Dissertations / Theses on the topic '(Na+,K+)-dependent ATPase'

Glutamate transporters play a role in removing extracellular excitatory neurotransmitter, L-glutamate into the cells. The rate of the uptake depends on the density of the transporters at the membrane. Some studies claimed that glutamate transporters could transit between the cytoplasm and the membrane on a time-scale of minutes. The present study examined the distribution of glutamate transporter GLAST predominantly expressed in rat cortical cultured astrocytes between the membrane and the cytoplasm by using deconvolution microscopy and then analyzing the images. The regulation of the distribution of GLAST was studied in the presence of glutamate transporter substrate (D-aspartate), purinergic receptor activators (α,β-methylene ATP, adenosine), neuroleptic drugs (clozapine, haloperidol), ammonia (hyperammonia) and Na+/K+-ATPase inhibitors (ouabain, digoxin and FCCP). It was demonstrated that the translocation of GLAST towards the plasma membrane was induced by D-aspartate, α,β-methylene ATP, adenosine, clozapine and ammonia (at 100 μM and very high concentrations of 10 mM). However, the inhibition of Na+/K+-ATPase activity had an opposite effect, resulting in redistribution of GLAST away from the membrane. It has previously been claimed that the membrane-cytoplasm trafficking of GLAST was regulated by phosphorylation catalysed by protein kinase C delta (PKC-delta). Involvement of this mechanism has, however, been put to doubt when rottlerin, a PKC-delta inhibitor, used to test the hypothesis showed to inhibit Na+/K+-ATPase-mediated uptake of Rb+, suggesting that rottlerin influenced the activity of Na+/K+-ATPase. As Na+/K+-ATPase converts ATP to energy and pumps Na+, K+ ions, thus helping to maintain normal electrochemical and ionic gradients across the cell membrane. Its inhibition also reduced D-aspartate transport and could impact on the cytoplasm-to-membrane traffic of GLAST molecules. Furthermore, rottlerin decreased the activity of Na+/K+-ATPase by acting as a mitochondrial inhibitor. The present study has focused on the inhibition of Na+/K+-ATPase activity by rottlerin, ouabain and digoxin in homogenates prepared from rat kidney and cultured astrocytes. The activity of Na+/K+-ATPase was measured by the absorption of inorganic phosphate product generated from the hydrolysis of ATP and the fluorescent transition of the dye RH421 induced by the movement of Na+/K+-ATPase. This approach has a potential to test whether the rottlerin effect on Na+/K+-ATPase is a direct inhibition of the enzyme activity. Rottlerin has been found to block the activity of Na+/K+-ATPase in a dose-dependent manner in both rat kidney and astrocyte homogenates. Therefore, rottlerin inhibited the activity of Na+/K+-ATPase directly in a cell-free preparation, thus strongly indicating that the effect was direct on the enzyme. In parallel experiments, ouabain and digoxin produced similar inhibitions of Na+/K+-ATPase activity in rat kidney while digoxin blocked the activity of Na+/K+-ATPase to a greater extent than ouabain in rat cortical cultured astrocytes. In a separate set of experiments, Na+/K+-ATPase in the astrocytic membrane was found to be unsaturated in E1(Na+)3 conformation in the presence of Na+ ions and this could explain the differences between the effects of digoxin and ouabain on the activity of Na+/K+-ATPase in rat astrocytes. In addition, it was found that at low concentrations of rottlerin, the activity of Na+/K+-ATPase was increased rather than inhibited. This effect was further investigated by studying rottlerin interactions with membrane lipids. The activity of Na+/K+-ATPase has been reported to be regulated by membrane lipids. The enzyme activity can be enhanced by increasing fluidity of the lipid membrane. I have, therefore, proposed that rottlerin binds to the membrane lipids and the effects of rottlerin on Na+/K+-ATPase are mediated by changes in the properties (fluidity) of the membrane. The hypothesis was tested by comparing rottlerin and a detergent, DOC (sodium deoxycholate), for their binding to the lipids by using a DMPC (1,2-Dimyristoyl-sn-Glycero-3-Phosphocholine) monolayer technique. DOC has been shown to both increase and inhibit activity of Na+/K+-ATPase in a manner similar to that displayed by rottlerin. The effects of rottlerin and DOC on the DMPC monolayers were studied by measuring the surface pressure of DMPC monolayers and surface area per DMPC molecule. I established that both rottlerin and DOC decreased the surface pressure of DMPC monolayers and increased the surface area per DMPC molecule. This indicates that both rottlerin and DOC penetrated into the DMPC monolayers. If rottlerin can interact with the lipids, changes in fluidity of the lipid membrane cannot be ruled out and should be considered as a possible factor contributing to the effects of rottlerin on the activity of Na+/K+-ATPase. Overall, the study demonstrates that rottlerin is not only a PKC-delta inhibitor but can have additional effects, both on the enzyme activities (Na+/K+-ATPase) and/or on lipid-containing biological structures such as membranes. The findings have implication not only for studies where rottlerin was used as a supposedly specific PKC-delta inhibitor but also for mechanisms of its toxicity.

Doctor of Philosophy (Medicine)
Glutamate transporters play a role in removing extracellular excitatory neurotransmitter, L-glutamate into the cells. The rate of the uptake depends on the density of the transporters at the membrane. Some studies claimed that glutamate transporters could transit between the cytoplasm and the membrane on a time-scale of minutes. The present study examined the distribution of glutamate transporter GLAST predominantly expressed in rat cortical cultured astrocytes between the membrane and the cytoplasm by using deconvolution microscopy and then analyzing the images. The regulation of the distribution of GLAST was studied in the presence of glutamate transporter substrate (D-aspartate), purinergic receptor activators (α,β-methylene ATP, adenosine), neuroleptic drugs (clozapine, haloperidol), ammonia (hyperammonia) and Na+/K+-ATPase inhibitors (ouabain, digoxin and FCCP). It was demonstrated that the translocation of GLAST towards the plasma membrane was induced by D-aspartate, α,β-methylene ATP, adenosine, clozapine and ammonia (at 100 μM and very high concentrations of 10 mM). However, the inhibition of Na+/K+-ATPase activity had an opposite effect, resulting in redistribution of GLAST away from the membrane. It has previously been claimed that the membrane-cytoplasm trafficking of GLAST was regulated by phosphorylation catalysed by protein kinase C delta (PKC-delta). Involvement of this mechanism has, however, been put to doubt when rottlerin, a PKC-delta inhibitor, used to test the hypothesis showed to inhibit Na+/K+-ATPase-mediated uptake of Rb+, suggesting that rottlerin influenced the activity of Na+/K+-ATPase. As Na+/K+-ATPase converts ATP to energy and pumps Na+, K+ ions, thus helping to maintain normal electrochemical and ionic gradients across the cell membrane. Its inhibition also reduced D-aspartate transport and could impact on the cytoplasm-to-membrane traffic of GLAST molecules. Furthermore, rottlerin decreased the activity of Na+/K+-ATPase by acting as a mitochondrial inhibitor. The present study has focused on the inhibition of Na+/K+-ATPase activity by rottlerin, ouabain and digoxin in homogenates prepared from rat kidney and cultured astrocytes. The activity of Na+/K+-ATPase was measured by the absorption of inorganic phosphate product generated from the hydrolysis of ATP and the fluorescent transition of the dye RH421 induced by the movement of Na+/K+-ATPase. This approach has a potential to test whether the rottlerin effect on Na+/K+-ATPase is a direct inhibition of the enzyme activity. Rottlerin has been found to block the activity of Na+/K+-ATPase in a dose-dependent manner in both rat kidney and astrocyte homogenates. Therefore, rottlerin inhibited the activity of Na+/K+-ATPase directly in a cell-free preparation, thus strongly indicating that the effect was direct on the enzyme. In parallel experiments, ouabain and digoxin produced similar inhibitions of Na+/K+-ATPase activity in rat kidney while digoxin blocked the activity of Na+/K+-ATPase to a greater extent than ouabain in rat cortical cultured astrocytes. In a separate set of experiments, Na+/K+-ATPase in the astrocytic membrane was found to be unsaturated in E1(Na+)3 conformation in the presence of Na+ ions and this could explain the differences between the effects of digoxin and ouabain on the activity of Na+/K+-ATPase in rat astrocytes. In addition, it was found that at low concentrations of rottlerin, the activity of Na+/K+-ATPase was increased rather than inhibited. This effect was further investigated by studying rottlerin interactions with membrane lipids. The activity of Na+/K+-ATPase has been reported to be regulated by membrane lipids. The enzyme activity can be enhanced by increasing fluidity of the lipid membrane. I have, therefore, proposed that rottlerin binds to the membrane lipids and the effects of rottlerin on Na+/K+-ATPase are mediated by changes in the properties (fluidity) of the membrane. The hypothesis was tested by comparing rottlerin and a detergent, DOC (sodium deoxycholate), for their binding to the lipids by using a DMPC (1,2-Dimyristoyl-sn-Glycero-3-Phosphocholine) monolayer technique. DOC has been shown to both increase and inhibit activity of Na+/K+-ATPase in a manner similar to that displayed by rottlerin. The effects of rottlerin and DOC on the DMPC monolayers were studied by measuring the surface pressure of DMPC monolayers and surface area per DMPC molecule. I established that both rottlerin and DOC decreased the surface pressure of DMPC monolayers and increased the surface area per DMPC molecule. This indicates that both rottlerin and DOC penetrated into the DMPC monolayers. If rottlerin can interact with the lipids, changes in fluidity of the lipid membrane cannot be ruled out and should be considered as a possible factor contributing to the effects of rottlerin on the activity of Na+/K+-ATPase. Overall, the study demonstrates that rottlerin is not only a PKC-delta inhibitor but can have additional effects, both on the enzyme activities (Na+/K+-ATPase) and/or on lipid-containing biological structures such as membranes. The findings have implication not only for studies where rottlerin was used as a supposedly specific PKC-delta inhibitor but also for mechanisms of its toxicity.

Thesis (Ph.D.)--University of Toledo, 2006.
"In partial fulfillment of the requirements for the degree of Doctor of Philosophy in Biomedical Sciences." Major advisor: Zi-Jian Xie. Includes abstract. Document formatted into pages: iii, 156 p. Title from title page of PDF document. Bibliography: pages 64-67, 97-100, 116-117, 125-155.

Thesis (Ph.D.)--University of Toledo, 2006.
"In partial fulfillment of the requirements for the degree of Doctor of Philosophy in Biomedical Sciences." Major advisor: Zi-Jian Xie. Includes abstract. Title from title page of PDF document. Bibliography: pages 64-70, 104-108, 121-158.

Le rein joue un role central dans le controle de l'homeostasie du milieu interieur. Il permet d'assurer la balance entre les entrees et sorties de differents ions, incluant le sodium (na), le potassium (k) et participe egalement au controle de l'equilibre acido-basique. Deux enzymes membranaires interviennent au premier plan. Il s'agit de la na,k-atpase et de la h,k-atpase renale. La na,k-atpase maintient le gradient electrochimique transmembranaire du na et du k. La presence d'une h,k-atpase specifiquement renale est une decouverte plus recente. Nous avons caracterise au point de vue moleculaire et fonctionnel la na,k-atpase et h,k-atpase exprimees dans la vessie du crapaud bufo marinus. La vessie du crapaud est consideree comme l'equivalent amphibien du canal collecteur cortical de mammifere, segment implique dans l'excretion finale du na et du k dans l'urine. L'utilisation de l'ovocyte comme systeme d'expression et la mise au point de tests fonctionnels adequats ont permis de mieux comprendre le role respectif des differentes sous-unites alpha et beta impliquees dans la formation d'un heterodimere fonctionnel. D'autre part, la caracterisation moleculaire et fonctionnelle de la h,k-atpase renale devrait permettre de mieux comprendre le role de cette enzyme dans le processus d'acidification urinaire ou la reabsorption de k

La Na/K ATPase est impliquée dans la physiopathologie de la neuropathie diabétique. Cette activité enzymatique est basse chez le patient diabétique de type 1, particulièrement chez ceux atteints de neuropathie, et chez certains sujets prédisposés à la neuropathie en cas de diabète. Les facteurs génétiques et environnementaux pouvant influencer l'activité Na/K ATPase ont été analysés. A cet effet, l'érythrocyte a été utilisé comme source enzymatique. Il semble que l'activité de la Na/K ATPase dans le globule rouge reflète celle dans le nerf. En effet, les activités Na/K ATPase nerveuses et érythrocytaires sont étroitement corrélées entre elles et aux vitesses de conduction nerveuses. D'autre part, la même isoforme al de la Na/K ATPase est exprimée dans ces deux types cellulaires. L'activité Na/K ATPase érythrocytaire est diminuée chez les sujets diabétiques de type 1 par rapport aux sujets contrôles et diabétiques de type 2. Cette activité est indépendamment corrélée au taux circulant de peptide C. L'activité Na/K ATPase est abaissée chez les sujets contrôles et diabétiques d'origine nord africaine. Les interventions thérapeutiques montrent que l'insuline et le peptide C, non sécrétés en cas de diabète insulinoprive, stimulent l'activitcNa/K ATPase. Une activité Na/K ATPase basse semble être un marqueur de neuropathie diabétique. Il pourrait exister une prédisposition génétique à développer une neuropathie. C'est pourquoi un polymorphisme de restriction, par l'enzyme Bgl Il, du gène ATPlAl codant pour !'isoforme al de la Na/K ATPase a été recherché. L'allèle clivé [R] du gène ATPlAl est associé à une activité Na/K ATPase abaissée, une diminution d'expression de al ainsi qu'à une prévalence accrue de la neuropathie. Cette influence du fond génétique n'est observable que chez les sujets insulinoprives. Ces faits suggèrent une interaction entre les facteurs génétiques (présence de l'allèle clivé [R]) et les facteurs environnementaux (absence de sécrétion de peptide C).

Mestrado em Biologia Molecular e Celular
O alumínio é o metal mais abundante na crosta terrestre sendo a sua exposição cada vez maior nas sociedades industrializadas. Numa sociedade cada vez mais envelhecida onde o número de casos de doenças neurodegenerativas tendem a aumentar, parece importante esclarecer os mecanismos celulares da neurotoxicidade do alumínio de forma a prevenir os seus efeitos. Estudos in vivo e in vitro indicam que a exposição a alumínio inibe a atividade da (Na+/K+)ATPase, proteína responsável pela manutenção e regulação dos gradientes eletroquímicos das membranas plasmáticas, e que a suscetibilidade à intoxicação por alumínio na dieta pode ser condicionada pelo colesterol, uma vez que, a exposição oral crónica a alumínio reduz a razão molar colesterol/fosfolípidos em sinaptossomas. Assim este trabalho, tem como objetivo esclarecer de que forma os diferentes níveis de colesterol afetam a inibição da (Na+/K+)ATPase sinaptossomal induzida pelo alumínio. Neste contexto foram utilizados como grupos de estudo, o controlo, em que não houve manipulação do teor de colesterol na membrana sinaptossomal; a incorporação, onde o teor de colesterol da membrana sinaptossomal foi aumentado; a depleção, em que o teor de colesterol da membrana sinaptossomal foi reduzido. As alterações do teor de colesterol da membrana dos sinaptossomas foram provocadas usando o composto metil-β-ciclodextrina e a atividade da (Na+/K+)ATPase sinaptossomal medida por colorimetria e por potenciometria. Neste trabalho, houve um aumento de 70% de colesterol sinaptossomal em relação ao controlo, e uma redução do teor de colesterol sinaptossomal em relação ao mesmo de 62%. A exposição in vitro dos sinaptossomas a AlCl3 300 μM inibiu a atividade da (Na+/K+)ATPase nos três grupos (controlo, incorporação e depleção). Nos sinaptossomas controlo onde o teor de colesterol foi de 183 ± 48 μg de colesterol/mg de proteína, a exposição in vitro a AlCl3 300 μM levou a uma inibição da atividade da (Na+/K+)ATPase de 76,39%, enquanto que, na presença de 279 ± 54 μg de colesterol/mg de proteína (incorporação de colesterol) o alumínio teve um efeito inibitório na atividade da (Na+/K+)ATPase de 72,88%. Por outro lado, para baixos níveis de colesterol (57 ± 16 μg de colesterol/mg de proteína) o alumínio teve um efeito inibitório na atividade da (Na+/K+)ATPase de 37,82%. Estes resultados, mostram que alterações do teor de colesterol nas membranas de sinaptossomas desempenham um importante papel na ação do alumínio sobre a (Na+/K+)ATPase. Assim, parece que baixos níveis de colesterol membranar atenuam o efeito inibitório do alumínio sobre a atividade da (Na+/K+)ATPase.
Aluminium is the most abundant metal in the earth's crust and its exposure increasing in industrialized societies. In an increasingly aging society where the number of cases of neurodegenerative diseases is increasing, it seems important to clarify the cellular mechanisms of the neurotoxicity of aluminium to prevent its effects. In vivo and in vitro studies indicate that exposure to aluminium inhibits the activity of (Na+/K+)ATPase, a protein responsible for the maintenance and regulation of the electrochemical gradients of the plasma membranes, and that susceptibility to intoxicaton by aluminium in the diet may be influenced by cholesterol, since chronic oral exposure to aluminium reduces the molar ratio of cholesterol/phospholipid in synaptosomes. Thus this work aims to clarify how the different levels of cholesterol affects the inhibition of synaptosomal (Na+/K+)ATPase induced by aluminium. In this context were used as the study groups, the control in which no manipulation of the content of cholesterol in synaptosomal membrane was made; incorporation, where the cholesterol content was increased synaptosomal membrane; depletion, wherein the cholesterol content of the membrane synaptosomal was reduced. The changes in membrane cholesterol content of synaptosomes were induced using the compound methyl-β-cyclodextrin and the activity of (Na+/K+) ATPase synaptosomal measured by colorimetry and potentiometry. In this study, there was a ~70% increase synaptosomal cholesterol relative to control, and reduced cholesterol content compared to the same synaptosomal ~62%. The in vitro exposure of synaptosomes to 300 μM AlCl3 inhibited the activity of (Na+/K+)ATPase in the three groups (control, incorporation and depletion). In control synaptosomes which the cholesterol content was 183 ± 48 μg cholesterol/mg protein, in vitro exposure to 300 μM AlCl3 led to an inhibition of the activity of (Na+/K+)ATPase of 76.39%, while in the presence of 279 ± 54 μg cholesterol/mg protein (incorporation of cholesterol) aluminium had an inhibitory effect on the activity of (Na+/K+)ATPase of 72.88%. Conversely, lowest cholesterol levels (57 ± 16 μg cholesterol/mg protein) aluminium had an inhibitory effect on the activity of (Na+/K+)ATPase of 37.82%. These results show that changes in cholesterol content of synaptosome membranes play an important role in the action of aluminium on the (Na+/K+)ATPase. Thus, it appears that low levels of membrane cholesterol attenuate the inhibitory effect of aluminium on the activity of (Na+/K+)ATPase.

The Na,K-ATPase activity of membrane-bound sodium pump exhibits non-Michaelis kinetics with respect to ATP. The enzyme consists of promoters (one plus one -subunit) that may be organised into higher oligomers. Each -subunit is believed (although it has not been proven) to posses only one ATP binding site. It is not understood how the low-affinity ATP effect arises. Experiments thus far have not been able to convincingly distinguish between the following possibilities: 1) negative co-operatively between the ATP binding sites of the two halves of a dimeric enzyme, 2) the single ATP binding site of a protomeric enzyme exhibiting variable affinity and function around the reaction cycle, and 3) a second, uncharacterised, ATP binding site on the -subunit. The experiments presented in this thesis investigate how the dual ATP effects are related to the subunit structure of the sodium pump. I solubilise Na,K-ATPase with dodecyl octaethyleneglycol monoether (C12E8). The aggregation state of the C12E8- solubilised enzyme is quantified by analytical ultracentrifugation and found to be predominantly protomeric. These soluble protomers retain dual responses towards ATP as determined from the substrate dependence curve of their Na,K-ATPase activity and their response to non-hydrolysable ATP analogs. Hence, the dual ATP responses are intrinsic to the protomer and do not arise from - interactions within an oligomeric enzyme. Furthermore, protomers that have their high-affinity ATP binding sites irreversibly blocked with fluorescein 5'-isothiocyanate can still bind 2'(3')-O-(2,4,6-trinitrophenyl)ADP indicating that each protomer possesses two nucleotide binding sites.

The Na+, K+ ATPase is an essential membrane protein in eukaryotic cells, which transports Na+ out of the cell in exchange for K+ into the cell. For this transport it hydrolyses one molecule of ATP for each cycle. The partial reactions of the ATPase cycle and the effects of posttranslational modifications on ATPase activity have been studied extensively. However, amalgamation of the reported rate constants for the partial reactions along with the effect of posttranslational modifications have never been attempted. We have designed a simplified four-state mathematical model of the Na+, K+ ATPase using published results for the partial reactions. We have incorporated the effect of the Na+ allosteric site and poise dependent glutathionylation and attempted to replicate K+ activated transient currents reported in voltage clamped cardiomyocytes. Our voltage clamped cardiomyocyte results indicate the K+ activated transient is an effect of poise dependent glutathionylation rather than the Na+ subsarcolemmal space. These results can be replicated to some extent by the proposed kinetic model. This is the first kinetic model of the Na+, K+ ATPase that incorporates both partial rate constants and a reported posttranslational modification which is able to reproduce voltage clamped cardiomyocyte data.

Os crustáceos são originariamente marinhos; ao longo da evolução, diversas espécies invadiram ambientes de salinidades menores, chegando à água doce. A capacidade dos crustáceos colonizarem com sucesso o ambiente dulcícola depende do desenvolvimento de mecanismos eficientes de hiperosmorregulação. A osmolalidade e a composição iônica da hemolinfa de um crustáceo, em meios diluídos, refletem o equilíbrio dinâmico entre a perda de íons por difusão e pela urina e sua reabsorção do meio externo, através das brânquias. A (Na,K)-ATPase branquial desempenha um papel chave no processo de captura de Na+ a partir de ambientes diluídos e suas características cinéticas vem sendo investigadas recentemente, embora as enzimas de caranguejos dulcícolas sejam pouco conhecidas. Segundo o modelo atual, a afinidade por Na+ é o parâmetro cinético mais variável entre as enzimas de diferentes espécies, refletindo a salinidade do habitat do animal, de modo que enzimas de espécies bem adaptadas à água doce apresentam afinidades maiores por Na+. Entretanto, vários resultados conflitantes têm sido relatados nos últimos anos. Recentemente, foi proposto que uma V-ATPase também desempenha papel essencial na captação de Na+ através das brânquias dos crustáceos dulcícolas. Esta enzima ainda é praticamente desconhecida: suas características cinéticas não foram estudadas e a relação entre a magnitude da sua atividade e a salinidade do meio externo não está estabelecida. Este projeto teve por objetivo a caracterização das enzimas (Na,K)-ATPase e V-ATPase das brânquias posteriores do caranguejo hololimnético Dilocarcinus pagei, considerado um antigo invasor da água doce. A (Na,K)-ATPase foi caracterizada em animais mantidos em água doce, a fim de comparar suas propriedades cinéticas com aquelas das enzimas de outras espécies de caranguejos, habitantes de meios mais salinos, visando melhorar o entendimento das adaptações bioquímicas associadas à invasão da água doce. A V-ATPase foi caracterizada em animais mantidos em água doce ou expostos por diferentes intervalos de tempo à salinidade de 21‰ ou ainda aclimatados por 10 dias a diferentes salinidades (5-21‰), visando estabelecer uma relação entre a magnitude da atividade e a salinidade do meio, além de investigar os mecanismos de regulação da atividade da enzima. A análise da fração microsomal branquial de D. pagei mantido em água doce em gradiente contínuo de sacarose mostrou dois picos protéicos (25-35% e 35-45% de sacarose), ambos com atividades K+-fosfatase, (Na,K)-ATPase e V-ATPase. Estes resultados indicam a presença de frações de membrana com densidades distintas, apresentando, em ambos os casos, as principais bombas de íons envolvidas na captação de Na+. Estas membranas podem ser originárias de locais distintos do epitélio branquial posterior assimétrico deste caranguejo. A análise por Western blotting revelou duas bandas imunoespecíficas (Mr 116 kDa e 105 kDa) correspondentes à subunidade α da (Na,K)-ATPase, sugerindo a presença de duas isoformas nas brânquias posteriores do animal. A estimulação da atividade K+-fosfatase da (Na,K)-ATPase pelo PNFF envolveu interações sítio-sítio (nH= 1,4), com V= 43,4 ± 2,2 U mg-1 e K0,5= 1,13 ± 0,06 mmol L-1. A estimulação da atividade da enzima por K+ (V= 39,9 ± 1,9 U mg-1 e K0,5= 4,2 ± 0,2 mmol L-1), Mg2+ (V= 45,0 ± 2,2 U mg-1, K0,5= 0,82 ± 0,04 mmol L-1) e NH4+ (V= 31,7 ± 1,6 U mg-1, K0,5= 19,0 ± 0,9 mmol L-1) também ocorreu por meio de interações sítio-sítio. A afinidade aparente da enzima pelo PNFF e Mg2+ foi similar às relatadas para enzimas de outros crustáceos, incluindo caranguejos habitantes de meios mais salinos. Entretanto, a enzima de D. pagei apresentou menor afinidade aparente por íons K+ que as outras espécies já estudadas. A atividade K+-fosfatase da (Na,K)-ATPase branquial de D. pagei mantido em água doce foi estimulada sinergicamente por K+ e NH4+ sugerindo a presença de dois sítios de ligação para estes íons na molécula da enzima. Ouabaína (4 mmol L-1) inibiu a atividade PNFFase total da preparação (≈ 89%), por meio de uma curva monofásica (KI= 225,6, ± 11,3 µ mol L-1), sugerindo que, se presentes na fração microsomal, as duas isoenzimas da (Na,K)-ATPase apresentam sensibilidades próximas para o inibidor. Ortovanadato (1µmol L-1) inibiu 95% da atividade PNFFase total por meio de uma curva bifásica, reforçando a sugestão da presença de duas isoenzimas na preparação. A hidrólise do ATP pela (Na,K)-ATPase branquial de D. pagei mantido em água doce ocorreu em sítios de alta (V= 6,4 ± 0,32 U mg-1 e K0,5 = 0,34 ± 0,02 µmol L-1) e baixa afinidade (V= 127,1 ± 6,2 U mg-1e KM = 84 ± 4,1 µmol L-1). Não foi encontrada uma correlação direta entre a afinidade pelo ATP e o habitat de diferentes espécies de caranguejos. A atividade (Na,K)-ATPase específica de D. pagei mantido em água doce foi cerca de 3 vezes menor que relatada para Potamon edulis, única espécie de caranguejo dulcícola para a qual este parâmetro foi relatado. Atividades específicas muito maiores foram encontradas para caranguejos estuarinos, particularmente quando aclimatados a salinidades baixas. A baixa atividade específica determinada para D. pagei pode ser atribuída ao baixo gradiente osmoiônico que este animal mantém entre a hemolinfa e o meio externo, comparado a outros caranguejos dulcícolas, que o caracteriza como uma espécie particularmente bem adaptada ao ambiente dulcícola. A estimulação da atividade da enzima por íons Na+ (V = 133,8 ± 7,3 U mg-1e K0,5= 4,7 ± 0,3 mmol L-1), Mg2+ (V= 136,5 ± 8,0 U mg-1, K0,5= 0,62 ± 0,04 mmol L-1), K+ (V = 131,7± 7,9 U mg-1 e K0,5= 0,47 ± 0,03 mmol L-1) e NH4+ (V= 125,6 ± 6,3 U mg-1, K0,5= 1,90 ± 0,09 mmol L-1) ocorreu por meio de interações sítio-sítio. A afinidade aparente por Na+ da enzima de D. pagei é baixa, se comparada às relatadas para outros animais dulcícolas, e similar às encontradas para espécies estuarino/marinhas. Em contraste, a afinidade aparente por K+ é 2,5 a 5 vezes maior que as determinadas para espécies habitantes de meios mais salinos e aparentemente está mais relacionada ao habitat do animal que a afinidade por Na+. Esta possibilidade é coerente com o fato da (Na,K)-ATPase branquial dos crustáceos apresentar os sítios de ligação de K+ expostos para a hemolinfa, o que possibilita a modulação da atividade da enzima pela concentração de K+ na hemolinfa. Ao contrário do observado para várias outras espécies de caranguejos, a atividade (Na,K)-ATPase branquial de D. pagei não foi estimulada sinergisticamente por K+ e NH4+. Entretanto, a presença de um dos íons no meio reacional provoca o aumento da afinidade aparente da enzima pelo outro em cerca de 3 vezes. Fisiologicamente, esta característica cinética pode ser importante para garantir o transporte de ambos os íons pela enzima, mesmo em presença de concentrações relativamente elevadas do outro. Ouabaína (3 mmol L-1) inibiu a atividade ATPase total (≈ 78%) por meio de uma curva bifásica (KI= 6,21 ± 0,32 µmol L-1 e 101,2 ± 5,1 µmol L-1), reforçando os resultados anteriores no sentido de demonstrar a existência de duas isoenzimas da (Na,K)-ATPase nas brânquias posteriores de D. pagei. Observou-se também uma inibição bifásica por ortovanadato (10 µmol L-1), que inibiu a atividade ATPase total em 85%. O pH ótimo para a atividade V-ATPase branquial de D. pagei foi de 7,5. A modulação da atividade V-ATPase do animal mantido em água doce por ATP (V= 26,5 ± 1,3 U mg-1; K0,5= 3,9 ± 0,2 mmol L-1) e Mg2+ (V = 27,9 ± 1,4 U mg-1; K0,5 =0,80 ± 0,04 mmol L-1) ocorreu por meio de interações cooperativas. Já a inibição da atividade ATPase insensível ao ortovanadato por bafilomicina A1 ocorreu segundo uma curva monofásica (KI= 55,0 ± 2,8 nmol L-1). Cerca de 44 % da atividade ATPase total foi inibida, correspondendo à V-ATPase. A atividade V-ATPase branquial de D. pagei diminuiu acentuadamente em resposta à exposição à salinidade de 21‰. Após 1h de exposição, a atividade diminuiu cerca de 3 vezes, chegando a 4 vezes após 24h, o que indica a atuação de mecanismos eficientes de regulação a curto prazo. Curiosamente, a atividade V-ATPase foi cerca de 2 vezes maior para um tempo de aclimatação de 120h a 21‰, comparado a 24 h, embora 2 vezes menor que a estimada em água doce. Passadas 240 h, a atividade voltou aos baixos níveis observados entre 1h e 24h, o que indica a ação de mecanismos de regulação a longo prazo. Além da diminuição da atividade específica também foi observado aumento da afinidade da enzima por ATP (12 vezes) e Mg2+ (3 vezes) em resposta à exposição dos animais a 21‰. Similarmente, ocorreu um aumento de até 190 vezes na afinidade da enzima por bafilomicina A1. Propõe-se que, em resposta à alteração de salinidade, ocorrem mudanças conformacionais tanto em V1 (onde se encontram os sítios de ligação de ATP e Mg2+) quanto V0 (onde se localiza o sítio de ligação de bafilomicina), resultando numa maior exposição do sítio para o inibidor e no aumento da afinidade por Mg2+ e ATP. Como os aumentos de afinidade são observados já após 1h de exposição, este mecanismo parece ser independente da expressão protéica e, portanto, não estaria relacionado à expressão de isoformas diferentes de alguma das subunidades da enzima. A diminuição da atividade V-ATPase branquial de D. pagei em resposta à exposição a uma salinidade elevada é compatível com os mecanismos propostos para a atuação desta enzima no processo de captura ativa de Na+ em crustáceos dulcícolas. Após 10 dias de aclimatação ainda se tem atividade V-ATPase detectável nas frações microsomais das brânquias posteriores do animal, possivelmente envolvida nas funções de regulação ácido-base e excreção de amônia. Os resultados obtidos para a aclimatação de D. pagei por um período de 10 dias a salinidades entre 5 e 21‰ mostraram também uma diminuição acentuada da atividade V-ATPase em resposta ao aumento da salinidade. Entretanto, com exceção da salinidade mais baixa (5‰) não se observou aumento da afinidade da enzima por bafilomicina, sugerindo que esta alteração seja limitada a tempos de aclimatação mais curtos. Entretanto, também se verificou um aumento acentuado da afinidade da enzima por ATP e Mg2+.
Crustacean arose in the sea but, during evolution, several species invaded lower salinity biotopes, reaching fresh water. The ability of crustaceans to successfully colonize the freshwater biotope depends on efficient mechanisms of hyperosmoregulation. In dilute media, crustaceans\' hemolymph osmolality and ionic composition reflect a balance between diffusive and urinary ion losses, and active ion capture through the gills. The gill (Na,K)- ATPase plays a pivotal role in Na+ capture from dilute environments and its kinetic characteristics are under investigation in recent years, although freshwater crab enzymes are poorly known. According to the most recent model, the apparent affinity for Na+ is the most variable kinetic parameter among gill enzymes from different species, and reflects the salinity of the species\' habitat. Thus, enzymes from species which are well adapted to freshwater usually present higher affinities for Na+. However, several recent results are incompatible with this model. On the other hand, it has been proposed that a V-ATPase is also involved in Na+ capture through the gills of hololimnetic crustaceans. This enzyme is almost completely unknown: its kinetic characteristics have not been studied yet and the relationship between the magnitude of its activity in the gills and the external medium salinity has not been established. This work aimed to characterize the (Na,K)-ATPase and V-ATPase from the posterior gill from the holimnetic crab Dilocarcinus pagei, considered an old fresh water colonizer. The (Na,K)- ATPase was characterized in animals maintained in fresh water, in order to establish a comparison of its kinetic properties with those of enzymes from other crab species that inhabit more saline media. This comparison may enhance our understanding of the biochemical adaptations associated to fresh water invasion. V-ATPase was characterized in animals kept in fresh water or exposed for varying time intervals to a medium of 21? salinity, or else acclimated for 10 days to media of different salinities (5-21?), aiming to establish a relationship between the enzyme specific activity in the gill tissue and the external salinity, and also investigate the mechanisms involved in enzyme activity regulation. The analysis of D. pagei gill microsomes in a continuous-density sucrose gradient revealed two protein peaks (25-35% and 35-45% sucrose), both showing K+-phosphatase, (Na,K)-ATPase and V-ATPase activities. These results indicate the presence of membrane fractions of distinct densities, both presenting the main ion pumps involved in Na+ capture. These membranes may originate from different places in the asymmetric posterior gill epithelium from this crab. Western compared to those reported for other freshwater animals, but similar to those found for estuarine/marine species. In contrast, the apparent affinity for K+ is 2.5 to 5-fold higher than those estimated for species that inhabit more saline media, and is apparently more related to the animals\' habitat than Na+ affinity. This possibility is consistent with the location of the (Na,K)-ATPase in crabs gill tissue, with K+ binding sites exposed to the hemolymph, allowing the direct modulation of enzyme activity by hemolymph K+ concentration. In contrast to data reported for other crab species, D. pagei gill (Na,K)-ATPase activity was not synergistically stimulated by K+ and NH4 +. However, the presence of one of these ions in the reaction medium results in an increase of about 3-fold in the apparent affinity of the enzyme for the other. This kinetic characteristic may be physiologically relevant to assure the transport of both ions, even in the presence of elevated concentrations of the other. Ouabain (3 mmol L-1) inhibited total ATPase activity (? 78%) through a biphasic curve (KI= 6.21 ± 0.32 mol L-1 and 101.2 ± 5.1 mol L-1) reinforcing previous results suggesting the presence of two isoenzymes in the microsomal preparations. A biphasic inhibition by orthovanadate (10 mol L-1) to about 15% residual activity was also observed. Optimal pH for D. pagei gill V-ATPase activity was 7.5. The modulation of enzyme activity of the animal kept in fresh water by ATP (V= 26.5 ± 1.3 U mg-1; K0.5= 3.9 ± 0.2 mmol L-1) and Mg2+ (V = 27.9 ± 1.4 U mg-1; K0.5 =0.80 ± 0.04 mmol L-1) occurred with positive cooperativity. The inhibition of the orthovanadate insensitive ATPase activity by bafilomycin A1 followed a monophasic curve (KI= 55.0 ± 2.8 nmol L-1). About 44 % of total ATPase activity was inhibited, corresponding to the V-ATPase. Dilocarcinus pagei gill V-ATPase activity substantially decreased in response to animal\'s exposure to 21? salinity. After 1h exposure, the activity diminished about 3-fold, reaching 4- fold after 24h, indicating the action of efficient short-time regulation mechanisms. Interestingly, V-ATPase activity was about 2-fold higher after 120h exposure, compared to 24h, although 2- fold lower compared to that estimated in fresh water. After 240h, the activity returned to the low levels observed for 1 and 24 h, indicating efficient long-term regulation. Besides the decrease in specific activity, it was also observed an increase in enzyme\'s apparent affinity for ATP (12 fold) and Mg2+ (3 fold) in response to animal\'s exposure to 21? salinity. Simultaneously, the enzyme\'s affinity for bafilomycin A1 increased up to 190-fold. We propose that, in response to salinity alteration, conformational changes take place both in V1 (in which the ATP and Mg2+ binding sites are located) and V0 (which contains the bafilomycin A1 bindind site), resulting in higher exposition of the inhibitor binding site and also higher affinity for Mg2+ and ATP. As the affinity increases are observed after just 1h exposure, this regulatory mechanism seems to be independent of protein expression and, thus, should not be related to the expression of distinct isoforms of some enzyme subunit. The lowering of gill V-ATPase activity in D. pagei in response to exposure to an elevated salinity is consistent with the mechanisms proposed for the role of this enzyme in active Na+ capture in hololimnetic crustaceans. After 10 days at 21, the gill microsomal fractions still show a little V-ATPase activity, possibly related to acid-base regulation and ammonia excretion processes. The results obtained for the acclimation of D. pagei for 10 days at salinities in the range 5 to 21? also showed a substantial decrease of V-ATPase activity in response to the increase in medium salinity. However, except for 5?, it was not observed an increase of enzyme\'s affinity for bafilomycin, suggesting that this alteration is limited to shorter periods of exposure. However, a significant increase in the enzyme\'s affinity for ATP and Mg2+ was also observed.

The Na/K pumps are essential for living system and widely expressed in all eukaryotic cell membranes. By actively transporting sodium ions out of and potassium ions into the plasma membrane, Na/K pumps creates both an electrical and a chemical gradient across the plasma membrane, which are crucial for maintaining membrane potential, cell volume, and secondary active transporting of other solutes, etc. Previously, oscillating electric field with a frequency close to the mean physiological turnover rate was used to synchronize and modulate the Na/K pump molecules. Results showed that the turnover rate of Na/K pumps can be accelerated by folds. However, this what we called first generation synchronization modulation (SM) technique can only synchronize sodium and potassium translocations into their corresponding half cycles. The detailed location of each sodium extrusion and potassium intrusion can not be determined. As a result, the synchronized pumps were uniformly distributed, generating steady-state macroscopic currents. Based on these studies, Dr.Chen developed a new generation synchronization modulation technique. The waveform of original SM by adding an overshoot pulse at the end of each half cycle. This overshoot pulse has a function of energy barrier which will force all of the Na/K pumps into the same state in the pumping cycle until the membrane polarity change. As a result, Na/K pump molecules are not only synchronized into half cycles of oscillating electric field, but individual steps of the pumping cycle. Accordingly, transient pump currents or so called 'pre-steady state' pump currents are generated, from which some detailed information abut the mechanism of Na/K pumps can be dissected. In this dissertation, we firstly characterized the synchronized pump currents by modified SM. The results showed that transient currents were induced at the beginning of each half cycle as expected. The ratio between positive and negative transient currents was close to 3:2, stoichiometric number of Na/K pump. Moreover, the transient currents were significantly reduced in the presence of ouabain in a time dependent manner. In addition, by gradually increasing the frequency of SM electric field in a step-wise fashion, the synchronized pump current can be modulated to the corresponding level. Next,we utilized this technique to study some detailed mechanisms of Na/K pump, including single channel configuration in transmembrane domain and extracellular D2O effect on the turnover rate. Lastly, we extended our study to applications of this new technique and found that the modified Synchronization Modulation technique can significantly hyperpolarize the membrane potential of skeletal muscle fiber in both physiological and high potasssium conditions. During intensive exercise, the interstitial potassium ions are accumulated and temporarily reach a high level, which will attenuate the contraction force and induce muscle fatigue. Na/K pumps are crucial in the maintenance of skeletal muscle excitability and contractility by restoring the Na and K concentration gradients. By accelerating the turnover rate of Na/K pumps, SM can efficiently re-establish the membrane potential and enhance skeletal muscle contractivity, which unleashes its potential in improving certain pathological conditions, such as exercise-induced hyperkalemia.