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by Paul Li Wai Ching. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1990. / Includes bibliographical references. / Acknowledgements --- p.i / Summary --- p.ii / Index to Figures --- p.V / Index to Tables --- p.vii / Abbreviations --- p.viii / CONTENTS / Chapter Chapter 1 --- INTRODUCTION --- p.1 / Chapter Chapter 2 --- LITERATURE REVIEW : SALT AND HYPERTENSION / Chapter 2.1. --- Summary of evidence linking salt and hypertension --- p.4 / Chapter 2.1.1. --- Epidemiological studies --- p.4 / Chapter 2.1.2. --- Dietary intervention studies --- p.7 / Chapter 2.1.3. --- Experimental studies --- p.9 / Chapter 2.2. --- Cellular sodium transport --- p.10 / Chapter 2.2.1. --- The Sodium Pump --- p.10 / Chapter 2.2.2. --- Defects in sodium transport in hypertension --- p.14 / Chapter 2.3. --- Hypothesis linking salt to the pathogenesis of hypertension --- p.15 / Chapter 2.4. --- Evidence for the presence of natriuretic Hormone --- p.18 / Chapter 2.4.1. --- Indirect evidence --- p.18 / Chapter 2.4.2. --- Direct evidence --- p.18 / Chapter 2.4.3. --- The source and properties of natriuretic hormone --- p.20 / Chapter 2.4.4. --- Other natriuretic factors --- p.21 / Chapter Chapter 3 --- REGULATION OF THE SODIUM PUMP / Chapter 3.1. --- General introduction --- p.24 / Chapter 3.2. --- Regulation of the sodium pump by intracellular sodium --- p.24 / Chapter 3.3. --- "Effects of ethanol on Na+,K+-ATPase activity" --- p.26 / Chapter 3.4. --- "Effects of potassium depletion on Na+,K+-ATPase activity" --- p.27 / Chapter 3.4.1. --- In vivo studies of sodium pump regulation by potassium --- p.27 / Chapter 3.4.2. --- In vitro studies of sodium pump regulation by potassium --- p.29 / Chapter 3.5. --- Effects of cardiac glycosides on the sodium pump --- p.30 / Chapter 3.5.1. --- In vivo studies of sodium pump regulation by cardiac glycosides --- p.31 / Chapter 3.5.2. --- In vitro studies of sodium pump regulation by cardiac glycosides --- p.33 / Chapter 3.6. --- Effects of dietary salt on the sodium pump --- p.35 / Chapter 3.6.1. --- Acute effects of salt-loading --- p.35 / Chapter 3.6.2. --- Chronic effects of salt-loading --- p.36 / Chapter Chapter 4 --- AIMS OF THE STUDY --- p.39 / Chapter Chapter 5 --- MEASUREMENT OF THE SODIUM PUMP ACTIVITY / Chapter 5.1. --- General introduction --- p.41 / Chapter 5.2. --- The measurement of sodium pump activity --- p.42 / Chapter 5.2.1. --- The sodium pump transport activity --- p.42 / Chapter 5.2.2. --- Quantitation of the number of sodium pump sites --- p.45 / Chapter 5.2.3. --- The measurement of enzyme activity --- p.47 / Chapter (a) --- Introduction --- p.47 / Chapter (b) --- Preparation of tissues and detergent treatment --- p.48 / Chapter (c) --- Measurement of ATPase activity by measuring the rate of release of inorganic phosphate --- p.49 / Chapter (d) --- The coupled-enzyme assay --- p.53 / Chapter (e) --- The K+-stimulated 3-0-MFPase assay --- p.54 / Chapter Chapter 6 --- METHODS - ESTABLISHMENT AND EVALUATION / Chapter 6.1. --- Chemicals --- p.57 / Chapter 6.2. --- "Measurement of Na+,K+-ATPase activity by the rate of release of inorganic phosphate" --- p.58 / Chapter 6.3. --- "Automated coupled-enzyme assay of Na+,K+-ATPase activity" --- p.62 / Chapter 6.4. --- "The measurement of Na+,K+-ATPase activity by the potassium-stimulated 3-0-MFPase assay" --- p.67 / Chapter 6.5. --- Determination of protein concentration --- p.70 / Chapter 6.6. --- Statistical analysis --- p.73 / Chapter 6.7. --- Results --- p.73 / Chapter 6.7.1. --- Evaluation of the inorganic phosphate release method --- p.73 / Chapter 6.7.2. --- Evaluation of the coupled-enzyme method --- p.78 / Chapter 6.7.3. --- Evaluation of the K+-stimulated 3-0-MFPase method --- p.89 / Chapter 6.7.4. --- Evaluation of the protein determination method --- p.94 / Chapter 6.8. --- Discussion --- p.96 / Chapter Chapter 7 --- "THE EFFECTS OF DIGOXIN TREATMENT ON Na+,K+-ATPase ACTIVITY OF DIFFERENT TISSUES" / Chapter 7.1. --- Introduction --- p.101 / Chapter 7.2. --- Materials and Methods --- p.103 / Chapter 7.2.1. --- Animals and diets --- p.103 / Chapter 7.2.2. --- Drugs --- p.103 / Chapter 7.2.3. --- Pharmacokinetics of digoxin --- p.103 / Chapter 7.2.4. --- The digoxin regimes --- p.104 / Chapter 7.2.5. --- Preparation and deoxycholate treatment of tissue homogenates --- p.105 / Chapter 7.2.6. --- "Measurement of Na+,K+-ATPase activity" --- p.107 / Chapter 7.2.7. --- Digoxin radioimmunoassay --- p.107 / Chapter 7.2.8. --- Measurement of plasma electrolytes --- p.109 / Chapter 7.3. --- Results --- p.110 / Chapter 7.3.1. --- The pharmacokinetics of digoxin in the rat --- p.110 / Chapter 7.3.2. --- Plasma digoxin levels --- p.110 / Chapter 7.3.3. --- Effects of digoxin treatment on body weight --- p.113 / Chapter 7.3.4. --- Effects of digoxin treatment on plasma electrolytes --- p.117 / Chapter 7.3.5. --- "Effects of digoxin treatment on tissue Na+,K+-ATPase activity" --- p.119 / Chapter 7.4. --- Discussion --- p.129 / Chapter Chapter 8 --- THE SALT-LOADING EXPERIMENT / Chapter 8.1. --- Introduction --- p.140 / Chapter 8.2. --- Materials and Methods --- p.142 / Chapter 8.2.1. --- Animals --- p.142 / Chapter 8.2.2. --- The salt-loading protocol --- p.142 / Chapter 8.2.3. --- Preparation of crude tissue homogenates --- p.143 / Chapter 8.2.4. --- "Measurement of Na+,K+-ATPase activity" --- p.144 / Chapter 8.2.5. --- Analysis of urinary electrolytes --- p.144 / Chapter 8.2.6. --- Measurements of body weight and wet weight of kidney --- p.145 / Chapter 8.3. --- Results --- p.145 / Chapter 8.3.1. --- Effects of salt loading on the body weight --- p.145 / Chapter 8.3.2. --- Effects of salt loading on 24-hour urinary sodium excretion --- p.148 / Chapter 8.3.3. --- Effects of salt loading on the wet weight of kidney --- p.152 / Chapter 8.3.4. --- "Effects of salt loading on tissue Na+,K+-ATPase activity" --- p.152 / Chapter 8.4. --- Discussion --- p.163 / Chapter Chapter 9 --- CONCLUSIONS AND FUTURE WORK --- p.175 / REFERENCES --- p.183

Copper and iron are trace elements that form an indispensable part of many proteins and are crucial for the well-being of all cells. At the same time, the intracellular levels of these metals require careful regulation, as excess or deficiency may be lethal. P1B-ATPases are key players in metal homeostasis. They belong to the superfamily of P-type ATPases, transmembrane proteins present in virtually all life forms, are responsible for solute translocation across biological membranes. The goal of this thesis is to improve our understanding of the structural and functional roles of P1B-ATPases in metal homeostasis by focusing on the host-microbe interaction. The thesis first describes the importance of Cu+ distribution in the outcome of host-microbe interaction. Copper is an important element in host-microbe interactions, acting both as a catalyst in enzymes and as a potential toxin. Cu+-ATPases drive cytoplasmic Cu+ efflux and protect bacteria against metal overload. Many pathogenic and symbiotic bacteria contain multiple Cu+-ATPase genes within particular genetic environments, suggesting alternative roles for each resulting protein. This hypothesis was tested by characterizing five homologous Cu+-ATPases present in the symbiotic organism Sinorhizobium meliloti. Mutation of each gene led to different phenotypes and abnormal nodule development in the alfalfa host. Distinct responses were detected in free-living S. meliloti mutant strains exposed to metal and redox stresses. Differential gene expression was detected under Cu+, oxygen or nitrosative stress. These observations suggest that CopA1a maintains the cytoplasmic Cu+ quota and its expression is controlled by Cu+ levels. CopA1b is also regulated by Cu+ concentrations and is required during symbiosis for bacteroid maturation. CopA2-like proteins, FixI1 and FixI2, are necessary for the assembly of two different cytochrome c oxidases at different stages of bacterial life. CopA3 is a phylogenetically distinct Cu+-ATPase that does not contribute to Cu+ tolerance. It is regulated by redox stress and required during symbiosis. We postulated a model where non-redundant homologous Cu+-ATPases, operating under distinct regulation, transport Cu+ to different target proteins. In its second part, the thesis describes the novel Fe2+-ATPases and their influence in the host-microbe interaction. Little is known about iron efflux transporters in bacterial systems. Recently, the participation of Bacillus subtilis PfeT, a P1B4-ATPase, in cytoplasmic Fe2+ efflux has been proposed. We report here the distinct roles of mycobacterial P1B4-ATPases in the homeostasis of Co2+ and Fe2+. Mutation of Mycobacterium smegmatis ctpJ affects the homeostasis of both ions. Alternatively, a M. tuberculosis ctpJ mutant is more sensitive to Co2+ than Fe2+, while mutation of the homologous M. tuberculosis ctpD leads to Fe2+ sensitivity but no alterations in Co2+ homeostasis. In vitro, the three enzymes are activated by both Fe2+ and Co2+ and bind one equivalent of either ion at their transport site. However, equilibrium binding affinities and activity kinetics show that M. tuberculosis CtpD has higher affinity for Fe2+ and twice the Fe2+ stimulated activity than the CtpJs. These parameters are accompanied by a lower activation by and affinity for Co2+. Analysis of Fe2+ and Co2+ binding to CtpD by X-ray spectroscopy shows that both ions are coordinated by 5-6 O/N atoms with similar geometry. Mutagenesis studies suggest the involvement of invariant Ser, His and Glu in metal coordination. Interestingly, replacement of Cys in the conserved CPS sequence at the metal binding pocket leads to a large reduction in Fe2+ but not Co2+ binding affinity. We propose that CtpJ ATPases participate in the control of steady state Fe2+ levels. CtpD, required for M. tuberculosis virulence, is a high affinity Fe2+ transporter involved in the rapid response to iron dyshomeostasis generated upon redox stress. These studies provide significant insights into the metal selectivity, regulation, transport kinetics and functional diversity of homologous P1B-ATPases in Cu+ and Fe2+ homeostasis. Moreover, these biochemical characterizations can be integrated with the structural-functional analysis to elucidate the complex metal distribution networks.

Protein folding and stability are essential for protein function. Changes in these characteristics can lead to altered physiological states and to the development of certain pathologies. While extensive research has focused on the stability of soluble proteins, membrane protein stability has received much less attention. Understanding the stability of membrane proteins can provide insight into folding mechanisms and the etiology of various pathologies. The purpose of this project is to prepare molecular tools to perform comparative studies of homologous membrane proteins that are found in various environments. To this end, thermophilic (Pyrococcus abyssi), mesophilic (Escherichia coli), and psychrophilic (Exiguobacterium 255-15) transmembrane Zn2+ transporting ATPases were cloned, expressed, and functionally characterized to correlate thermostability with optimal functional temperatures. In addition, the lipid environments and composition (rigid or fluid lipids) may also be involved in determining the stability of membrane proteins. Toward exploring the role of extremophilic lipids, Archaeoglobus fulgidus and Thermotoga maritima were grown and lipids were extracted. Availability of these molecular tools will enable physical-chemical studies toward understanding the structural factors that determine functional stability.

Cu+-ATPases receive Cu+ from specific chaperones via ligand exchange and subsequently drive the metal efflux from the cell cytoplasms. Cu+-ATPases have two transmembrane metal binding/transport sites (TM-MBS) and various cytoplasmic domains: the actuator (A-domain) and ATP binding domains (ATPBD), and regulatory N-terminal metal binding domains (N-MBD). Archaeoglobus fulgidus CopA, the Cu+-ATPase used in these studies, contains a single N-MBD and an apparently non-functional C-terminal MBD. The Cu+ dependent interaction of N-MBD and ATPBD was postulated as a possible mechanism for enzyme regulation. The Cu+ transfer from the chaperone to CopA is independent of the N-MBD capability to bind Cu+. Therefore, we hypothesized that ligand (Cu+ or nucleotide) binding to cytoplasmic domains might affect the interactions between the cytoplasmic domains. To test these ideas, the interactions among isolated cytoplasmic domains were characterized. Studies using isolated domains showed that while the N-MBD interacts with ATPBD, the presence of Cu+ or nucleotide (ADP) prevents this interaction. The N-MBD does not interact with the A domain. Alternatively, the C-MBD interacts with both ATPBD and A-domains in a ligand independent fashion. Only one Cu+ is transferred to CopA in absence of nucleotides, while the presence of ADP allows full loading of TM-MBS. These results suggest that the ligand binding affects the interactions between the cytoplasmic domains, and also change the conformation of CopA to help it accept the second Cu+ from chaperone.

Submitted by Marcos Anselmo (marcos.anselmo@unipampa.edu.br) on 2016-04-18T19:43:16Z No. of bitstreams: 1 Juliana Bernera Ramalho.pdf: 1079700 bytes, checksum: 8fe6895eb29d1faefd61f672d9c35b58 (MD5) / Made available in DSpace on 2016-04-18T19:43:16Z (GMT). No. of bitstreams: 1 Juliana Bernera Ramalho.pdf: 1079700 bytes, checksum: 8fe6895eb29d1faefd61f672d9c35b58 (MD5) Previous issue date: 2014-03-19 / O glutamato é um dos mais importantes neurotransmissores excitatórios presentes no sistema nervoso central e participa de uma variedade de processos fisiológicos, desempenhando papel importante na plasticidade sináptica, aprendizagem e memória. Seu excesso leva a uma ativação excessiva dos seus receptores, desencadeando excitação das células nervosas podendo levá-las à morte e, essa tem sido apontada como causa de diversas doenças neurodegenerativas. O glutamato monossódico (MSG) é um realçador de sabor amplamente utilizado na indústria de alimentos e, embora vários agonistas glutamatérgicos específicos possam imitar os efeitos tóxicos do glutamato, o MSG é possivelmente o agente mais comumente utilizado para caracterizar os mecanismos celulares e moleculares envolvidos na excitotoxicidade induzida pelo glutamato. Ainda, diversos estudos têm demonstrado prejuízos de aprendizagem e memória em animais expostos ao MSG. Já está estabelecido que o selênio (Se) é eficaz na prevenção de uma série de condições degenerativas, incluindo doenças inflamatórias e neurológicas e, estudos têm relatado que os compostos orgânicos de Se são capazes de melhorar a memória em roedores. O Selenofuranosídeo é um composto orgânico de Se e sua capacidade neuroprotetora foi demonstrada recentemente em um modelo de demência esporádica em camundongos. O objetivo deste trabalho foi avaliar o possível efeito protetor do selenofuranosídeo frente ao prejuízo à memória de longa duração, atividades das enzimas Na+, K+-ATPase e AChE e estresse oxidativo em ratos Wistar machos com 5 semanas de idade expostos ao MSG. O MSG (2g/kg) e o selenofuranosídeo (5 mg/kg) foram administrados por via oral durante 10 dias. Nos dias 11 e 12, os animais foram submetidos aos testes comportamentais (open-field e esquiva inibitória) e posterior eutanásia. Sangue, fígado, rim, córtex e hipocampo foram removidos para determinação dos parâmetros de estresse oxidativo e bioquímicos. Ainda, os tecidos cerebrais foram utilizados para determinar a atividade das enzimas Na+, K+-ATPase e AChE. Os resultados obtidos demonstram que a exposição ao MSG levou a uma alteração de memória, observada através da diminuição no tempo de latência no teste de esquiva inibitória, acompanhada da diminuição da atividade da Na+, K+-ATPase no hipocampo e córtex. O tratamento com selenofuranosídeo mostrou-se eficaz em proteger contra o prejuízo de memória associado à exposição ao MSG, através do aumento na latência no teste de esquiva inibitória e da atividade da enzima Na+, K+-ATPase no hipocampo. Não houve alteração nos parâmetros de estresse oxidativo avaliados e na atividade da AChE. Nossos resultados sugerem que o tratamento com selenofuranosídeo foi efetivo em melhorar a alteração de memória apresentada pelos animais expostos ao MSG e que esse composto pode ser um potencial agente terapêutico alternativo para o tratamento de doenças neurodegenerativas. / Glutamate is the major excitatory neurotransmitter present in the central nervous system. It participates in a variety of physiological processes and plays an important role in synaptic plasticity, learning and memory. Its excess leads to excessive activation of its receptors, triggering excitation of nerve cells and may lead them to death, and this has been identified as the cause of several neurodegenerative diseases. Monosodium glutamate (MSG) is a flavor enhancer widely used in the food industry and, although several specific glutamatergic agonists can mimic the toxic effects of glutamate, MSG is possibly the most commonly used agent to characterize the cellular and molecular mechanisms involved in glutamate-induced excitotoxicity. Also, several studies have shown learning and memory loss in animals exposed to MSG. It is already established that the selenium (Se) is effective in preventing a number of degenerative conditions, including inflammatory and neurological diseases, and studies have reported that organoselenium compounds are able of enhancing memory function in rodents. The Selenofuranoside is a new organoselenium compound and their neuroprotective capacity was recently demonstrated in a sporadic dementia model in mice. The aim of this study was to evaluate the possible protective effect of selenofuranoside against impairment to long-term memory, the enzymes Na+, K+-ATPase and AChE activities and oxidative stress in male Wistar rats at 5 weeks of age exposed to MSG. The MSG (2g/kg) and selenofuranoside (5mg/kg) were orally administered for 10 days. In the days 11 and 12, the animals were subjected to behavioral tests (open-field and inhibitory avoidance) and subsequent euthanasia. Blood, liver, kidney cortex and hippocampus were removed for determination of oxidative stress and biochemical parameters. Also, the brain tissues were used to determine enzymes Na+, K+-ATPase and AChE activities. The results showed that exposure to MSG led to memory loss observed by decreasing the latency time in the inhibitory avoidance test, accompanied by decreased activity of Na+, K+-ATPase in the hippocampus and cortex. Treatment with selenofuranoside proved effective in protecting against memory loss associated with exposure to MSG by increasing the latency in the inhibitory avoidance test and the enzyme activity of Na+, K+ -ATPase in the hippocampus. There was no change in the evaluated parameters of oxidative stress and the AChE activity. Our results suggest that treatment with selenofuranoside was effective in improving the memory impairment presented by animals exposed to MSG and that this compound can be a potential alternative therapeutic for the treatment of neurodegenerative diseases.

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The ARR4 gene from Saccharomyces cerevisiae was highly homological with E. coli arsA. The defective nucleotide-binding domain of ARR4[G30R] exhibited negative dominance from the growth curve experiment, showing that ARR4 protein might act as a dimer in vivo. On the nonreducing gel, the purified 40 kDa ARR4 presented as monomer, dimer and oligomer conformational states. Besides, the chemical cross-linking experiments confirmed the positional size of dimeric ARR4. The metal ions of Co2+, Sb3+, As3+ and As5+ have not effect on the ATPase activity and ARR4 oligomerization. ARR4 exhibit a low level of Mg2+-ATPase activity in the range of 50-70 and 100-120 nmol/min/mg respectively as estimated by phosphate released and NADH-coupled assay. The maximal activity was obtained at a ratio of ATP:Mg2+ of 2:1 and the ATPase activity of ARR4[G30R] was about 50% of that of the ARR4 from both ATPase assays. In the presence of ATP or Mg2+, ARR4 has similar ratios of dimer and monomer. When incubated with Mg2+-ATP, ARR4 tended to form more dimer than with ATP and Mg2+ individually. ARR4[G30R] exhibited less dimer formation. Mixing ARR4 and ARR4[G30R] at a constant total protein concentration with different molar ratios (1:1, 1:2 and 1:4), a near linear relationship of activity versus amount of the ARR4 protein was observed. This observation suggests that ATP hydrolysis takes places in one ATP binding site independent of the other site of NBD. The proportional ATPase activities were consistent with the ratios of ARR4-ARR4[G30R] dimerization on the nonreducing gels. In conclusion, the conformational changes induced by Mg2+-ATP is related to the dimerization of ARR4 and both its NBDs would hydrolyze ATP independently.

Thesis (Ph.D.)--Ohio University, November, 2009. / Release of full electronic text on OhioLINK has been delayed until December 1, 2014. Title from PDF t.p. Includes bibliographical references.