Thesis (Ph. D.)--University of Texas at Austin, 2003. / Vita. Includes bibliographical references. Available also from UMI Company.

This study describes the characterisation and identification of two species of sulphate-reducing bacteria isolated from marine environments. The isolate coded Ind 1 was recovered from the heavily corroded hull of an oil storage vessel moored off the Indonesian coast. An isolate, referred to as Al 1, originated from a soured oil reservoir in Alaska. Observations using microscopy (light, scanning electron and atomic force) revealed that cells were Gram-negative, rod-shaped and very motile. Physiological characterisation, analysis of the fatty acid profiles and partial and full 16S rRNA sequencing demonstrated strong similarities between the two species and members of the Desulfovibrio genus. The position of the strains within phylogenetic trees showed Al 1 clustering closely with Desulfovibrio vietnamensis. Ind 1 revealed a high degree of similarity with both Desulfovibrio gigas and Desulfovibrio gabonensis and these three strains formed a separate cluster in the delta subdivision of the Proteobacteria. However, whole-cell protein profiles and Fourier-transform infrared spectroscopy studies showed that there is enough dissimilarity between the two isolates and the remaining species of the genus Desulfovibrio to consider Al 1 and Ind 1 as new separate species. Purification, physico-chemical and spectroscopic characterisation of the key enzymes involved in the sulphate metabolism was carried out for both isolates. Nuclear magnetic resonance and electron paramagnetic resonance studies revealed that the proteins of Al 1 and Ind 1 exhibited various features in common with their counterparts from other members of the genus Desulfovibrio. In particular, proteins from Ind 1 showed many similarities with the enzymes previously described for D. gigas. Based on the obtained results, the classification of Ind 1 as Desulfovibrio indonensiensis sp. nov. and Al 1 as Desulfovibrio alaskensis sp. nov. are proposed. The overall results highlight the complexity of the relationship between cell physiology and the organisms' environmental impact.

Diss. (sammanfattning) Uppsala : Sveriges lantbruksuniv., 2006. / Härtill 4 uppsatser.

It has been observed that thermophilic enzymes are often more sluggish at lower temperatures but comparable active as their mesophilic homologues at their corresponding living temperatures. Although these thermophilic enzymes exhibit high structural stability, the increased stability leads to a decreased flexibility of the thermophilic enzymes in return. To yield further advances in analysis of the interrelationships between flexibility and activity of enzymes, also the molecular basis of enzyme adaptation, we used a pair of thermo-meso acylphosphatase homologues with high level of similarity isolated from hyperthermophilic archeaon Pyrococcus horikoshii (PhAcP) and human (HuAcP) as model to study this issue. Despite the fact that their active-site residues are highly conserved, activity (kcat) of PhAcP is remarkably reduced compared with HuAcP at low temperatures. Based on crystal structure comparison, an extra salt bridge was formed between active site residue and C-terminus of PhAcP. To examine the role of salt bridge plays in catalytic reaction of AcPs, we designed a mutant PhG91A to disrupt the salt bridge in thermophilic PhAcP. In parallel, a salt bridge was re-engineered into mesophilic HuAcP to create HuA99K. Interestingly, the thermophilic variant PhG91A exhibited a more mesophilic-like manner in terms of activity and thermodynamic parameters. On the contrary, mesophilic HuA99K displayed a more thermophilic-like character. This is supplemented by detailed molecular dynamics (MD) simulations, revealing good qualitative agreement with experimental findings. Both theory and experiment results had provided evidences that the presence of a specific salt bridge is directly associated with the temperature adaptation of AcPs by reducing the catalytic site flexibility. / Lam, Yan. / Adviser: K. B. Wong. / Source: Dissertation Abstracts International, Volume: 70-06, Section: B, page: 3364. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2008. / Includes bibliographical references (leaves 120-127). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts in English and Chinese. / School code: 1307.

Thesis (Ph. D.)--Carleton University, 1997. / Also available in electronic format on the Internet.

ATP-difosfohidrolases, comumente denominadas de apirases, descritas pela primeira vez à cerca de quarenta anos atrás, são enzimas que hidrolisam nucleosídeos di ou trifosfatados por um mecanismo que parece ser sequencial, mas sem a formação de um intermediário energético, e de funções metabólicas ainda desconhecidas. Elas tem sido encontradas nas células dos mais diversos pró- e eucariotos investigados e também em tumores. A vasta abundância dessas enzimas e a sua alta atividade enzimática sugerem que as mesmas estariam envolvidas em importantes funções do metabolismo celular. No presente trabalho, identificamos e caracterizamos a presença de uma nova enzima em Trypanosoma cruzi, o agente causador da doença de Chagas, uma apirase, com características semelliantes à outras apirases já descritas em diversos organismos. Diversos ensaios enzimáticos foram feitos mostrando uma atividade enzimática que não foi anulada pelos inibidores clássicos de ATPases tipo-P, tipo-F ou tipo-V. Esta enzima mostrou depende~n·cla para Mg2+, na~o acontecendo o mesmo para Ca2+. Experimentos usando soros imunes contra apirases de outras fontes, reveleram não apenas reação cruzada, mas também sugeriram que a massa molecular dessa enzima no T cruzi, estaria em tomo de 57,5 kDa. Ensaios citoquímicos (imunoflorescência) com o soro imune anti- NTPase de Toxoxplasma gondii, apresentou fluorescência contra diferentes estágios desse protozoa. No entanto, a localização subcelular dessa enzima no T cruzi não ficou muito clara, tomando-se necessário uma microscopia eletrônica para resolver esse problema. Um dado interessante e particular nesse traballio, foi a presença dessa enzima nos diversos estágios do ciclo de vida do parasita, com atividades enzimáticas diferentes para esses mesmos estágios. / ATP-diphosphohydrolases which are generally called apyrases were described for the first time some forty years ago. These are enzymes which hydrolyze di- and triphosphate nucleosides through a mechanism which seems to be sequential, without forming a high-energy enzyme intermediate. Their metabolic function is yet unknown. ATP-diphosphohydrolases have been found in a number of pro- and eucariotic cells and also in tumor cells. The vast abundance ofthese enzymes and their high enzymatic activity suggest that they are involved in important functions in the cell metabolism. In the present work we have identified and characterized the presence of a new enzyme in Trypanosoma cruzi, the agent of Chagas disease, namely an apyrase with characteristics which are similar to other apyrases already described in different organisms. A number of enzymatic assays showed that the enzyme is not inhibited by the classical inhibitors of P-type, F-type or V-type ATPases. The enzyme exhibited a marked dependence on Mg2+ and not on Ca2+. Experiments using immune sera against apyrases of other sources showed cross- reactivity and suggested that the molecular mass of the enzyme in cruzi is around 57.5 kDa. Immunofluorescence cytochemistry using antiToxoplasma gondii NTPase antibodies showed that fluorescence was present in different stages of the protozoa. However, the exact sub-cellular localization of the enzyme in T cruzi was not clear and electron microscopy will be necessary to answer this question. An interesting result of our work is the identification of the enzyme in different stages of the life cycle of the parasite, with different enzymatic activities among the stages.

The main aim of this thesis was to examine the hypothesis that the A/J strain variant of H55N substitution affects citrate synthase (CS) enzyme activity and metabolic health in mice fed a high fat diet (HFD). C57BL/6J (B6) mice and congenic B6.A-(rs3676616-D10Utsw1)/KjnB6 (B6.A) mice, a strain which carries the A/J allele of Cs on the B6 strain background, were fed a HFD (45% kcal from fat) for 12 weeks. CS activity, but not that of ß-hydroxyacyl-coenzyme dehydrogenase was lower in the gastrocnemius muscle of B6.A mice compared to B6 mice (P< 0.001). During HFD feeding the glucose tolerance of mice decreased progressively and to a greater extent in B6.A females compared to B6 females, with males showing a similar trend. Interestingly, after 12 weeks of HFD feeding only B6.A males showed increases (P< 0.05) in their resting metabolic rate; moreover; core body temperature were also increased (P< 0.05) for congenic B6.A of both sexes by the end of the study. However, body weight and fat gain did not differ between B6.A and B6 mice. The second aim of the thesis was to test the hypothesis that low CS activity promotes palmitate-induced lipotoxicity in muscle cells. After 18 hours of incubation in 0.8 mM palmitate, C2C12 muscle cells with a ~50% reduction in CS activity showed low (P< 0.001) viability, increased (P< 0.001) levels of cleaved Caspase-3, high levels of AMP-activated protein kinase and acetyl-CoA carboxylase phosphorylation (P< 0.05), low levels of protein kinase B phosphorylation, high mitogen-activated protein kinases activation (P< 0.001) compared to the control shRNA cells. This was coupled with higher levels of mitochondrial proteins (P< 0.05), which are involved in oxidative phosphorylation. C2C12 cells with reduced CS activity also showed high reactive oxygen species production (P< 0.05), low intracellular ATP levels (P< 0.05), and lower basal mitochondrial respiration (P< 0.001). In summary, the A/J strain variant of H55N is associated with low CS enzyme activity and impaired metabolic health when fed HFD. Palmitate has a lipotoxic effect on Cs shRNA transfected cells and can lead to cell death.

The enzymatic reduction of a high-valence form of metals to a low-valence reduced form has been proposed as a strategy to treat water contaminated with a range of metals and radionuclides. Metal reduction by sulphate reducing bacteria (SRB) is carried out either chemically (involving reduction by hydrogen sulphide) or enzymatically (involving redox enzymes such as the hydrogenases). While reduction of metal ions by hydrogen sulphide is well known, the enzymatic mechanism for metal reduction is poorly understood. The aims of this study were to investigate the role of SRB in facilitating platinum removal, and to investigate the role of a hydrogenase in platinum reduction in vitro. In order to avoid precipitation of platinum as platinum sulphide, a resting (non-growing) mixed SRB culture was used. The maximum initial concentration of platinum (IV), which SRB can effectively remove from solution was shown to be 50 mg.l⁻¹. Electron donor studies showed high platinum (IV) uptake in the presence of hydrogen, suggesting that platinum (IV) uptake from solution by SRB requires careful optimization with respect to the correct electron donor. Transmission electron microscopy (TEM) and energy dispersive X-ray (EDX) analysis indicated that platinum was being precipitated in the periplasm, a major area of hydrogenase activity in SRB. Purification of the hydrogenase by ammonium sulphate precipitation (65%), Toyopearl-Super Q 650S ion exchange and Sephacry 1 S-100 size exclusion chromatography revealed that the hydrogenase was monomeric with a molecular weight of 58 KDa, when analyzed by 12% SDS-PAGE. The purified hydrogenase showed optimal temperature and pH at 35°C and 7.5 respectively, and a poor thermal stability. In vitro investigation of platinum reduction by purified hydrogenase from mixed SRB culture showed that hydrogenase reduces platinum only in the presence of hydrogen. Major platinum (IV) reduction was observed when hydrogenase was incubated with cytochrome C₃ (physiological electron carrier in vivo) under hydrogen. The same observations were also noted with industrial effluent. Collectively these findings suggest that in vitro platinum reduction is mediated by hydrogenase with a concerted action of cytochrome C₃ required to shuttle the electron from hydrogenase.

ATP-difosfohidrolases, comumente denominadas de apirases, descritas pela primeira vez à cerca de quarenta anos atrás, são enzimas que hidrolisam nucleosídeos di ou trifosfatados por um mecanismo que parece ser sequencial, mas sem a formação de um intermediário energético, e de funções metabólicas ainda desconhecidas. Elas tem sido encontradas nas células dos mais diversos pró- e eucariotos investigados e também em tumores. A vasta abundância dessas enzimas e a sua alta atividade enzimática sugerem que as mesmas estariam envolvidas em importantes funções do metabolismo celular. No presente trabalho, identificamos e caracterizamos a presença de uma nova enzima em Trypanosoma cruzi, o agente causador da doença de Chagas, uma apirase, com características semelliantes à outras apirases já descritas em diversos organismos. Diversos ensaios enzimáticos foram feitos mostrando uma atividade enzimática que não foi anulada pelos inibidores clássicos de ATPases tipo-P, tipo-F ou tipo-V. Esta enzima mostrou depende~n·cla para Mg2+, na~o acontecendo o mesmo para Ca2+. Experimentos usando soros imunes contra apirases de outras fontes, reveleram não apenas reação cruzada, mas também sugeriram que a massa molecular dessa enzima no T cruzi, estaria em tomo de 57,5 kDa. Ensaios citoquímicos (imunoflorescência) com o soro imune anti- NTPase de Toxoxplasma gondii, apresentou fluorescência contra diferentes estágios desse protozoa. No entanto, a localização subcelular dessa enzima no T cruzi não ficou muito clara, tomando-se necessário uma microscopia eletrônica para resolver esse problema. Um dado interessante e particular nesse traballio, foi a presença dessa enzima nos diversos estágios do ciclo de vida do parasita, com atividades enzimáticas diferentes para esses mesmos estágios. / ATP-diphosphohydrolases which are generally called apyrases were described for the first time some forty years ago. These are enzymes which hydrolyze di- and triphosphate nucleosides through a mechanism which seems to be sequential, without forming a high-energy enzyme intermediate. Their metabolic function is yet unknown. ATP-diphosphohydrolases have been found in a number of pro- and eucariotic cells and also in tumor cells. The vast abundance ofthese enzymes and their high enzymatic activity suggest that they are involved in important functions in the cell metabolism. In the present work we have identified and characterized the presence of a new enzyme in Trypanosoma cruzi, the agent of Chagas disease, namely an apyrase with characteristics which are similar to other apyrases already described in different organisms. A number of enzymatic assays showed that the enzyme is not inhibited by the classical inhibitors of P-type, F-type or V-type ATPases. The enzyme exhibited a marked dependence on Mg2+ and not on Ca2+. Experiments using immune sera against apyrases of other sources showed cross- reactivity and suggested that the molecular mass of the enzyme in cruzi is around 57.5 kDa. Immunofluorescence cytochemistry using antiToxoplasma gondii NTPase antibodies showed that fluorescence was present in different stages of the protozoa. However, the exact sub-cellular localization of the enzyme in T cruzi was not clear and electron microscopy will be necessary to answer this question. An interesting result of our work is the identification of the enzyme in different stages of the life cycle of the parasite, with different enzymatic activities among the stages.

A alga marinha Gracilaria tenuistipitata var. liui Zhang et Xia (Gracilariales, Rhodophyta) tem grande importância econômica por ser a principal fonte para a produção de ficocolóides. Esta linhagem tem sido extensivamente cultivada em tanques ou diretamente no mar na China e Taiwan por exibir grande tolerância a variação de fatores ambientais. A maior fonte de nitrogênio no oceano está na forma de nitrato que é o principal fator limitante no crescimento das macroalgas. A assimilação de nitrogênio é um processo que ocorre em duas etapas catalisadas sequencialmente pelas enzimas nitrato redutase (NR) e nitrito redutase (NiR), respectivamente. A NR catalisa a redução de nitrato a nitrito usando NAD(P)H como fonte doadora de elétrons. O nitrito é reduzido a amônio (pela NiR) e é imediatamente assimilado em compostos orgânicos nitrogenados, como aminoácidos e bases nitrogenadas. A NR é considerada o primeiro passo regulador no processo de assimilação de nitrato. Neste trabalho descrevemos a oscilação circadiana da atividade da NR bem como do seu conteúdo protéico em G. tenuistipitata. A atividade da NR e os níveis da proteína NR apresentam um pico no meio do dia em algas mantidas sob regime de claro:escuro (12:12h). A atividade de NR é 30 vezes maior durante o período de claro em algas crescendo sob condições de luz:escuro, e o nível protéico de NR é cerca de 40 vezes maior em extratos do período de claro. A oscilação observada está sob controle do relógio biológico uma vez que a flutuação da atividade da NR persiste após 10 dias sob condições de claro constante. Nenhuma oscilação na atividade de NR foi encontrada quando a alga foi mantida sob escuro constante, demonstrando que a luz é um fator de grande importância no controle desta enzima. Experimentos de absorção de nitrato do meio realizados em culturas de algas crescendo sob condições de claro:escuro mostrou que a absorção de nitrato ocorre preferencialmente no período noturno, ocorrendo o pico máximo de absorção no meio da noite. Daí concluirmos que os processos de assimilação de nitrato e tomada do mesmo do meio estejam ocorrendo de maneira independente. A NR de G. tenuistipitata pertence a classe das NRs específicas para o NADH, com constante de Michaelis-Menten aparente de 95 &#181;M. O KM aparente para o nitrato foi estimado em 197 &#181;M, valor esse razoável com os descritos para outras algas. A focalização isoelétrica revelou que a NR é uma proteína básica com pI de 8,66. A NR desta espécie foi purificada em 4 etapas: cromatografia de troca iônica, precipitação com sulfato de amônio, filtração em gel e cromatografia de afinidade em resina Affigel-blue. A proteína não desnaturada apresenta massa molecular de aproximadamente 420 kDa, e 4 subunidades idênticas de 110 kDa, baseado em SDS-PAGE. A localização intracelular da NR de G. tenuistipitata foi examinada utilizando técnicas de imunofluorescência e \"immuno-gold\" e revelaram que a NR está associada com os cloroplastos. / The marine red alga Gracilaria tenuistipitata var. liui Zhang et Xia) is economically important, being the main source for the production of phycocolloids. This strain is extensively cultivated in ponds in southern China and Taiwan and exhibits a wide tolerance to environmental factors. The major source of nitrogen in the ocean is in the form of nitrate being the main limiting macroalgal production. The nitrogen assimilation process occurs in a two-step reaction catalyzed by 2 enzymes working sequentially, nitrate reductase (NR) and nitrite reductase (NiR). NR catalyzes the reduction of nitrate to nitrite using NAD(P)H as electron donor. Nitrite is reduced to ammonium (by NiR) and it is immediately assimilated in organic nitrogen compounds, such as amino acids and bases. NR is considered the first and regulatory step in this assimilating process. We report the circadian oscillation of NR activity as well as NR protein content in G. tenuistipitata. Both NR activity and NR protein levels peaks at midday in algae maintained under light:dark cycle (12:12h). Toe NR activity is 30 fold higher in light period for alga growing under light-dark conditions, and the NR protein level is about 40 fold higher in extracts from the light period. The oscillation observed is under biological clock control since the fluctuation of NR activity persists after 10 days under constant light conditions. No oscillation in the NR activity is found when the algae were maintained under constant dark condition. The experiments on nitrate uptake carried out in algae growing under light:dark conditions had shown that the absorption peaks at the middle of the dark periods. So we can conclude that the process of nitrate assimilation and nitrate uptake is occurring in such an independent ways. NR from G. tenuistipitata is a NADH specific enzyme with Michaelis-Menten apparent constant of 95 &#181;M. Toe apparent KM for nitrate was estimated to be 197 &#181;M, which is reasonable with the values described to another algae. The isoeletric focusing revealed NR is a basic protein with pi of 8.66. The NR from was purified in four steps: ion exchange chromatography, ammonium sulfate precipitation, gel filtration chromatography, and Affigel-blue affinity column. Non-denaturated protein shows a molecular mass of about 420 kDa, and 4 identical subunits of 110 kDa, based on SDS-PAGE. Toe intracellular localization of NR in G. tenuistipitata was examined by applying both immunofluorescence and immunogold methods revealing NR associated with chloroplasts.