimento foi realizado com dois genótipos - RB92579 (tolerante ao déficit hídrico) e SP80-3280 (suscetível ao déficit hídrico) submetidos a quatro tratamentos: controle (condições normais de suprimento de água), estresse moderado (-1,5 a -1,8 MPa), estresse severo (abaixo de -2,0 MPa) e recuperação (48 hrs após a reidratação). Foram realizadas análises de potencial de água foliar, trocas gasosas foliares e biomassa para caracterização fisiológica das plantas. As alterações nas respostas transcricionais dos genes codificadores de enzimas do ciclo C4 (NADP-ME, NAD-ME, PEPCK, AspAT, AlaAT, PEPC, NADP-MDH e PPDK) e atividades das enzimas NADP-ME, NAD-ME, PEPCK, AspAT e AlaAT, foram analisadas por RT-qPCR e espectrofotometria, respectivamente. Em condições de déficit hídrico, o genótipo SP80-3280 foi mais sensível ao estresse por seca em termos de aumento da perda de biomassa e menor potencial de água foliar. Também mostrou menor capacidade de recuperação fotossintética após reidratação do que o genótipo tolerante RB92579. As análises transcricionais e das atividades das enzimas envolvidas na via fotossintética C4 mostraram que a cana-de-açúcar utiliza a via PEPCK como mecanismo de descarboxilação, além da NADP-ME, mais evidente em condições de déficit hídrico tanto para genótipos tolerantes à seca como para suscetíveis. Finalmente, os resultados aqui obtidos, juntamente com a informação existente, não suportam a classificação estabelecida da cana-de-açúcar (Saccharum spp.) como um subtipo C4 NADP-ME clássico, mas considerá-la como uma espécie NADP-ME + PEPCK.

Le cytoplasme des cellules eucaryotes contient de nombreux compartiments membranaires fermes, capables de fusionner entre eux afin de maintenir leurs proprietes et leur composition. Ces fusions sont etroitement regulees dans le temps et l'espace. Ce manuscrit decrit l'etude des mecanismes moleculaires permettant cette regulation au sein de la voie endocytaire de l'amibe dictyostelium discoideum. Afin de realiser cette etude, un protocole permettant de reconstituer in vitro les fusions des compartiments endocytaires a ete mis au point. Il repose sur le marquage de la voie endocytaire par de la peroxydase de raifort biotinylee ou par de l'avidine, deux marqueurs dont l'internalisation par pinocytose a ete caracterisee chez dictyostelium discoideum. Grace a cette approche, il a ete possible de demontrer que chez cette amibe, seuls les compartiments endocytaires precoces ne contenant pas d'enzymes hydrolytiques sont capables de fusionner. Cette fusion est controlee par la petite proteine g rab7 dont l'implication dans le recyclage des enzymes depuis les post-lysosomes a ete recemment evoquee dans la litterature. Sur la base de ces resultats, la voie endocytaire de dictyostelium discoideum serait constituee d'une etape precoce de fusions permettant le melange du materiel nouvellement ingere et d'enzymes recyclees depuis la fin de la voie endocytaire, suivie d'une etape de maturation. Il a egalement ete possible de demontrer que l'atp soluble n'est pas necessaire aux fusions des endosomes precoces de dictyostelium discoideum. Paradoxalement le nem et l'a485 sont capables d'inhiber la fusion de ces endosomes, donnees dans la litterature comme dependant de l'atpase nsf. Pris ensemble, ces resultats suggerent un recrutement tres precoce de nsf lie a de l'atp sur les membranes allant fusionner, precedant meme leur association (<<<>docking<>>>). Le role de nsf reste neanmoins posterieur a celui de rab7.

La proteine-kinase ck2 est une serine/threonine proteine-kinase ubiquiste dans le monde eucaryote. Elle se compose de deux sous-unites catalytiques differentes alpha et alpha' et d'une sous-unite regulatrice beta qui s'assemblent sous la forme d'heterotetrameres. La ck2 utilise l'atp comme le gtp, phosphoryle des proteines et peptides substrats acides et necessite une concentration extraphysiologique d'ions magnesiums pour pouvoir exhiber son activite catalytique maximale. Bien que plusieurs evidences suggerent qu'in vivo, la ck2 joue un role indispensable, aucun mode de regulation cellulaire n'a pu etre propose pour cette kinase a l'heure actuelle. Parmi les candidats susceptibles de porter une telle fonction regulatrice, les polyamines sont des composes vis-a-vis desquels de nombreuses etudes ont ete realisees. Presentes en concentrations submillimolaires, ces molecules sont capables de stimuler l'activite catalytique de la ck2 d'un facteur 10 a 20 dans des conditions de concentrations physiologiques d'ions magnesiums. Lors de ces travaux, les parametres cinetiques regissant l'activation de la ck2 en presence de polyamines, ont ete definis. L'effet de la spermine sur la ck2 a ete analyse par dichroisme circulaire. L'interaction spermine-ck2 a ete caracterisee en presence de molecules analogues de la spermine ainsi qu'en ce qui concerne le type et le nombre de liaisons mises en jeu. L'utilisation d'un analogue photoactivable de la spermine a permis d'identifier un site majeur de liaison des polyamines sur la sous-unite beta au niveau du peptide t72-m78. Deux autres sites ont ete detectes au niveau des residus h108 de cette sous-unite et l220 de la sous-unite alpha. La construction, l'expression et la purification de proteines de fusion impliquant le domaine d51-p110 de la sous-unite beta ou la sous-unite beta entiere ont permis de confirmer les resultats du marquage de photoaffinite d'une part et de definir ce domaine comme autonome et fonctionnel d'autre part. Son utilisation a conduit entre autre a la mise en evidence des domaines impliques dans l'interaction des proteines fgf-2 et ck2. Aujourd'hui, la poursuite de ce travail par une approche cellulaire pourrait permettre d'apporter des elements de reponse quant a l'existence d'un role de regulateurs physiologiques joue par les polyamines vis-a-vis de la ck2

Les proteoglycanes composes de chaines de chondroitine sulfate isoles de la media-intima d'aorte de porc possedent une affinite pour la lipoproteine lipase purifiee a partir du lait de vache. Etude du complexe enzyme-proteoglycanes marque a l'iode 125

[EN] Carbamoyl phosphate synthetase 1 (CPS1), a 1462-residue mitochondrial enzyme, catalyzes the entry of ammonia into the urea cycle, which converts ammonia, the neurotoxic waste product of protein catabolism, into barely toxic urea. The urea cycle inborn error and rare disease CPS1 deficiency (CPS1D) is inherited with mendelian autosomal recessive inheritance, being due to CPS1 gene mutations (>200 mutations reported), and causing life-threatening hyperammonemia. We have produced recombinantly human CPS1 (hCPS1) in a baculovirus/insect cell expression system, isolating the enzyme in active and highly purified form, in massive amounts. This has allowed enzyme crystallization for structural studies by X-ray diffraction (an off-shoot of the present studies). This hCPS1 production system allows site-directed mutagenesis and enzyme characterization as catalyst (activity, kinetics) and as protein (stability, aggregation state, domain composition). We have revealed previously unexplored traits of hCPS1 such as its domain composition, the ability of glycerol to replace the natural and essential CPS1 activator N-acetyl-L-glutamate (NAG), and the hCPS1 protection (chemical chaperoning) by NAG and by its pharmacological analog N-carbamyl-L-glutamate (NCG). We have exploited this system to explore the effects on the activity, kinetic parameters and stability/folding of the enzyme, and to test the disease-causing nature, of mutations identified in patients with CPS1 deficiency (CPS1D). These results, supplemented with those obtained with other non-clinical mutations, have provided novel information on the functions of three non-catalytic domains of CPS1. We have introduced three CPS1D-associated mutations and one trivial polymorphism in the glutaminase-like domain of CPS1, supporting a stabilizing and an activity-enhancing function of this non-catalytic domain. Two mutations introduced into the bicarbonate phosphorylation domain have shed light on bicarbonate binding and have directly confirmed the importance of this domain for NAG binding to the distant (in the sequence) C-terminal CPS1 domain. The introduction of 18 CPS1D-associated missense mutations mapping in a clinically highly eloquent central non-catalytic domain have proven the disease-causing nature of most of these mutations while showing that in most of the cases they trigger enzyme misfolding and/or destabilization. These results, by proving an important role of this domain in the structural integration of the multidomain CPS1 protein, have led us to call this domain the Integrating Domain. Finally, we have examined the effects of eight CPS1D-associated mutations, of one trivial polymorphism and of five non-clinical mutations, all of them mapping in the C-terminal domain of the enzyme where NAG binds, whereas we have re-analyzed prior results with another four clinical and five non-clinical mutations affecting this domain. We have largely confirmed the pathogenic nature of the clinical mutations, predominantly because of decreased activity, in many cases due to hampered NAG binding. A few mutations had substantial negative effects on CPS1 stability/folding. Our analysis reveals that NAG activation begins with a movement of the final part of the ß4-¿4 loop of the NAG site. Transmission of the activating signal to the phosphorylation domains involves helix ¿4 from this domain and is possibly transmitted by the mutually homologous loops 1313-1332 and 778-787 (figures are residue numbers) belonging, respectively, to the carbamate and bicarbonate phosphorylation domains. These two homologous loops are called from here on Signal Transmission Loops.
[ES] La carbamil fosfato sintetasa 1 (CPS1), una enzima mitocondrial, cataliza la entrada del amonio en el ciclo de la urea, que convierte esta neurotoxina derivada del catabolismo de las proteínas en urea, mucho menos tóxica. El déficit de CPS1 (CPS1D) es un error innato del ciclo de la urea, una enfermedad rara autosómica recesiva, que se debe a mutaciones en el gen CPS1 (>200 mutaciones descritas) y que cursa con hiperamonemia. Hemos producido CPS1 humana recombinante (hCPS1) en un sistema de expresión de células de insecto y baculovirus, y la hemos aislado en forma activa, muy pura y en cantidad elevada. Este sistema de producción de hCPS1 permite la realización de mutagénesis dirigida y la caracterización de la enzima como catalizador (actividad, cinética) y como proteína (estabilidad, estado de agregación y composición de dominios). Hemos revelado características de la hCPS1 antes no exploradas como es la composición de dominios, la capacidad que tiene el glicerol para reemplazar al activador natural y esencial de la CPS1, N-acetil-L-glutamato (NAG), y la protección de la hCPS1 por NAG y por su análogo farmacológico N-carbamil-L-glutamato (NCG) (chaperonas químicas). Hemos utilizado este sistema para explorar los efectos en actividad, parámetros cinéticos y estabilidad/plegamiento de la enzima, y para comprobar la naturaleza patogénica de mutaciones identificadas en pacientes con CPS1D. Estos resultados, junto con los obtenidos con otras mutaciones no clínicas, han aportado información novedosa sobre tres de los dominios no catalíticos de CPS1. Las observaciones realizadas tras introducir en el dominio de tipo glutaminasa de la enzima tres mutaciones asociadas a CPS1D y un polimorfismo trivial, apoyan la contribución de este dominio no catalítico a la estabilidad y a aumentar la actividad de la enzima. Dos mutaciones introducidas en el dominio de fosforilación de bicarbonato han arrojado luz sobre el modo de unión del bicarbonato (un sustrato). Los resultados de estas mutaciones también han confirmado la contribución de este dominio para la unión de NAG, cuyo sitio de unión se encuentra en el dominio C-terminal de CPS1, bastante alejado (en la secuencia) del dominio de fosforilación de bicarbonato. Además, hemos introducido 18 mutaciones de cambio de sentido asociadas a CPS1D, las cuales están localizadas en un dominio no catalítico, central y de elevada elocuencia clínica. Estos resultados han demostrado la naturaleza patogénica de estas mutaciones, ya que en la mayoría de los casos estas mutaciones producen un mal plegamiento o/y desestabilización de la enzima. Debido a que estos resultados han puesto de manifiesto el importante papel de este dominio en la integración estructural de la proteína multidominio CPS1, lo hemos llamado Dominio Integrador. Finalmente, hemos examinado los efectos de 8 mutaciones asociadas a CPS1D, de un polimorfismo trivial y de 5 mutaciones no clínicas, todas localizadas en el dominio C-terminal de la enzima, donde se une NAG. Además, hemos reanalizado resultados anteriores con otras 4 mutaciones clínicas y 5 no clínicas afectando a este dominio. Hemos confirmado el carácter patogénico de las mutaciones clínicas, las cuales predominantemente causan una disminución en la actividad enzimática, en muchos casos debida a que la unión de NAG se encuentra obstaculizada. Unas pocas mutaciones mostraron efectos negativos en la estabilidad/plegamiento de CPS1. Nuestros análisis revelan que la activación por el NAG empieza con un movimiento de la parte final del bucle ß4-¿4 del sitio de NAG. La transmisión de la señal activadora a los dominios de fosforilación implica a la hélice ¿4 de este dominio y posiblemente se transmite a través de los bucles homólogos 1313-1332 y 778-787 (numeración de residuos) pertenecientes, respectivamente, a los dominios de fosforilación de carbamato y bicarbonato. Por ello, hemos llamado a ambos bucles Bucles de
[CAT] La carbamil fosfat sintetasa 1 (CPS1), un enzim mitocondrial, catalitza l'entrada d'amoni en el cicle de la urea, que convertix l'amoni, producte neurotòxic del catabolisme de les proteïnes, en urea, una molècula molt poc tòxica. El dèficit de CPS1 (CPS1D) és un error innat del cicle de la urea, una malaltia rara autosòmica recessiva, que es deu a mutacions en el gen CPS1 (>200 mutacions descrites) i que cursa amb hiperamonièmia. Hem produït CPS1 humana recombinant (hCPS1) en un sistema d'expressió de cèl·lules d'insecte i baculovirus, i l'hem aïllada en forma activa, molt pura i en gran quantitat. Això ha permés la cristal·lització de l'enzim per a estudis estructurals amb difracció de raios-X (treball no inclòs en esta tesi Aquest sistema de producció de hCPS1 permet la realització de mutagènesi dirigida i la caracterització de l'enzim com a catalitzador (activitat, cinètica) i com a proteïna (estabilitat, estat d'agregació i composició de dominis). Hem revelat característiques de la hCPS1 no explorades abans com és la composició de dominis, la capacitat que té el glicerol per a reemplaçar l'activador natural i essencial de CPS1, N-acetil-L-glutamat (NAG), i la protecció de la hCPS1 per NAG i pel seu anàleg farmacològic N-carbamil-L-glutamat (NCG) (xaperones químiques) . Hem utilitzat aquest sistema per a explorar els efectes en l'activitat, els paràmetres cinètics i l'estabilitat/plegament de l'enzim, i per a comprovar la naturalesa patogènica de mutacions identificades en pacients amb CPS1D. Aquestos resultats, junt amb els obtinguts amb altres mutacions no clíniques, han aportat informació nova sobre tres dels dominis no catalítics de la CPS1. Les observacions, després d'introduir tres mutacions associades a CPS1D i un polimorfisme trivial en el domini tipus glutaminasa de CPS1, recolzen la contribució d'aquest domini no catalític a l'estabilitat i a l'optimització de l'activitat enzimàtica. Dues mutacions introduïdes en el domini de fosforilació de bicarbonat han esclarit el mode d'unió de bicarbonat. Els resultats d'aquestes mutacions també han confirmat la contribució d'aquest domini per a la unió de NAG, el lloc d'unió de la qual es troba en el domini C-terminal de CPS1, prou allunyat (en la seqüència) del domini de fosforilació de bicarbonat. A més, hem introduït 18 mutacions de canvi de sentit associades a CPS1D, les quals estan localitzades en un domini no catalític, central i d'elevada eloqüència clínica. Aquestos resultats han demostrat la naturalesa patogènica d'aquestes mutacions, ja que, en la majoria dels casos produïxen un mal plegament o/i desestabilització de l'enzim. Pel fet que aquestos resultats han posat de manifest l'important paper d'aquest domini en la integració estructural de la proteïna multidomini CPS1, l'hem anomenat Domini Integrador. Finalment, hem examinat els efectes de huit mutacions associades a CPS1D, un polimorfisme trivial i cinc mutacions no clíniques, totes elles localitzades en el domini C-terminal de l'enzim, on s'unix NAG. A més, hem reanalitzat resultats anteriors amb altres quatre mutacions clíniques i cinc no clíniques que afecten aquest domini. Hem confirmat el caràcter patogènic de les mutacions clíniques, les quals predominantment causen una disminució en l'activitat enzimàtica, en molts casos pel fet que la unió de NAG es troba obstaculitzada. Unes poques mutacions van mostrar efectes negatius substancials en l'estabilitat/plegament de CPS1. Les nostres anàlisis revelen que l'activació de NAG comença amb un moviment de la part final del bucle ß4-¿4 del lloc de NAG. La transmissió del senyal activadora als dominis de fosforilació involucra l'hèlix ¿4 d'aquest domini i es transmet, possiblement, a través dels bucles homòlegs 1313-1332 i 778-787 (numeració dels residus), pertanyents, respectivament, als dominis de fosforilació de carbamato i bicarbonat. Per això, hem anomenat a ambd
Díez Fernández, C. (2015). USING RECOMBINANT HUMAN CARBAMOYL PHOSPHATE SYNTHETASE 1 (CPS1) FOR STUDYING THIS ENZYME'S FUNCTION, REGULATION, PATHOLOGY AND STRUCTURE [Tesis doctoral no publicada]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/52855
TESIS

Le biocarbone est un biofiltre aéré utilise dans l'épuration des eaux usées ayant des grains de biodagéne comme support. La flore bactérienne fixée sur ce support a été isolée, identifiée et son activité hydrolytique mesurée. Des souches ayant des activités importantes ont été sélectionnées et ont été marquées par résistance à certains antibiotiques et substrats suicides. Ce marquage a permis de suivre l'implantation et l'évolution de ces souches sur le support. L'étude de la régulation des activités protéasiques et lipasiques chez une des souches sélectionnées (serratia marcescens 532 s) a été effectuée, ainsi que l'étude de la régulation du métabolisme carbone de klebsiella oxytoca (sélectionnée à partir du filtre). Cette bactérie présente un métabolisme branché dont nous avons étudié les flux carbonés en fonction du pourcentage de saturation du milieu de culture en oxygène ainsi que de la concentration en sulfate

La 5-aminolévulinate deshydratase (E. C. 4. 2. 1. 24 ; 5-ALAD) est une enzyme oligomérique qui permet la synthèse du porphobilinogène à partir de l'acide aminolévulinique. Son étude après différents traitements par des antibiotiques, des plantules de radis, montre après une analyse par des techniques immunochimiques, une relation étroite entre l'activité enzymatique et la quantité de protéines. Le traitement par la gabaculine induit une inhibition d'environ 75% de l'activité 5-ALAD, ainsi qu'une augmentation de la concentration endocellulaire de l'α-cétoglutarate par rapport à un témoin non traité. Ce cétoacide est un précurseur de l'acide glutamique dans les chloroplastes. Un rôle régulateur de ce cétoacide in vivo sur la synthèse des chlorophylles, est envisagé. L'analyse de la 5-ALAD par des techniques d'immunoempreintes fait apparaître des sous-unités de tailles différentes (37 et 40 kDa) aussi bien chez le radis que chez le tabac. Cependant, seul le tabac sauvage montre une bande immunodétectée a 55 kDa. Différentes hypothèses sur son rôle et son identité sont envisagées en relation avec les travaux récents. L'étude de la 5-ALAD extraite des deux souches de tabac montrent, chez le mutant albinos une activité enzymatique supérieure d'un facteur 6 à celle du tabac sauvage.

La threonine synthase de plante est specifiquement activee par la s-adenosylmethionine, produit directement derive de la methionine. Afin de mieux comprendre le mecanisme de cette interaction de la s-adenosylmethionine avec l'enzyme, l'adnc codant la threonine synthase d'arabidopsis a ete isole pour la premiere fois chez les plantes par complementation fonctionnelle d'un mutant bacterien. Dans une seconde etape, l'adnc codant la threonine synthase de plante a ete utilise pour surproduire chez e. Coli la forme nature de la threonine synthase. L'enzyme a ete purifiee a homogeneite et s'est averee etre identique a l'enzyme purifiee a partir de plante. Des experiences de cinetiques a l'etat-stationnaire et de liaison a l'equilibre nous ont permis de deduire que la fixation de la sam a l'enzyme augmente de 8 fois sa vitesse de catalyse et augmente considerablement son affinite apparente pour le substrat o-phosphohomoserine (diminution de 25 fois du k#m). Un modele cinetique a alors ete etabli, permettant de rendre compte a la fois des donnees de cinetique a l'etat stationnaire et des donnees de fixation a l'equilibre de la sam sur l'enzyme libre. Nous suggerons de plus que les modifications considerables des proprietes cinetiques de l'enzyme resultent d'une transition allosterique cooperative induite par la s-adenosylmethionine. Des analyses en cinetiques rapides indiquent que cette transition se deroule a une vitesse plus elevee en presence du substrat. Recemment, des cristaux de l'enzyme diffractant a environ 3 angstroms ont ete obtenus en presence de s-adenosylmethionine. Afin de mieux comprendre la fonction physiologique de l'activation de la threonine synthase par la s-adenosylmethionine, le controle du partage du flux d'o-phosphomoserine entre la threonine synthase et l'enzyme competitrice cystathionine -synthase a ete simule numeriquement par integration des parametres cinetiques de ces enzymes.

Utilisation de methodes d'immunochimie, d'immunocytochimie (or colloidal) et de dosages immunoenzymatiques. L'ensemble de ces methodes, grace a la preparation d'anticorps monospecifiques, a permis de montrer qu'il existe chez la truite au moins deux isoenzymes de l'anhydrase carbonique (ac): l'isoenzyme erythrocytaire (ace) et l'isoenzyme branchiale (acb). L'ace est present dans le cytosol des globules rouges a l'exclusion de tout autre tissu. Son activite n'est pas affectee par le milieu d'adaptation (eau de mer, eau douce). Il est implique dans la deshydratation du bicarbonate plasmatique. L'acb est present dans les branchies, les cellules a chlorures sont particulierement riches, notamment chez les truites d'eau de mer, ce qui suggere que l'acb est implique dans les echanges ioniques. L'acb est present dans les cellules de l'epithelium "respiratoire", ou il est concentre au niveau de la membrane plasmique (villosites) des cellules pavimenteuses et dans les grains de secretion de cellules a mucus. Selon ces observations, on pense que l'acb agit au niveau de l'eau respiree, en accelerant l'hydratation du co::(2) metabolique et en protonant l'ammoniaque. L'acb a ete localise dans deux epitheliums extra-branchiaux au moins: la pseudobranchie et la vessie urinaire. L'acb representerait la forme tissulaire, non erythrocytaire, de l'ac chez les poissons. L'existence d'un acb dans la vessie suggere que cet organe, chez les poissons, participe a l'acidification de l'urine

L'exposition de cellules d'érable à des concentrations sublétales de cuivre (50 m) s'accompagne d'un arrêt de l'augmentation de la masse fraîche au bout du 5#e#m#e jour de culture. Cet arrêt de croissance est précédé par une diminution de la respiration couplée et découplée, avec diminution concomitante des marqueurs mitochondriaux (fumarase, cytochrome oxydase, cardiolipide). Le cuivre provoque ainsi une dilution progressive des mitochondries dans la population cellulaire, due à un arrêt de la division des mitochondries précédent l'arrêt de la division des cellules. Lorsque le cuivre est soustrait du milieu, la division des mitochondries, puis des cellules, sont relancées. Nous disposons ainsi d'un levier pour analyser le contrôle du nombre de mitochondries dans les cellules. Par ailleurs, nous avons montré que l'oxydase alternative est étroitement contrôlée par le niveau de cuivre dans le milieu de culture des cellules (activité et quantité de protéine). Les effets de l'excès de cuivre sur la photosynthèse ont été étudiés en utilisant des chloroplastes purifiés présentant un haut degré d'intégrité morphologique et fonctionnelle (épinard et pois). La teneur de 1. 8 m de cuivre donne une inhibition (ic50) de 50% du dégagement d'oxygène, alors que sur les thylacoides purifiés on observe un ic50 apparent de 54 m. Ceci suggère que le cuivre agit essentiellement sur les enzymes du cycle de Benson. Enfin, nous avons observé une interférence entre le cuivre et le manganèse au niveau de la photosynthèse des chloroplastes intacts, et pas au niveau des thylacoides. Les analyses par oxygraphie et par fluorescence modulée suggèrent qu'il y a une compétition entre le cuivre et le manganèse au niveau de leur transport à travers l'enveloppe plastidiale.

Au cours de ce travail de these nous avons caracterise un adnc correspondant a la proteine t (1560 pb) du complexe de la glycine decarboxylase. Il possede un cadre de lecture ouvert de 1220 pb codant pour un peptide mature de 378 acides amines. La sequence primaire ainsi que la masse deduite (40 961 da) ont ete confirme respectivement par microsequencage et par mesure au spectrometre de masse. Dans la perspective d'une etude structurale, nous avons reussi a surexprimer chez e. Coli la proteine t en construisant un vecteur d'expression produisant un arnt rare chez les bacteries. L'etude de l'expression de la proteine t ainsi que de ses transcrits a montre qu'ils sont principalement presents dans les tissus folaires et semblent subir une forte induction a la lumiere. De plus, nous avons demontre que les transcrits correspondant aux proteines de la gdc (p, h et t) sont exprimes des le 4#i#e#m#e jours de developpement de la plante avec un pic au 7#i#e#m#e jour. . A ce stade, la faible representation des proteines de la gdc (p, h et t), suggere l'existence d'un controle post-transcriptionnel de l'expression de leurs genes. Nous avons egalement isole et sequence le gene codant pour la proteine t. Ce gene, compose de quatre exons, possede deux sites d'initiations dont l'un presente une sequence riche en pyrimidine proche de la sequence inr (initiator element). Enfin, l'analyse de la region promotrice a permis de caracteriser trois regions consensus qui semblent etre impliquees dans la regulation a la lumiere et dans la specificite tissulaire

L'accumulation des formes libres et liees de l'acc (amino-cyclopropane carboxylique acide) pose le probleme des mecanismes enzymatiques impliques tels que: efe (enzyme formant l'ethylene), acc synthetase et mabonyl-acc transferase. L'etude de tissus excises maintenus en survie sur milieu gelose contenant un inhibiteur tel que: le cordycepine, le cycloheximide et le cobalt ont permis de mettre en evidence certains mecanismes de regulation des emissions ethyleniques

碩士
國立中山大學
生物科學系研究所
99
A major sweet potato leaf-type catalase was detected and identified from fullyexpandedmature leaves using in-gel activity staining assay with native- andSDS-PAGEs. The putative catalase activity band was inhibited by a catalaseinhibitor 3-amino-1,2,4-triazole. The major leaf-type catalase activity wasoptimal over 8, and was significantly repressed by β-mercaptoethanol. However,its activity was much less affected by temperature within the range of 5 to 450C.Temporal and spatial expression showed that it was specifically detected inleaves, but not in roots and stems. Its activity increased from the immature L2leaves, and reached the maximal at the fully-expanded mature L3 leaves, thenslightly decreased in partial yellowing senescent L4 leaves, and was almost notdetected in completely yellowing L5 leaves similar to folding unopenedimmature L1 leaves. The catalase level showed approximately inversecorrelation with the H2O2 amounts in leaves of different developmental stages.Dark and ethephon, an ethylene-releasing compound, also temporarily enhancedthe catalase activities from 6 h to 24 h, however, the enhanced activitydecreased from 24 h to 48 h in detached leaves after treatment. The catalaselevel also showed approximately negative correlation with the H2O2 amounts intreated leaves. The major leaf-type catalase activity was repressed by EGTA,and the repression can be reversed by exogenous CaCl2. The major leaf-typecatalase activity was also repressed by calmodulin inhibitor chlorpromazine,and the repression can be reversed by exogenous purified SPCAM calmodulinfusion protein. Chlorpromazine-treated leaves also elevated H2O2 amount.Based on these data we conclude that a major leaf-type catalase with maximalactivity in L3 leaf was identified in sweet potato. Its activity was temporarilyenhanced by dark and ethephon, and was modulated by external calcium ion(Ca2+) and calmodulin. A possible physiological role and function in associationwith cellular H2O2 homeostasis in cope with developmental and environmentalcues in sweet potato leaves is suggested.

Physical biology offers powerful tools for quantitatively dissecting the various aspects of cellular life that one cannot attribute to inanimate matter. Signature examples of living matter include adaptation, self-organization, and division. In this thesis, we explore different interconnected facets of these processes using statistical mechanics, nonequilibrium thermodynamics, and biophysical modeling.

One of the key mechanisms underlying physiological and evolutionary adaptation is allosteric regulation. It allows cells to dynamically respond to changes in the state of the environment often expressed through altered levels of different environmental cues. The first thread of our work is dedicated to exploring the combinatorial diversity of responses available to allosteric proteins that are subject to multi-ligand regulation. We demonstrate that proteins characterized through the Monod-Wyman-Changeux model of allostery and operating at thermodynamic equilibrium are capable of eliciting a wide range of response behaviors which include the kinds known from the field of digital circuits (e.g., NAND logic response), as well as more sophisticated computations such as ratiometric sensing.

Despite the fact that biomolecules at thermodynamic equilibrium are able to orchestrate a variety of fascinating behaviors, the cell is ultimately 'alive' because it constantly metabolizes nutrients and generates energy to drive functions that cannot be sustained in the absence of energy consumption. One prominent example of such a function is nonequilibrium error correction present in high-fidelity processes such as protein synthesis, DNA replication, or pathogen recognition. We begin the second thread of our work by providing a conceptual understanding of the prevailing mechanism used in explaining this high-fidelity behavior, namely that of kinetic proofreading. Specifically, we develop an allostery-based mechanochemical model of a kinetic proofreader where chemical driving is replaced with a mechanical engine with tunable knobs which allow modulating the amount of dissipation in a transparent way. We demonstrate how varying levels of error correction can be attained at different regimes of dissipation and offer intuitive interpretations for the conditions required for efficient biological proofreading.

We then extend the notion of error correction to equilibrium enzymes not endowed with structural features typically required for proofreading. We show that, under physiological conditions, purely diffusing enzymes can take advantage of the existing nonequilibrium organization of their substrates in space and enhance the fidelity of catalysis. Our proposed mechanism called spatial proofreading offers a novel perspective on spatial structures and compartmentalization in cells as a route to specificity.

In the last thread of the thesis, we make a transition from molecular-scale studies to the mesoscopic scale, and explore the principles of self-organization in nonequilibrium structures formed in reconstituted microtubule-motor mixtures. In particular, we develop a theoretical framework that predicts the spatial distribution of kinesin motors in radially symmetric microtubule asters formed under various conditions using optogenetic control. The model manages to accurately recapitulate the experimentally measured motor profiles through effective parameters that are specific for each kind of kinesin motor used. Our theoretical work of rigorously assessing the motor distribution therefore offers an avenue for understanding the link between the microscopic motor properties (e.g., processivity or binding affinity) and the large-scale structures they create.

In all, the thesis encompasses a series of case studies with shared themes of allostery and nonequilibrium, highlighting the capacity of living matter to perform remarkable tasks inaccessible to nonliving materials.

碩士
實踐大學
食品營養與保健生技學系碩士班
99
Osteoporosis and hypertension are the health problem in recent years. Egg is a very important livestock products in our country. Egg yolk includes the rich lipid and the protein. In order to understand the function of health care in egg yolk protein hydrolysate to the human body, we cooked egg yolks than extracted with acetone and 95 % ethanol to remove most of lipid content, and residues were delipid egg yolk protein (DEYP). DEYP powders were individually hydrolyzed with papain, alcalase and flavourzyme. The optimal hydrolysis parameter : substrate concentration = 1.5 % ; enzyme and substrate ratio, E / S = 1：1000 ; pH = 7.0 ; temperature = 55 ℃ ; obtain the hydrolysates P4, P2F2, A2F2. We feed the SD rats with P4, P2F2, A2F2 to observe the bone mineral density, blood sugar, insulin, ALP activity, serum calcium, total cholesterol, LDL cholesterol, HDL cholesterol, triglyceride. Observed the experiment group and baseline group’s bone mineral density, blood sugar, insulin, ALP activity, serum calcium, showed no significantly with each other. This three enzyme hydrolysates had inhibition of ACE activity, IC50 values 17 ~ 92 µg /mL ; and the three hydrolysates were separated from ultrafiltration membrane with MWCO 5.0 KDa. The molecular weight lower than 5.0 KDa enzyme hydrolysates had inhibition of ACE activity, IC50 values 15 ~ 78 µg /mL. And the three enzyme hydrolysates were separated from ultrafiltration membrane and HPLC analysis .The molecular weight lower than 5.0 KDa enzyme hydrolysates had inhibition of ACE activity, IC50 values 21 ~ 350 µg /mL.