Le cuivre est un métal de transition essentiel pour le fonctionnement des organismes vivants. Chez la plante Arabidopsis thaliana, la moitié du contenu en cuivre est localisé dans le chloroplaste. Cet organite, spécifique des cellules végétales, est constitué d'une enveloppe délimitant le stroma, un compartiment aqueux au sein duquel se trouve un système membranaire complexe, les thylacoïdes. Dans les chloroplastes d'Arabidopsis, le cuivre est le cofacteur de deux protéines essentielles : la superoxyde dismutase Cu/Zn, impliquée dans la défense contre des espèces réactives de l'oxygène au niveau du stroma et la plastocyanine, une protéine du lumen des thylacoïdes, impliquée dans la chaine de transfert des électrons photosynthétiques. Des études de génétique inverse ont démontré que le transport du cuivre à la plastocyanine impliquait deux protéines membranaires appartenant à la famille des ATPases-PIB-1 : HMA6, localisée dans l'enveloppe et HMA8, localisée dans la membrane des thylacoïdes. Une étude fonctionnelle in vitro a montré que HMA6 était un transporteur de haute affinité de cuivre monovalent présentant les caractéristiques générales des ATPases-P. Afin de comparer les propriétés enzymatiques de ces deux ATPases-PIB-1 et de mieux comprendre leur rôle respectif dans l'homéostasie du cuivre au sein du chloroplaste, nous avons déterminé in vitro les propriétés enzymatiques de HMA8.La stratégie employée pour la caractérisation de HMA8 a été similaire à celle utilisée pour la caractérisation de HMA6. Dans un premier temps, la sélectivité ionique de HMA8 a été évaluée à l'aide de tests phénotypiques dans la levure Saccharomyces cerevisiae. Les propriétés enzymatiques de HMA8 ont ensuite été déterminées in vitro après expression dans la bactérie Lactoccocus lactis, par des expériences de phosphorylation par l'ATP. Cette analyse a permis de démontrer que HMA8 présentait une plus forte affinité apparente pour le cuivre mais une activité catalytique plus lente que HMA6. L'analyse de modèles tridimensionnels de HMA6 et HMA8 a montré que ces différences pourraient être expliquées par des différences de charges au niveau de la cavité où le métal est libéré et/ou par la nature des partenaires interagissant avec ces ATPases. Ces différences pourraient expliquer les fonctions distinctes de ces deux transporteurs dans le chloroplaste : HMA6 régulerait la concentration en cuivre dans le stroma en interagissant avec différentes protéines cibles (notamment des chaperonnes à cuivre), alors que HMA8 aurait un rôle plus précis pour la distribution du cuivre à la plastocyanine.Pour mieux comprendre le mécanisme de libération du cuivre par HMA6 et HMA8, nous avons effectué une étude fonctionnelle de mutants de la région reliant les deux premières hélices transmembranaires (TMA et TMB). Dans cette étude, nous avons ciblé les cystéines et histidines qui de par leurs propriétés chimiques sont les résidus les plus à même d'interagir avec le métal. Les mutants d'intérêts ont été sélectionnés par criblage phénotypique dans la levure puis exprimés dans la bactérie L. lactis. La caractérisation biochimique in vitro de leurs propriétés enzymatiques a été réalisée par des tests de phosphorylation par l'ATP et le Pi. Cette étude nous a permis d'identifier deux résidus, une cystéine et une histidine, impliqués la libération du cuivre et de proposer un modèle de cheminement du métal dans la partie extracytoplasmique du site de transport de HMA6 / Copper is an essential transition metal for living organisms. In the plant Arabidopsis thaliana, half the copper content is localized in the chloroplast. This organelle specific of plant cells, consists of an envelope delimiting the stroma, an aqueous compartment within which there is a complex membrane system, the thylakoids. In chloroplasts of Arabidopsis, copper is the cofactor of two essential proteins: the superoxide dismutase Cu / Zn, involved in defense against reactive oxygen species in the stroma and plastocyanin, a protein of the thylakoid lumen involved in the chain transfer photosynthetic electron. Reverse genetics studies have demonstrated that copper transport in plastocyanin involved two membrane proteins belonging to the family of ATPases-PIB-1: HMA6, located in the envelope and HMA8, localized in the thylakoid membranes. A functional in vitro study showed that HMA6 was a monovalent high affinity copper transporter showing the general characteristics of P-ATPases. To compare the enzymatic properties of these two ATPases and better understand their respective role in copper homeostasis in the chloroplast, we in vitro determined the enzymatic properties of HMA8.The strategy employed for the characterization of HMA8 was similar to that used for the characterization of HMA6. Initially, the ion selectivity of HMA8 was evaluated using phenotypic tests in the yeast Saccharomyces cerevisiae. The enzymatic properties of HMA8 were then determined in vitro after expression in the bacterium Lactoccocus lactis, by phosphorylation experiments by ATP. This analysis demonstrated that HMA8 had a stronger apparent affinity for copper but a slower catalytic activity than HMA6. The analysis of three-dimensional models of HMA6 and HMA8 showed that these differences could be explained by differences in the electrostatic potential at the cavity where the metal is released and/or by the nature of the partners interacting with these ATPases. These differences might explain the distinct functions of the two carriers in the chloroplast: HMA6 would regulate the copper concentration in the stroma by interacting with various target proteins (including copper chaperone), while HMA8 would have a more specific role for the distribution of copper plastocyanin.To better understand the mechanism of copper release by HMA6 and HMA8, we conducted a functional study of mutants of the region connecting the first two transmembrane helices (TMA and TMB). In this study, we specifically targeted cysteines and histidines because of their chemical properties that make them very strong metal ligands. The mutants of interest were selected by phenotypic screening in yeast and then expressed in the bacterium L. lactis. The in vitro biochemical characterization of their enzymatic properties was carried out by phosphorylation tests by ATP and Pi. This study allowed us to identify two residues, one cysteine and one histidine, involved the release of copper and to propose a metal path model in extracytoplasmic part of the transport site of HMA6

CoordenaÃÃo de AperfeiÃoamento de Pessoal de NÃvel Superior / This work reports the biochemical characterization, cytotoxic and allergenic effects of a lipid transfer protein isolated from M. citrifolia seeds (McLTP1), with trypsin and alpha-amylase inhibition properties. McLTP1 was purified with a procedure involving trichloroacetic acid precipitation and gel filtration chromatography. This protein showed significant inhibitory activities against trypsin (767,10  8,36 TIU/mgP), chymotrypsin (25,36  0,86 IU/mgP), papain (65,419  0,152 IU/mgP) and alpha-amylase (24,40%). Atomic force microscopy displayed that McLTP1 oligomerized in tetramers showing a central channel. Fluorescence and CD assays revealed that the McLTP1 structure is highly stable, regardless of pH and temperature levels. In vitro, McLTP1 presented a selective cytotoxic effect to human ovarian cancer cells (OVCAR-8; IC50 of 16,6 μg/mL) and demonstrated hemolytic effect against fresh rabbit red blood cels. Similarly to other non-specific lipid transfer protein reported, McLTP1 showed allergenic properties in mice, being considered as a true food allergen since it was able to sensitize the animals via the gastrointestinal tract. / Morinda citrifolia L. à uma espÃcie nativa do Sudeste da Ãsia intensamente investigada em funÃÃo de suas propriedades terapÃuticas reportadas hà mais de 2.000 anos. Recentemente, uma proteÃna transferidora de lipÃdeos denominada McLTP1 (UniProt Accession Number: C0HJH5) foi isolada de sementes de noni pelo nosso grupo de pesquisa. McLTP1 à uma proteÃna termoestÃvel de massa molecular 9,4 kDa, resistente à proteÃlise e dotada de atividades moduladoras da inflamaÃÃo e da dor pela via oral, promissoras e inÃditas para esse grupo de molÃculas. Este trabalho objetivou caracterizar bioquimicamente McLTP1, bem como avaliar o seu potencial alergÃnico em camundongos, como etapas bÃsicas para o seu uso racional e seguro do ponto de vista farmacolÃgico. Em adiÃÃo, as propriedades terapÃuticas de McLTP1 foram tambÃm ampliadas, atravÃs da investigaÃÃo de seu efeito citotÃxico em diferentes linhagens de cÃlulas tumorais. A proteÃna em estudo foi isolada utilizando o protocolo jà estabelecido, envolvendo as etapas de precipitaÃÃo seletiva de proteÃnas do extrato total das sementes de noni com Ãcido tricloroacÃtico 2,5% e cromatografia de exclusÃo molecular. O ensaio de alergenicidade in vivo foi conduzido apÃs prÃvia aprovaÃÃo pelo Comità de Ãtica para Uso de Animais da Universidade Federal do Cearà e utilizou fÃmeas nulÃparas com massa corporal entre 25 e 30 g. McLTP1 apresentou in vitro atividades inibitÃrias de tripsina (767,10  8,36 UIT/mgP), quimotripsina (25,36  0,86 UI/mgP), papaÃna (65,419  0,152 UI/mgP) e alfa-amilase (24,40%). A atividade inibitÃria de tripsina de McLTP1 foi reduzida significativamente em temperaturas superiores a 37 ÂC, apresentando atividade residual de apenas 5,91% quando aquecida a 100 ÂC por 30 min. Essa atividade foi tambÃm influenciada pelo pH, sendo de apenas 30,13% e 39,05% quando a proteÃna foi incubada em tampÃes de pH 3,0 e 12,0. O padrÃo de oligomerizaÃÃo de McLTP1 demonstrou a formaÃÃo de agregados dimÃricos/tetramÃricos delimitando um canal central de diÃmetro de 4,4 nm. As anÃlises espectroscÃpicas mostraram que McLTP1 apresenta espectro de CD similar Ãquele apresentado por outras proteÃnas transferidoras de lipÃdeos e caracterÃstico de proteÃnas ricas em alfa-hÃlice. Espectro de CD de McLTP1 nÃo mostrou alteraÃÃes significativas em diferentes temperaturas e pHs, corroborando com os dados de estabilidade obtidos anteriormente. Diferentemente, em condiÃÃes redutoras (DTT 1 mM) o espectro de CD mostrou alteraÃÃo na estrutura secundÃria da proteÃna e os mÃnimos e mÃximos de elipticidade molar foram tambÃm alterados na presenÃa de micelas iÃnicas de SDS (10 mM). McLTP1 apresentou atividade citotÃxica seletiva contra cÃlulas de cÃncer de ovÃrio (Ovcar-8; CI50: 16,6 μg/mL), nÃo sendo citotÃxica para as cÃlulas tumorais de cÃlon humano (HCT-116), leucemia humano (HL-60) e glioblastoma humano (SF-295) testadas. McLTP1 foi capaz de promover hemÃlise significativa em hemÃcias de coelho a partir da concentraÃÃo de 0,005 mgP/mL. McLTP1 apresentou potencial efeito alergÃnico in silico e em camundongos imunizados pela via oral, induzindo a sÃntese de anticorpos IgG e IgG1. Tal como descrito na literatura para outras LTPs, anticorpos anti-McLTP1 produzidos em coelho foram tambÃm capazes de reconhecer proteÃnas presentes em extratos de Rosaceae, Cucurbitaceae e na polpa do fruto de noni. Os dados obtidos permitiram caracterizar parcialmente a proteÃna em estudo, bem como avaliar o seu potencial imunogÃnico apÃs administraÃÃo oral. Novos testes serÃo conduzidos objetivando avaliar a importÃncia clÃnica dessas respostas, uma vez que testes de toxicidade demonstraram que McLTP1 nÃo foi capaz de promover reaÃÃes adversas em camundongos, mesmo apÃs administraÃÃo da dose de 8 mg/kg por 28 dias.

Submitted by (lucia.rodrigues@ufrpe.br) on 2016-06-08T14:47:27Z No. of bitstreams: 1 Julio Cezar dos Santos Nascimento.pdf: 1976053 bytes, checksum: 53410347ad1578d486d39f21c31e19aa (MD5) / Made available in DSpace on 2016-06-08T14:47:27Z (GMT). No. of bitstreams: 1 Julio Cezar dos Santos Nascimento.pdf: 1976053 bytes, checksum: 53410347ad1578d486d39f21c31e19aa (MD5) Previous issue date: 2011-02-28 / During ripening, vegetable seeds and grain accumulated substantial amount of phytic acid, representing over 60% of total phosphorus in them. Phytate acts as an energy source for seed germination and is rarely available for non-ruminants, since they do not synthesize the enzyme. Due to the unavailability of biological organic phosphorus in many vegetable foods, research has sought alternative sources of phosphorus in order to better use. Incorporated in this context are the phytases, which form a group of enzymes that catalyze reactions gradual dephosphorylation of phytate. For enzyme purification the aqueous two-phase systems (ATPS) have been widely used for protein separation due to its low cost compared to other purification processes. The aim of this study was to evaluate the variables that influence production and purification of phytase by aqueous two-phase systems, as well as the biochemical characteristics of phytase produced by Aspergillus niger var. phoenicis URM 4924. The culture medium for the production of phytase were studied using factorial design and response surface methodology. The best condition for the production of phytase (8.80 U/mL) was found matching 1.25% of rice bran and 3.0% corn steep liquor. The optimum pH was 5.0, and remains at over 80% of residual activity at pH 5.0 to 9.0 for 15 hours. The phytase showed affinity constant of 0.12 mM and maximum velocity of 7.9 ηmol.s-1. The best conditions of extraction in aqueous two-phase systems were obtained with 26% (w/w) PEG 8000 (g/mol), pH 8.0 and 12% (w/w) citrate thus promoting purification factor of 7.58, partition coefficient of 3.62 and yield of 113.4%. The extraction using ATPS PEG/citrate proved to be promising for purification of phytase produced by A. niger var. phoenicis URM 4924 and may be applied in the composition of animal feed non-ruminants. / Durante o amadurecimento, sementes de legumes e cereais acumulam quantidade substancial de ácido fítico, representando mais de 60% do fósforo total dos mesmos. O fitato serve como fonte de energia para a germinação da semente, sendo pouco disponível para animais não-ruminantes, pois esses não sintetizam a fitase. Devido à indisponibilidade biológica do fósforo fítico em diversos alimentos de origem vegetal, as pesquisas têm buscado por fontes alternativas de fósforo, visando um melhor aproveitamento. Inseridas neste contexto estão as fitases, que formam um grupo de enzimas que catalisam reações graduais de defosforilação do fitato. Para a purificação de enzimas os sistemas de duas fases aquosas (SDFA) têm sido amplamente usados para separação de proteínas por conta do seu baixo custo quando comparado a outros processos de purificação. O objetivo deste trabalho foi a avaliação das variáveis que influenciam a produção e purificação da enzima por sistemas de duas fases aquosas, bem como a determinação das características bioquímicas de fitase produzidas por Aspergillus niger var. phoenicis URM 4924. Os meios de cultivo para a produção de fitases foram estudados utilizando planejamento fatorial e metodologia de superfície de resposta. A melhor condição para a produção de fitase (8,80 U/mL) foi encontrada combinando 1,25% de farelo de arroz e 3,0% de milhocina. A fitase apresentou temperatura ótima a 60°C e manteve 38,4% da atividade residual a 90°C por 120 minutos. O pH ótimo foi 5,0, e permaneceu com mais de 80% da atividade residual na faixa de pH 5,0 a 9,0 por 15 horas. A fitase apresentou constante de afinidade de 0,12 μM e velocidade máxima de 7,9 ηmol.s-1. As melhores condições de extração em sistemas de duas fases aquosas foram obtidas com 26% (m/m) de PEG 8000 (g/mol), pH 8,0 e 12% (m/m) de citrato promovendo assim, aumento de pureza de 7,58, coeficiente de partição de 3,62 e recuperação de 113,4%. A extração utilizando SDFA PEG/citrato demonstrou ser promissora para purificação de fitase produzida por A. niger var. phoenicis URM 4924, podendo ser aplicada na composição de rações de animais não-ruminantes.

Le Rovabio Excel ™ est un cocktail enzymatique complexe sécrété par le champignon filamenteux Penicillium funiculosum. La société ADISSEO commercialise cet additif alimentaire destiné à la nutrition animale car les principales enzymes qui le constituent dégradent les polymères contenus dans les céréales, tels que les polysaccharides non amylacés. Ainsi, le Rovabio Excel™ permet d’améliorer la digestibilité et d’augmenter la valeur nutritionnelle des matières premières agricoles en réduisant la viscosité du bol alimentaire des animaux. Dans le but d’augmenter sa compétitivité, ADISSEO a fait conduire des études sur cette solution pour la caractériser biochimiquement et optimiser son potentiel xylanolytique.Ces travaux de thèse s’inscrivent dans ces projets industriels et ont poursuivi deux objectifs distincts. Le premier correspondait à l’augmentation de la thermostabilité de la protéine XynB du Rovabio Excel™, pour lui permettre de résister à la granulation. Le second concernait XynA, la protéine majoritaire de la solution multienzymatique, qui a été caractérisée biochimiquement. Les premiers résultats de caractérisation biochimique de XynA ont montré que la protéine était 100 fois plus active sur β-1,4-glucane que sur xylane. Des tests complémentaires sur pNP-cellobiose et pNP-β-D-Lactopyranose ont révélé que XynA était 5,2 fois plus active sur pNP-cellobiose et possédait une activité « exo ». Enfin, l’analyse des produits d’hydrolyse d’oligosaccharides composés de 2 à 5 unités de glucose a confirmé que la protéine XynA était une cellobiohydrolase de type I, très sensible à l’inhibition par le cellobiose (IC50 - C2 = 17,7 µM). L’étude la thermostabilité de XynB a confirmé que cette protéine n’était pas naturellement thermostable. Les résultats des travaux d’ingénierie avec l’ajout d’un pont disulfure pour rigidifier la structure 3D de la protéine n’ont pas été probants. En revanche, la création de protéines chimères à partir de protéines plus thermostables (TfxA de Thermomonospora fusca et XynII de Trichoderma reesei) a permis d’améliorer la stabilité thermodynamique de XynB avec des Tm augmentés de plus de 10°C / The Rovabio Excel™ is a complex enzymatic cocktail secreted by the filamentous fungus Penicillium funiculosum. The ADISSEO company sells it as food additive for animal feed because the main enzymes degrade polymers contained in grains, such as non-starch polysaccharides. Thus, the Rovabio Excel™ improves the digestibility and increases the nutritional value of agricultural raw materials by reducing the viscosity of the diet of animals. In order to increase its competitiveness, ADISSEO did conduct studies on this solution to characterize it biochemically and maximize its xylanolytic potential.This thesis takes part of this industrial project and have pursued two distinct objectives. The first corresponds to the increase in the thermostability of the protein XynB from the Rovabio Excel™, to enable it to resist at the granulation process. The second was XynA, the major protein of the multienzyme solution, which was characterized biochemically.Initial results of biochemical characterization of XynA showed that the protein was 100 times more active on β-1,4-glucan on xylan. Additional tests on pNP-cellobiose and pNP-β-D-Lactopyranose revealed that XynA was 5.2 times more active on pNP-cellobiose and possess an "exo-acting" activity. Finally, the analysis of products from oligosaccharides hydrolysis, composed of 2 to 5 units of glucose, confirmed that the protein XynA was a type I cellobiohydrolase, very sensitive to inhibition by cellobiose (IC50-C2 = 17.7 µM).The thermostability of XynB study has confirmed that this protein was not thermostable naturally. The results of the engineering work with the addition of a disulfide bridge to rigidify the 3D structure of the protein were not conclusive. However, the creation of chimeric proteins with more thermostable proteins (TfxA from Thermomonospora fusca and XynII from Trichoderma reesei) has improved the thermodynamic stability of XynB with Tm increased by more than 10°C

Esta tese apresenta o desenvolvimento de metodologias analíticas em sistemas de solução em fluxo com detecção amperométrica e espectrofotométrica explorando a utilização de tecidos vegetais como fonte enzimática para a biocatálise de reações. Desempenhos satisfatórios foram obtidos com a utilização do mesocarpo fibroso do coco (Cocus nucifera, L.) e com a casca e/ou polpa de frutos da palmeira leque (Latania sp), fontes de polifenol oxidase. Uma metodologia, simples e rápida, para a determinação dos parâmetros bioquímicos da polifenol oxidase foi desenvolvida ao longo do presente trabalho com a enzima naturalmente imobilizada no tecido do coco, pela eliminação das etapas de extração e de purificação e associando-se análise em fluxo e espectrofotometria. Parâmetros cinéticos importantes como o efeito do pH, a constante de Michaelis-Menten (Km), a velocidade máxima (Vmax),a energia de ativação (Ea) e os parâmetros térmicos (valores D, z e Q10)foram determinados utilizando como substrato o catecol. A atividade relativa junto a diferentes substratos e o efeito de inibidores foram também avaliados. Aplicações envolvendo estes tecidos vegetais em reatores empacotados (associados com FIA) ou incorporados em eletrodos de pasta de carbono (medidas realizadas em batelada) para a determinação de produtos fenólicos como catecol, fenol e dopamina, inibidores da atividade enzimática como o sulfito, bem como para a quantificação do conteúdo de água, demonstram a sua versatilidade. Os produtos fenólicos foram quantificados com boa sensibilidade (na faixa de µmol L-1) pela redução amperométrica das respectivas quinonas, produzidas pela oxidação enzimática. Aplicações com amostras reais, dentre as quais água de rio e resíduos de uma fábrica de papel puderam ser implementadas. A determinação do conteúdo de água em meio aquo-restrito explorou a estimulação, pela água, da atividade catalitica da polifenoI oxidase naturalmente imobilizada no mesocarpo fibroso do coco na presença de catecol. Esta propriedade foi utilizada para a determinação rápida e reprodutível do conteúdo de água em amostras comerciais de álcool, utilizando acetonitrila como carregador. A quantificação de sulfito baseou-se no seu efeito inibidor sobre a polifenol oxidase naturalmente imobilizada em relação à catálise oxidativa do catecol. Diversos interferentes comumente presentes em amostras de vinho foram também investigados. Durante este trabalho, foi desenvolvida uma simples e interessante maneira de conservar o mesocarpo fibroso do coco como fonte de polifenol oxidase naturalmente imobilizada. O tecido desta fruta foi desidratado e moído. O pó deste tecido, estocado a mais de dois anos à temperatura ambiente, ainda apresenta boa atividade enzimática. / The development of analytical methodologies exploring plant tissues as enzymatic source for biocatalysis of many reactions is presented in this thesis. Amperometry and spectrophotometry was associated with flow analysis for analytical purposes and for biochemical characterization of naturally immobilized polyphenol oxidase. Good performance was achieved with tissues from the fruits of two palm trees: the fibrous mesocarp of green coconut fruits, Cocus nucifera, L., and the skin and the pulp of green fruits from Latania sp. Both tissues are very effective in the biotransformation of o-diphenols to the corresponding o-quinones. A simple, fast, and new methodology for the determination of the biochemical parameters of the poyphenol oxidase naturally immobilized on the fibrous tissue was developed (eliminating the extraction and purification of enzymes) by association of flow injection analysis and spectrophotometry. For coconut tissue, cinetic parameters like pH effect, Michaelis-Menten constant (Km) and maximum rate (Vmax), activation energy (Ea) and thermal parameters (D, z and Q10values) on catechol biotransformation were determined. Also the response for several substrates as too for various inhibitors was explored. Amperometric quantification of phenolic compounds was made using the vegetal tissue in form of packed reactors (associated with FIA) or incorporated in carbon paste electrodes (measurements in batch). Very good response for catechol, phenol and dopamine was registered for this compounds, with very high sensitivity (µmol L-1 range). Applicability to real samples as for river water and for a paper plant waste water was verified. The same amperometry-FIA system was employed for enzymology in non-aqueous medium. The activity of the polyphenol oxidase contained in coconut tissues are strongly dependent of water. The strategy involves the stimulation by the content of water on the biocatalytic activity of the enzyme in the presence of catechol substrate. This strategy was used for the determination of water content in alcohol samples, in medium of dry acetonitrile. The inhibition of the enzymatic activity produced by many compounds can be explored for an indirect quantification of the inhibitor. A flow injection amperometric procedure was developed for the determination of sulphite ion based in its inhibitory effect on the activity of polyphenol oxidase on the oxidation of catechol. The effect on the bioreactor performance of potential interferents commonly present in wine samples were also investigated. During this work, it was developed a simple and interesting way to preserve coconut tissue. The mesocarp of this fruit can be dried and grounded. These tissues still with very good activity after more than two years in \"shelf temperature\".

As fosfolipases A2 (PLA2s) de peçonhas de serpentes compreendem um grupo de enzimas de massas moleculares variáveis entre 14.000 e 18.000, e são responsáveis por vários efeitos tóxicos induzidos pela peçonha destes animais, tornando-se necessária a busca por inibidores naturais de PLA2¬s. O presente trabalho propôs a caracterização bioquímica, farmacológica e estrutural de duas proteínas inibitórias isoladas do plasma da serpente Bothrops jararacussu (BjussuMIPs), que neutralizam as atividades enzimáticas, tóxicas e farmacológicas de diferentes PLA2s. Estes inibidores foram isolados por cromatografia de afinidade em miotoxina-Sepharose, demonstrando que ambos são glicoproteínas com massas moleculares de 24.000 (BjussuMIP) e 23.500 (BjussuMIP) para os monômeros e de 120.000 (BjussuMIP) e 160.000 (BjussuMIP) para os oligômeros. O tratamento dos BjussuMIPs com a N-glicosidase F reduziram os seus pesos moleculares para aproximadamente 18.000, mas não afetaram suas atividades inibitórias sobre PLA2s, sugerindo que os carboidratos tem pouco ou nenhum papel na associação dos BjussuMIPs com estas enzimas. A análise do BjussuMIP por dicroísmo circular mostrou 44% de -hélice, 18% de folhas , 10% de voltas e 28% de estruturas aleatórias. O cDNA obtido por PCR a partir do fígado desta serpente revelou 432 pb (BjussuMIP) e 543 pb (BjussuMIP) que codificam para 144 e 181 resíduos de aminoácidos, respectivamente. O alinhamento da sequência de BjussuMIP com a de outros inibidores do tipo , denominados de PLIs, apresentou 73-92% de similaridade e o BjussuMIP mostrou 89-94% com inibidores do tipo PLIs. Os BjussuMIPs demonstraram ser relativamente estável a variações de pH (6-12) e temperatura, entretanto, perderam atividade inibitória quando submetido a altas temperaturas. A caracterização funcional indica que os BjussuMIPs apresentaram propriedades inibitórias sobre diferentes PLA2s isoladas de peçonhas de serpentes dos gêneros Bothrops e Crotalus. Ambos BjussuMIPs revelaram propriedades farmacológicas como a inibição das atividades fosfolipásica, anticoagulante, miotóxica, indução de edema, citotóxica, bactericida e letal. Os resultados obtidos demonstram que o BjussuMIP mostra maior afinidade sobre as PLA2s homólogas Lys49 como BthTX-I e PrTX-I, enquanto que o BjussuMIP apresenta-se mais específico para PLA2s Asp49, sugerindo uma especificidade entre os BjussuMIPs e tipos de PLA2s. Além disso, ambos os inibidores mostraram ser eficazes na suplementação do antiveneno botrópico em diferentes concentrações, resultando no aumento da capacidade do soro em neutralizar toxinas de serpentes. Os aspectos abordados neste trabalho poderão trazer informações complementares sobre possíveis mecanismos de ação, podendo resultar no melhor entendimento dos efeitos inibitórios exercidos pelos BjussuMIPs, assim como auxiliar o tratamento do envenenamento ofídico pela suplementação da soroterapia tradicional. / Phospholipases A2 (PLA2s) from snake venoms comprise a group of enzymes with molecular weights varying from 14,000 to 18,000, and are responsible for several toxic effects induced by the venom of these animals, making important the search for natural inhibitors of PLA2s. The present work proposed the biochemical, pharmacological and structural characterization of two protein inhibitors isolated from the plasma of Bothrops jararacussu snake (BjussuMIPs), which neutralize the enzymatic, toxic and pharmacological activities of different PLA2s. These inhibitors were isolated by an affinity chromatography on myotoxin-Sepharose, showing that both are glycoproteins with molecular weights of 24,000 (BjussuMIP) and 23,500 (BjussuMIP) for the monomers and 120,000 (BjussuMIP) and 160,000 (BjussuMIP) for the oligomers. The treatment of BjussuMIPs with N-glucosidase F reduced their molecular weights to about 18,000, but did not affect their inhibitory activity on PLA2s, suggesting that the carbohydrates have little or no role in the association of these BjussuMIPs with these enzymes. The analysis of BjussuMIP by circular dichroism showed 44% of -helix, 18% of sheets, 10% of turns and 28% of random structures. The cDNA obtained by PCR from the snake liver showed 432 bp for BjussuMIP and 543 bp for BjussuMIP, which encode for 144 and 181 amino acid residues, respectively. The alignment of the sequence of BjussuMIP with those from other -inhibitors (PLIs) showed 73-92% of similarity and 89-94% for the BjussuMIP compared to other PLIs. The BjussuMIPs showed to be relatively stable to changes in pH (6-12) and temperature, however lost of its activity when submitted to high temperatures. The functional characterization indicates that both BjussuMIPs presented inhibitory properties on different snake venom PLA2s from the genera Bothrops and Crotalus. Both BjussuMIPs showed pharmacological properties such as inhibition of phospholipase, anticoagulant, myotoxic, cytotoxic, bactericidal, edema-inducing and lethal activities. The results show that BjussuMIP presents higher affinity to Lys49-PLA2 homologous, such as BthTX-I and PrTX-I, while BjussuMIP is more specific to Asp49-PLA2s, suggesting specificity between BjussuMIPs and types of PLA2s. Moreover, both inhibitors proved effective in the supplementation of Bothrops antivenom at different concentrations, resulting in an increased capacity of serum in neutralizing snake toxins. The issues reported in this work could bring additional information on possible mechanisms of action and may result in better understanding of the inhibitory effects exerted by these BjussuMIPs, as well as assist the treatment of ophidian envenomations by supplementation of the traditional serum therapy.

Esta tese apresenta o desenvolvimento de metodologias analíticas em sistemas de solução em fluxo com detecção amperométrica e espectrofotométrica explorando a utilização de tecidos vegetais como fonte enzimática para a biocatálise de reações. Desempenhos satisfatórios foram obtidos com a utilização do mesocarpo fibroso do coco (Cocus nucifera, L.) e com a casca e/ou polpa de frutos da palmeira leque (Latania sp), fontes de polifenol oxidase. Uma metodologia, simples e rápida, para a determinação dos parâmetros bioquímicos da polifenol oxidase foi desenvolvida ao longo do presente trabalho com a enzima naturalmente imobilizada no tecido do coco, pela eliminação das etapas de extração e de purificação e associando-se análise em fluxo e espectrofotometria. Parâmetros cinéticos importantes como o efeito do pH, a constante de Michaelis-Menten (Km), a velocidade máxima (Vmax),a energia de ativação (Ea) e os parâmetros térmicos (valores D, z e Q10)foram determinados utilizando como substrato o catecol. A atividade relativa junto a diferentes substratos e o efeito de inibidores foram também avaliados. Aplicações envolvendo estes tecidos vegetais em reatores empacotados (associados com FIA) ou incorporados em eletrodos de pasta de carbono (medidas realizadas em batelada) para a determinação de produtos fenólicos como catecol, fenol e dopamina, inibidores da atividade enzimática como o sulfito, bem como para a quantificação do conteúdo de água, demonstram a sua versatilidade. Os produtos fenólicos foram quantificados com boa sensibilidade (na faixa de µmol L-1) pela redução amperométrica das respectivas quinonas, produzidas pela oxidação enzimática. Aplicações com amostras reais, dentre as quais água de rio e resíduos de uma fábrica de papel puderam ser implementadas. A determinação do conteúdo de água em meio aquo-restrito explorou a estimulação, pela água, da atividade catalitica da polifenoI oxidase naturalmente imobilizada no mesocarpo fibroso do coco na presença de catecol. Esta propriedade foi utilizada para a determinação rápida e reprodutível do conteúdo de água em amostras comerciais de álcool, utilizando acetonitrila como carregador. A quantificação de sulfito baseou-se no seu efeito inibidor sobre a polifenol oxidase naturalmente imobilizada em relação à catálise oxidativa do catecol. Diversos interferentes comumente presentes em amostras de vinho foram também investigados. Durante este trabalho, foi desenvolvida uma simples e interessante maneira de conservar o mesocarpo fibroso do coco como fonte de polifenol oxidase naturalmente imobilizada. O tecido desta fruta foi desidratado e moído. O pó deste tecido, estocado a mais de dois anos à temperatura ambiente, ainda apresenta boa atividade enzimática. / The development of analytical methodologies exploring plant tissues as enzymatic source for biocatalysis of many reactions is presented in this thesis. Amperometry and spectrophotometry was associated with flow analysis for analytical purposes and for biochemical characterization of naturally immobilized polyphenol oxidase. Good performance was achieved with tissues from the fruits of two palm trees: the fibrous mesocarp of green coconut fruits, Cocus nucifera, L., and the skin and the pulp of green fruits from Latania sp. Both tissues are very effective in the biotransformation of o-diphenols to the corresponding o-quinones. A simple, fast, and new methodology for the determination of the biochemical parameters of the poyphenol oxidase naturally immobilized on the fibrous tissue was developed (eliminating the extraction and purification of enzymes) by association of flow injection analysis and spectrophotometry. For coconut tissue, cinetic parameters like pH effect, Michaelis-Menten constant (Km) and maximum rate (Vmax), activation energy (Ea) and thermal parameters (D, z and Q10values) on catechol biotransformation were determined. Also the response for several substrates as too for various inhibitors was explored. Amperometric quantification of phenolic compounds was made using the vegetal tissue in form of packed reactors (associated with FIA) or incorporated in carbon paste electrodes (measurements in batch). Very good response for catechol, phenol and dopamine was registered for this compounds, with very high sensitivity (µmol L-1 range). Applicability to real samples as for river water and for a paper plant waste water was verified. The same amperometry-FIA system was employed for enzymology in non-aqueous medium. The activity of the polyphenol oxidase contained in coconut tissues are strongly dependent of water. The strategy involves the stimulation by the content of water on the biocatalytic activity of the enzyme in the presence of catechol substrate. This strategy was used for the determination of water content in alcohol samples, in medium of dry acetonitrile. The inhibition of the enzymatic activity produced by many compounds can be explored for an indirect quantification of the inhibitor. A flow injection amperometric procedure was developed for the determination of sulphite ion based in its inhibitory effect on the activity of polyphenol oxidase on the oxidation of catechol. The effect on the bioreactor performance of potential interferents commonly present in wine samples were also investigated. During this work, it was developed a simple and interesting way to preserve coconut tissue. The mesocarp of this fruit can be dried and grounded. These tissues still with very good activity after more than two years in \"shelf temperature\".

Made available in DSpace on 2014-07-29T12:03:35Z (GMT). No. of bitstreams: 1 Tese_Cristine_Cysneiros.pdf: 1284054 bytes, checksum: c3e0f947790961725c6b67bf30fac547 (MD5) Previous issue date: 2009-04-03 / The objective of this work was to produce and characterize four multi enzyme complexes from the fungus Humicola grisea var. thermoidea, maintained for 96 hours at 42oC in growth media containing different carbon sources: Marandu grass; soybean seedcoats; Tifton 85 hay; and corn forage. Different amounts of cellulase, xylanase and -glucosidase enzymes were obtained depending on the different carbon sources. Cellulase presented increased activity in temperatures between 40ºC and 50ºC. The observed optimum temperature range for xylanase and β-glycosidase was from 50ºC and 60ºC. Optimum pH for cellulase activity was 6.0 when fungus growth occurred in Tifton hay, corn forage, and soybean seedcoats. When the enzyme was obtained from medium containing Marandu grass, optimum enzyme activity occurred at pH 5.5. Regardless of the carbon source, xylanase activity was higher at pH 6.0. As for β-glucosidase, optimum activity was observed at pH 5.5 for Tifton media as compared to pH 6.5 for corn and soybean containing media. For grass Marandu, the activity of the enzyme was maximum in the range 5.5 to 6.5. Cellulase produced from all growth media were maintained stable after incubation for 60 minutes at 39°C. Xylanase presented thermal stability during 240 minute incubation period at 50°C. Activity stability of β-glycosidase varied according to carbon source and presented 66.7 to 125.75% activity at 50°C for 240 minutes. / Enzimas fibrolíticas exógenas são produzidas por cultura específica de bactérias ou fungos. São essenciais aos animais por estarem envolvidas na hidrólise dos componentes complexos das dietas em moléculas orgânicas mais simples como glicose, celobiose, xilose, aminoácidos, ácidos graxos, que são então usadas pelos microrganismos do rúmen e/ou pelo animal. Melhoras no desempenho dos ruminantes devido ao uso de enzimas fibrolíticas são atribuídas principalmente à maior degradação da fibra no rúmen, o que resulta em aumento da ingestão de energia disponível pelos animais. Os objetivos deste trabalho foram os de produzir e caracterizar quatro soluções enzimáticas, utilizando o fungo Humicola grisea var. thermoidea e avaliar seus efeitos por meio da digestibilidade verdadeira in vitro da matéria seca de quatro substratos: capim Marandu, casquinha de soja, feno de Tifton-85 e forragem de milho. As soluções enzimáticas foram produzidas a partir de quatro meios de culturas diferentes, contendo a fonte de carbono específica, durante 96 horas de cultivo, a 42°C. Foi observado que o fungo produziu as enzimas celulases, xilanase e -glicosidase em diferentes concentrações, o que foi dependente da fonte de carbono. A caracterização bioquímica mostrou que a celulase produzida apresentou maior atividade em temperatura entre 40ºC e 50°C. A temperatura ótima de xilanase e β-glicosidase foi entre 50 e 60°C. O pH ótimo da enzima celulase foi 6,0, quando o fungo cresceu em feno de Tifton, forragem de milho e casquinha de soja. Para o capim Marandu, a enzima apresentou atividade ótima em pH 5,5. Para as quatro fontes de carbono, a xilanase produzida apresentou pH ótimo de 6,0. Em relação a β-glicosidase, a atividade enzimática foi maior em pH 5,5, no meio com feno de Tifton. Para capim Marandu, a atividade da enzima foi máxima na faixa de 5,5 a 6,5. Quanto à forragem de milho e casquinha de soja, a enzima exibiu maior atividade em pH 6,5. A celulase produzida, nas quatro fontes de carbono, permaneceu estável após a incubação por 60 minutos, a 39°C. Xilanase produzida apresentou estabilidade térmica durante 240 minutos de incubação, a 50°C. A β-glicosidase, dependendo da fonte de carbono, manteve de 66,7 a 125,75% de sua atividade, a 50°C, durante 240 minutos. Para avaliar o potencial das soluções enzimáticas sobre a digestibilidade in vitro dos substratos, 2,5; 5,0 e 10 mL de cada solução foram aplicados, por aspersão, em 17 g dos seus respectivos substratos, moídos em peneira com malha de 1 mm de diâmetro. Após aspersão, as enzimas ficaram em contato com os substratos por 2 e 24 h (tempo de reação enzima-substrato), antes de serem incubados no rúmen. A digestibilidade in vitro da MS foi avaliada em líquido ruminal tamponado, durante o período de 12, 24, 48 e 96 h. Para cada substrato, foram incubados 34 sacos (4 níveis de enzimas x 4 períodos de incubação x 2 repetições x 1 branco x 1 testemunha). As soluções enzimáticas, em qualquer nível de enzimas, quando comparados aos tratamentos controle, aumentaram a digestibilidade da MS dos substratos, nos tempos de reação enzima-substrato e período de incubação no rúmen. Este estudo mostrou que enzimas fibrolíticas exógenas produzidas por H. grisea tem potencial para uso como aditivo em dietas de ruminantes

As fosfolipases A2 (PLA2s) de peçonhas de serpentes compreendem um grupo de enzimas de massas moleculares variáveis entre 14.000 e 18.000, e são responsáveis por vários efeitos tóxicos induzidos pela peçonha destes animais, tornando-se necessária a busca por inibidores naturais de PLA2¬s. O presente trabalho propôs a caracterização bioquímica, farmacológica e estrutural de duas proteínas inibitórias isoladas do plasma da serpente Bothrops jararacussu (BjussuMIPs), que neutralizam as atividades enzimáticas, tóxicas e farmacológicas de diferentes PLA2s. Estes inibidores foram isolados por cromatografia de afinidade em miotoxina-Sepharose, demonstrando que ambos são glicoproteínas com massas moleculares de 24.000 (BjussuMIP) e 23.500 (BjussuMIP) para os monômeros e de 120.000 (BjussuMIP) e 160.000 (BjussuMIP) para os oligômeros. O tratamento dos BjussuMIPs com a N-glicosidase F reduziram os seus pesos moleculares para aproximadamente 18.000, mas não afetaram suas atividades inibitórias sobre PLA2s, sugerindo que os carboidratos tem pouco ou nenhum papel na associação dos BjussuMIPs com estas enzimas. A análise do BjussuMIP por dicroísmo circular mostrou 44% de -hélice, 18% de folhas , 10% de voltas e 28% de estruturas aleatórias. O cDNA obtido por PCR a partir do fígado desta serpente revelou 432 pb (BjussuMIP) e 543 pb (BjussuMIP) que codificam para 144 e 181 resíduos de aminoácidos, respectivamente. O alinhamento da sequência de BjussuMIP com a de outros inibidores do tipo , denominados de PLIs, apresentou 73-92% de similaridade e o BjussuMIP mostrou 89-94% com inibidores do tipo PLIs. Os BjussuMIPs demonstraram ser relativamente estável a variações de pH (6-12) e temperatura, entretanto, perderam atividade inibitória quando submetido a altas temperaturas. A caracterização funcional indica que os BjussuMIPs apresentaram propriedades inibitórias sobre diferentes PLA2s isoladas de peçonhas de serpentes dos gêneros Bothrops e Crotalus. Ambos BjussuMIPs revelaram propriedades farmacológicas como a inibição das atividades fosfolipásica, anticoagulante, miotóxica, indução de edema, citotóxica, bactericida e letal. Os resultados obtidos demonstram que o BjussuMIP mostra maior afinidade sobre as PLA2s homólogas Lys49 como BthTX-I e PrTX-I, enquanto que o BjussuMIP apresenta-se mais específico para PLA2s Asp49, sugerindo uma especificidade entre os BjussuMIPs e tipos de PLA2s. Além disso, ambos os inibidores mostraram ser eficazes na suplementação do antiveneno botrópico em diferentes concentrações, resultando no aumento da capacidade do soro em neutralizar toxinas de serpentes. Os aspectos abordados neste trabalho poderão trazer informações complementares sobre possíveis mecanismos de ação, podendo resultar no melhor entendimento dos efeitos inibitórios exercidos pelos BjussuMIPs, assim como auxiliar o tratamento do envenenamento ofídico pela suplementação da soroterapia tradicional. / Phospholipases A2 (PLA2s) from snake venoms comprise a group of enzymes with molecular weights varying from 14,000 to 18,000, and are responsible for several toxic effects induced by the venom of these animals, making important the search for natural inhibitors of PLA2s. The present work proposed the biochemical, pharmacological and structural characterization of two protein inhibitors isolated from the plasma of Bothrops jararacussu snake (BjussuMIPs), which neutralize the enzymatic, toxic and pharmacological activities of different PLA2s. These inhibitors were isolated by an affinity chromatography on myotoxin-Sepharose, showing that both are glycoproteins with molecular weights of 24,000 (BjussuMIP) and 23,500 (BjussuMIP) for the monomers and 120,000 (BjussuMIP) and 160,000 (BjussuMIP) for the oligomers. The treatment of BjussuMIPs with N-glucosidase F reduced their molecular weights to about 18,000, but did not affect their inhibitory activity on PLA2s, suggesting that the carbohydrates have little or no role in the association of these BjussuMIPs with these enzymes. The analysis of BjussuMIP by circular dichroism showed 44% of -helix, 18% of sheets, 10% of turns and 28% of random structures. The cDNA obtained by PCR from the snake liver showed 432 bp for BjussuMIP and 543 bp for BjussuMIP, which encode for 144 and 181 amino acid residues, respectively. The alignment of the sequence of BjussuMIP with those from other -inhibitors (PLIs) showed 73-92% of similarity and 89-94% for the BjussuMIP compared to other PLIs. The BjussuMIPs showed to be relatively stable to changes in pH (6-12) and temperature, however lost of its activity when submitted to high temperatures. The functional characterization indicates that both BjussuMIPs presented inhibitory properties on different snake venom PLA2s from the genera Bothrops and Crotalus. Both BjussuMIPs showed pharmacological properties such as inhibition of phospholipase, anticoagulant, myotoxic, cytotoxic, bactericidal, edema-inducing and lethal activities. The results show that BjussuMIP presents higher affinity to Lys49-PLA2 homologous, such as BthTX-I and PrTX-I, while BjussuMIP is more specific to Asp49-PLA2s, suggesting specificity between BjussuMIPs and types of PLA2s. Moreover, both inhibitors proved effective in the supplementation of Bothrops antivenom at different concentrations, resulting in an increased capacity of serum in neutralizing snake toxins. The issues reported in this work could bring additional information on possible mechanisms of action and may result in better understanding of the inhibitory effects exerted by these BjussuMIPs, as well as assist the treatment of ophidian envenomations by supplementation of the traditional serum therapy.

Plants are constantly exposed to pathogens and various environmental stresses, such as cold, salinity and drought. Plants normally respond rapidly to these biotic and abiotic stresses. Efficient perception of biotic and abiotic stresses and cell programmed signaling mechanisms for appropriate responses are important for growth and survival of plants. Calcium is an important second messenger in signaling pathways that respond to environmental stresses, pathogen attack as well as hormonal stimuli (For review, see DeFalco et al., 2010; Reddy and Reddy, 2004; Sanders et al., 2002). The transient increase of cytosolic free calcium concentration has been shown in a variety of external signals (Reddy, 2001), which in turn triggers many signal transduction pathways leading to a variety of cellular responses (Bush, 1995). Any calcium mediated signal transduction process involves generation of signal-specific calcium signature in the cytosol (Scrase-Field and Knight, 2003). These changes in cytosolic calcium level or ‘calcium signatures’ are sensed by the specific group of proteins called the ‘calcium sensors’. Different calcium sensors recognize specific calcium signatures and transduce them into downstream effects, including altered protein phosphorylation and gene expression patterns. In plants the protein kinases are a large and differentiated group of calcium sensors. After analyzing 1264 protein kinase sequences, a superfamily of protein kinase called CDPK/SnRK family of protein kinase were defined (Hrabak et al., 2003). CDPK/SnRK family of protein kinases encompasses five subfamilies viz., calcium-dependant protein kinases, (CPKs), calcium/calmodulin dependant protein kinases (CCaMKs), calmodulin-dependant protein kinases (CaMKs), CPKrelated kinases (CRKs), and SnF1 related kinase 3 (SnRK3) and are regulated by calcium directly or indirectly. Among these, in plants, calcium-dependant protein kinases (CPKs) are predominant calcium sensors, which are shown to be involved in myriads of physiological responses. They are Ser/Thr family of protein kinases typically made up of five domains with an Nterminal variable domain followed by catalytic protein kinase domain, an autoinhibitory/ junction domain, a regulatory calmodulin-like domain (CaMLD) and a Cterminal domain of variable length. The CPKs are unique due to the presence of CaMLD which couples the calcium sensor directly to its responder (kinase domain). Although CPKs are highly conserved, there are several features that distinguish different members of the plant CDPK family. In an attempt to investigate the role of a CPK isoform, in the present work we bring out the results and inferences on isolation and characterization of a novel cDNA encoding a calcium-dependant protein kinase isoform ScCPK1 from Swainsonacanescens, a pharmaceutically important Australian herb known to produce an anticancer drug, swainsonine. Initially, we have cloned an 800 bp partial CPK cDNA from S. canescens by reverse transcription polymerase chain reaction (RTPCR) using degenerate oligonucleotide primers designed based on conserved regions of the other known CPKs. A 2.1 kb full length CPK was obtained using 5` and 3` RACE which was designated as ScCPK1. An open reading frame (ORF) of 1659 bp was detected that encodes a protein of 552 amino acids with a calculated molecular mass of 61.8 kDa. Comparison of the deduced amino acid sequence of ScCPK1 with sequences of other CPKs revealed the highest identity (>90%) to Glycine max and Vigna radiate CPKs. As described for other CPKs, ScCPK1 has a long variable domain (88 aa), an auto-inhibitory domain (31 aa) and a C-terminal calmodulin domain (145 aa) containing four EF-hand calcium binding motifs, which is found in many CPKs. Phylogenetic tree analysis showed that ScCPK1 was closely related to StCPK4 , CmCPK1 and CmCPK2. The entire coding region of ScCPK1 was cloned into pRSETA expression vector and expressed as fusion protein in E.coli. The recombinant ScCPK1 protein was purified to homogeneity by NiNTA affinity chromatography. The recombinant purified ScCPK1 was catalytically active in a calcium-dependent manner. The recombinant ScCPK1 phosphorylated itself and histone IIIS as substrate only in the presence of Ca2+. Phosphoaminoacid analyses showed that ScCPK1 phosphorylates serine and threonine residues of histone IIIS and its autophosphorylation also occurs on serine and threonine residues. ScCPK1 has a pH and temperature optima of 7.5 and 37 °C, respectively. It showed high affinity to histone III-S with a Km of 4.8 µM and had a Vmax of 4.700 pmoles of γ32P incorporation/min/mg at saturating substrate concentrations. The ScCPK1 is ~100fold active and showed 10fold higher affinities to histone III-S than CaCPK1 and CaCPK2, CPKs which were characterized from Cicer arietinum previously in our laboratory (Prakash and Jayabaskaran, 2006). From literature it is known that many CPKs are activated or inhibited by metal ions. (PutnamEvans etal., 1990; Anil and Rao, 2001). The influence of Na+ and Mg2+on the in vitro substrate phosphorylation activity of the recombinant ScCPK1 was tested in this work. Addition of NaCl strongly inhibited ScCPK1 activity. The inhibition of substrate phosphorylation activity by salt implies ionic interactions between the positively charged substrate and the enzyme’s active site. The optimum concentration of Mg2+ for ScCPK1 substrate phosphorylation activity was found to be 810 mM, similar to CaCPK1 and CaCPK2 (Prakash and Jayabaskaran, 2006). However, the activity was inhibited above 10 mM Mg2+suggesting the disruption of ionic interactions between the enzyme and the substrate. The kinase and autophosphorylation activities of the recombinant ScCPK1 were calmodulin independent and sensitive to CaM antagonists’ calmidazolium and W7 (N(6aminohexyl)5chloronaphthalene sulphonate). This indicates that the activation was supported by calmodulin-like domain, which is typical of CPK family. Farmer and Choi (1999), showed that DcCPK1 activity was inhibited by polyamines vizspermine and spermidine, and polylysine. We found that substrate phosphorylation activity of ScCPK1 was inhibited by polyLLysine with an IC50 of 8 M but not the polyamines, spermine and spermidine. An interesting aspect that makes CPKs attractive for research is their functional similarity to mammalian PKCs. There are no structural PKC analogues found in plant genomic data. Similar to PKCs, CPKs are regulated by intracellular Ca2+ signals. There is also experimental evidence that some of the CPKs are additionally activated by phospholipids (Farmer and Choi, 1999; Szczegielniak etal., 2000). We investigated the effects of lipid molecules on the activity of ScCPK1. Phosphorylation of histone IIIS by ScCPK1 was stimulated by phosphatidylethanolamine, phosphatidylserine and phosphatidylinositol in the presence of Ca2+, where as lysophosphatidylcholine, phosphatidylcholine and phosphatidic acid did not increase the enzyme activity. Our data that shows interaction of ScCPK1 with phospholipids supports the idea that this protein kinase could be associated with the membrane. The work from Farmer and Choi (1999), with DcCPK1 suggested that some of the PKClike activities observed in plants may be attributed to CPKs. They also demonstrated that DcCPK1 phosphorylated PKC pseudosubstrate peptide and also was sensitive to staurosporine inhibition. However, the protein kinase inhibitor, staurosporine inhibited the substrate phosphorylation activity of ScCPK1 completely with an IC50 value of 700 nM invitro. But PKC inhibitor PMA was less effective, inhibiting the substrate phosphorylation activity of ScCPK1 to a maximum of 50%, but at a very high concentration (200 nM). Our data suggests that ScCPK1 may not have any features attributable to PKC. We investigated subcellular localization of the ScCPK1. To gain a better understanding of the subcellular localization of the ScCPK1, we generated GFP fusion protein of ScCPK1 and transiently expressed it in Agrobacterium-mediated transformed tobacco BY2 cells. Analysis of the GFP expression patterns in transformed tobacco BY2 cells revealed ScCPK1 localization in the plasma membrane of the transformed tobacco BY2 cells despite lacking consensus myristoylation and palmitoylation motifs (as per in silico analyses). Taking together, our data have demonstrated that ScCPK1 is a Ser/Thr protein kinase and its sub-cellular localization studies revealed that it is localized to plasma membrane. We propose that ScCPK1 is a key component of one or more signaling pathways and plays vital roles in plant development, responses to environmental stimuli and/ or in secondary metabolite biosynthetic gene expression. The involvement of the ScCPK1 as a component of signaling pathways warrants further studies.