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Structural studies of three glycosidases /Larsson, Anna, January 2006 (has links)
Diss. (sammanfattning) Uppsala : Uppsala universitet, 2006. / Härtill 5 uppsatser.
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Implication du chaperome de la protéine F508del-CFTR dans son transport intracellulaire et/ou sa dégradation : rôle des lectines EDEMs et la mannosidase du RE / Involvement of F508del-CFTR protein chaperome in its trafficking defect : role of EDEMs lectins and ER mannosidase ISidelarbi, Khadidja 24 November 2017 (has links)
De nombreuses maladies génétiques sont directement liées à la reconnaissance de protéines mal repliées par le contrôle qualité du réticulum endoplasmique (RE), conduisant à leur rétrotranslocation vers le cytosol puis leur dégradation. Dans le cas des glycoprotéines mutées, comme la protéine F508del-CFTR (Cystic Fibrosis Transmembrane conductance Regulator), la démannosylation extensive de leur résidus mannoses constitue une étape clé dans ce processus. L’objectif de ce travail a été d’identifier et de caractériser des molécules capables de rétablir l’expression membranaire de F508del-CFTR en ciblant cette activité enzymatique. Dans un premier temps, nous avons testé des dérivés multivalents basés sur le Deoxymannojirimycin (DMJ), un inhibiteur spécifique de la classe I des α-mannosidases et révélé leur effet correcteur puissant sur F508del-CFTR. Nous avons par la suite mis en évidence leur mécanisme d’action et tenté d’expliquer l’augmentation d’efficacité observée entre les monovalents et les multivalents. Nous nous sommes enfin focalisés sur le rôle des principales mannosidases dans le RE, l’α1,2-mannosidase I du RE (ERManI) et la famille des lectines EDEM (ER degradation-enhancing α-mannosidase-like protein). Nous avons montré par une stratégie de siRNA qu’ERManI, EDEM1 et EDEM2 sont impliquées dans la rétention réticulaire du F508del-CFTR.Notre étude ouvre ainsi de nouvelles perspectives quant à l’identification de nouveaux agents pharmacologiques ciblant ces protéines. / Numerous genetic diseases are directly associated to the recognition of misfolded proteins by the endoplasmic reticulum (ER) quality control, leading to their retention and subsequently their retrotranslocation to the cytosol for degradation. In the case of mutated glycoproteins such as F508del-CFTR (Cystic Fibrosis Transmembrane conductance Regulator), causing CF pathology, mannose trimming is a key step of this process. Our objective was to identify and characterize molecules targeting ER-mannosidases activity, with the goal to restore F508del-CFTR to the plasma membrane.First, we tested multivalent derivatives, based mainly on Deoxymannojirimycin (DMJ), a specific inhibitor of α-mannosidasesI and revealed their better corrective effect on F508del-CFTR and their mechanism of action. Then we explored the mechanism explaining the higher efficiency of the multivalents compared to the monovalent. Finally, we focused on the role of key players of mannose trimming, ER-α1,2-mannosidase I (ERManI) and EDEMs (ER degradation-enhancing α-mannosidase-like protein) proteins on F508del-CFTR trafficking defect. We showed the implication of ERManI, EDEM1 and EDEM2 in F508del-CFTR retention, using a siRNA strategy. In conclusion, our study highlights these proteins as potential pharmacological targets to develop correctors for the F508del-CFTR trafficking defect.
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Conception et synthèse d’iminosucres multivalents bioactifs à motif glycoimidazole : développement d’une méthode de déshydroxylation sélective / Conception and synthesis of bioactive multivalent iminosugars with glycoimidazole motif : development of a selective dehydroxylation methodPichon, Maëva 23 November 2018 (has links)
La multivalence est reconnue en tant qu’outil permettant d’augmenter le pouvoir d’inhibition d’un inhibiteur. La mise en évidence, en 2010, d’un effet multivalent puissant sur l’inhibition des glycosidases par un iminosucre multivalent a ouvert la voie vers la synthèse de clusters toujours plus puissants. En 2016, un composé multivalent a permis d’atteindre une inhibition 173000 fois meilleure que le composé monovalent correspondant, ce qui équivaut à une augmentation record du pouvoir d’inhibition par iminosucre de 4800. La synthèse d’une nouvelle génération d’iminosucres multivalents à ligand affin pour sa cible, pour parvenir à augmenter encore l’effet multivalent, était mon objectif de thèse. Au cours de ce travail, de nouveaux iminosucres multivalents à motif gluco- ou mannoimidazole, très affin des mannosidases, ont été synthétisés. Ces composés multivalents ont permis de travailler sur deux projets parallèles : un projet fondamental et un projet appliqué. Le premier a consisté en l’étude de l’influence de l’affinité du ligand sur l’effet multivalent par mesure du pouvoir d’inhibition sur l’enzyme commerciale répondant le mieux à l’effet multivalent, l’α-mannosidase de Jack-bean. Les résultats montrent l’existence d’un effet multivalent notable mais sa valeur n’est pas aussi importante comparée aux études précédentes. Les iminosucres multivalents pouvant également cibler les glycosidases d’intérêt thérapeutique, nous nous sommes intéressés, dans le cadre du projet appliqué, au traitement de la mucoviscidose. L’idée était de viser les mannosidases I et II du réticulum endoplasmique pour permettre de secourir la protéine CFTR déficiente impliquée dans cette maladie. Malheureusement, aucun effet correcteur ni potentiateur n’a pu être observé pour nos composés. En marge de ces axes principaux, une méthode de déshydroxylation sélective a été optimisée et développée en élargissant son champ d’application et en apportant des preuves mécanistiques. / Multivalency is known to be a tool to drive inhibitors more potent. The multivalent effect, evidenced in 2010 with multivalent iminosugar on glycosidase inhibition, paved the way to the synthesis of multimeric inhibitors even more powerful. In 2016, an iminosugar cluster was reported to be 173 000 times more potent inhibitor compared to the monovalent reference. This is equal to an inhibitory potency enhancement of 4800 per iminosugar. The goal of my PhD was the synthesis of a new generation of multivalent iminosugars very potent towards their target, to keep increasing multivalent effect. New multivalent iminosugars with glycoimidazole motifs, with high affinity towards mannosidases, were synthetised. Those multivalent compounds allowed us to work on two parallel projects: a fundamental and an applied one. The first one focused on the influence of ligand affinity on multivalent effect by measuring the inhibition potency on the most sensitive to multivalent effect commercially available enzyme: the Jack-bean α-mannosidase. Results showed that multivalent effect was maintained but at a lower level than those reported previously. Multivalent iminosugars being also able to target glycosidases of therapeutic interest, we were interested in the treatment of cystic fibrosis. Targeting endoplasmic reticulum mannosidases I and II could restore the CFTR protein whose deficiency is the root cause of the disease. Unfortunately, no corrective or potentiation effect were observed. Aside those principal lines, a selective dehydroxylation method was optimized and developed widening the scope and providing mechanistic proofs.
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Enzymes hydrolysing wood polysaccharides : a progress curve study of oligosaccharide hydrolysis by two cellobiohydrolases and three [beta]-mannanases /Harjunpaa, Vesa. January 1900 (has links) (PDF)
Thesis (Academic dissertation)--University of Helsinki, 1998. / Includes bibliographical references. Also available on the World Wide Web.
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Synthèse d'oligosaccharides pour applications biologiques : aminoéthyle glycoside de sialyl Lewis X et glycosyl-carbasucres / Synthesis of oligosaccharides for biological applications : aminoethyl glycoside of sialyl Lewis X and glycosyl-carbasugarsLu, Dan 25 November 2014 (has links)
Les glucides, les biomolécules les plus abondants dans la nature, sont essentiels pour le maintien et la survie des organismes. Certaines molécules à base de glucides à la fois d'origine synthétique et naturelle sont connues pour leurs activités pharmacologiques et utilisés pour traiter différentes maladies. Deux projets ont été réalisés sur la base d'oligosaccharides biologiquement actifs dans cette thèse. Sialyl Lewis X (sLex), un tétrasaccharide exprimé sur la surface cellulaire d'un tissu embryonnaire et un tissu tumoral, a été découvert pour jouer un rôle important dans l'adhésion entre les cellules tumorales et les cellules endothéliales dans les métastases tumorales à travers la liaison de E-sélectine avec une forte affinité et sélectivité. E-sélectine est un membre de famille des sélectines des molécules d'adhésion cellulaire, exprimée sur l'endothélium tumoral qui est activé par la cytokine. En conséquence, un type de sonde moléculaire basé sur sLex a été conçu et synthétisé dans le but de diagnostiquer le cancer. Aminoéthyle glycoside de sLex 36 a été efficacement préparé à partir de quatre synthons monosaccharidiques par sept étapes d’une manière hautement stéréosélective et régiosélective avec un rendement total de 18%. La sonde moléculaire conjuguant 36 avec une protéine marqué a été montré capable de visualiser une tumeur implantée chez la souris par imagerie in vivo, alors que le contrôle négatif a donné seulement un profil de circulation vasculaire. Ce travail est actuellement dans le processus d'un dépot de brevet. D'autre part, le mécanisme de traitement des N-glycanes par l'endo-α-mannosidase et les inhibiteurs de cette enzyme ont été étudiés. Décorations de N-glycane existent sur la plupart des protéines eucaryotes et leurs compositions aberrantes sont associés à diverses maladies, y compris les infections virales et le cancer. Endo-α-mannosidase hydrolyse la liaison α-1,2-mannosidique entre le mannose en glucose-substitué et le reste de la N-glycane dans l'appareil de Golgi, en fournissant une voie indépendante de glucosidase I et II pour la maturation de N-glycane. Le but de présente étude est de confirmer le mécanisme catalytique nouvellement proposé pour endo-α-mannosidase: la participation du groupe voisin via un 1,2-anhydro intermédiaire de sucre. Etant donné que cet intermédiaire est instable, le glycosyl-1,2-β-anhydro carbasucre 53 a été conçu et synthétisé pour soutenir l'existence de cet intermédiaire pour l'hydrolyse catalysée par glycosidase. De plus, le glycosyl-1,2-β-aziridine carbasucre 64 et le glycosyl-1,2-oléfinique carbasucre 63 ont été conçus et synthétisés comme nouveaux inhibiteurs d’endo-α-mannosidase pour le développement d'agents thérapeutiques. En outre, le glycosyl-1,2-α-anhydro carbasucre 52 et le glycosyl-1,2-α-aziridine carbasucre 65 ont également été synthétisés comme références. Tous les composés cibles ont été préparés de manière efficace à partir du même glycosyl-carbasucre qui a été obtenu de D-(+)-maltose par plusieurs étapes de protections, de modifications, du réarrangement de Claisen. L'évaluation biologique est en cours. / As the most abundant biomolecules found in nature, carbohydrates are essential for sustaining and survival of organisms. Some carbohydrate-based molecules both of synthetic and natural origin are known for their pharmacological activities and used to treat different diseases. Two projects were carried out based on biologically active oligosaccharides in this thesis. Sialyl Lewis X (SLex), which is a tetrasaccharide expressed on cell surface of embryonic tissue and tumor tissue, was discovered to play an important role in the adhesion between tumor cells and blood endothelial cells in tumor metastases through binding E-selectin with high affinity and selectivity. E-selectin is a member of the selectin family of cell adhesion molecules, expressed on cytokine activated tumoral endothelium. Accordingly, a kind of molecular probe based on sLex was designed and synthesized for the purpose of diagnosing cancer. Aminoethyl glycoside of sLex 36 was efficiently prepared from four monosaccharide building blocks through seven steps with high stereoselectivity and regioselectivity in 18% total yield. The molecular probe conjugating 36 with a labelled scaffold protein was shown to visualize an implanted tumor in the mouse by in vivo imaging, whereas the negative control gave only a vascular circulation profile. This work is currently in the process of a patent application. On the other hand, N-linked glycans processing mechanism by endo-α-mannosidase and this enzyme’s inhibitors were studied. N-glycan decorations exist on the majority of eukaryotic proteins and their aberrant compositions are associated with various diseases, including viral infection and cancer. Endo-α-mannosidase hydrolyzes the α-1,2-mannosidic bond between the glucose-substituted mannose and the rest of the N-glycan in Golgi apparatus, providing a glucosidase I and II independent pathway for the maturation of N-glycan. The aim of the present study is to confim the newly proposed catalytic mechanism for endo-α-mannosidase: neighboring group participation via a 1,2-anhydro sugar intermediate. Since this intermediate is unstable, glycosyl-1,2-β-anhydro carbasugar 53 was designed and synthesized to support the existence of this intermediate for glycosidase-catalyzed hydrolysis. Moreover, glycosyl-1,2-β-aziridine carbasugar 64 and glycosyl-1,2-olefinic carbasugar 63 were designed and synthesized as novel endo-α-mannosidase inhibitors for the development of therapeutic agents. In addition, glycosyl-1,2-α-anhydro carbasugar 52 and glycosyl-1,2-α-aziridine carbasugar 65 were also synthesized as compare. All the target compounds were prepared efficiently from the same glycosyl-carbasugar that was obtained from D-(+)-maltose through several steps of protections, modifications, and Claisen rearrangement. The biological evaluation is underway.
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Novel Biologically Active Chalcogenides : Synthesis And ApplicationsSivapriya, K 07 1900 (has links)
The thesis is divided in to five chapters.
Chapter I: Synthesis of New thiolevomannosan derivatives
In this chapter, a general and efficient method for the synthesis of conformationally locked thiosugars has been discussed. An unprecedented synthesis of a novel thioorthoester and its synthetic utility in glycosylation has been demonstrated.
Chapter II: Studies on -Mannosidase Inhibitors
The synthesis and evaluation of novel, conformationally locked glycomimic, thiolevomannosan and its analogs sulfoxide and sulfone starting from readily available D-mannose is discussed in this chapter. This is the first report of thiosugar derivatives with enhanced potency compared to kifunensine. Docking and biochemical studies have been discussed.
Chapter III: Studies on Novel Cyclic Tetraselenides of Mannose
In this chapter, the syntheses and structural properties of novel cyclic tetraselenides starting from mannose have been discussed. These tetraselenides are the first of their kind where all four selenium atoms are arranged in a cyclic manner as the backbone of mannose. X-ray structures have been reported for the tetraselenides.
Chapter IV: Novel Chalcogenides of Uridine and Thymidine: Synthesis and Applications
An efficient and simple method to synthesise disulfides and diselenides of thymidine and uridine derivatives has been demonstrated in this chapter. The utility of these disulfides in various ring opening reactions as well in Michael addition reactions has been demonstrated.
Chapter V: Studies on New, Potent Urease Inhibitors
In this chapter, a facile one-pot synthesis of thio and selenourea derivatives under mild conditions by the reaction of amines and commercially available Viehe’s iminium salt in the presence of benzyltriethylammonium tetrathiomolybdate as sulfur transfer reagent and tetraethylammonium tetraselenotungstate as selenium transfer reagent has been discussed. A few of the urea derivatives have shown potent inhibitor activity in the nanomolar range for jackbean urease.
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α-Manosidases intestinais da larva de Tenebrio molitor (Coleoptera) / α-Mannosidases intestinal from Tenebrio molitor (Coleoptera) larvaeNathália Ramalho Moreira 19 September 2008 (has links)
Os estudos da função intestinal foram particularmente estimulados após a conscientização de que o tubo digestivo é uma enorme interface relativamente pouco protegida entre o inseto e o meio ambiente e pode ser usado como alvo para controle de pragas. Neste contexto, nosso trabalho envolve a purificação e caracterização de uma α-manosidase solúvel e a detecção de uma α-manosidase de membrana. As α-manosidases pertencem a uma família de exoglicosidases as quais hidrolisam resíduos de α-D-manosil a partir de terminais não redutores de oligossacarídeos. Estas enzimas são implicadas no catabolismo de carboidratos e na via de N-glicosilação protéica em insetos, mas pouco se sabe sobre a bioquímica destas glicosidases. O Tenebrio molitor é um Coleoptera bastante estudado pelo nosso laboratório devido a sua relevância como praga agrícola e o seu posicionamento em um ponto estratégico da árvore filogenética de insetos. O estudo de distribuição desta enzima mostrou que a α-manosidase encontra-se, principalmente, como uma enzima solúvel no conteúdo anterior e médio do intestino médio, mas também existe uma atividade significante na fração de membrana. Para confirmar a existência desta enzima de membrana, microvilosidades foram purificadas por precipitação diferencial com cálcio. A enzima aminopeptidase foi utilizada como marcadora, uma vez que sabe-se que esta enzima é uma típica de membrana microvilar. Como a α-manosidase solúvel é majoritária demos início a sua purificação e posterior caracterização. A sua purificação foi realizada utilizando uma combinação de quatro passos de cromatografia: Uma de troca iônica em Hitrap Q XL (Amersham/Bioscience), duas filtrações em gel, uma em Superdex 75 e outra em Superdex 200 (Amersham/Bioscience) usando o sistema AKTA, e o último passo é uma hidrofóbica em Phenyl Superose. Nós observamos a presença de dois picos de atividade nomeados de Man 1 e Man 2, sugerindo a existência de duas α- manosidases solúveis, que se diferem quanto a hidrofobicidade. O pH ótimo das α-manosidases é de 5,6 e sua massa molecular, determinada por cromatografia de 8 filtração em gel, é de 123 kDa, e no SDS-PAGE observamos uma única banda de 70 KDa, indicando a existência de duas subunidades. Em um gel nativo revelado com o substrato fluorescente (metilumbelliferil-α-D-manopiranosídeo) nota-se somente uma banda de atividade. A Man 2 possui pI de 3,38. α-manosidases de T. molitor seguem a cinética de Michaelis-Menten com Km para o substrato p-nitrofenil-α-D-manopiranosídeo de 0,84 mM para Man 1 e 0,62 mM para Man 2. Também foram feitos ensaios de inibição com dois inibidores que sabidamente inibem carboidrases, um é o deoximanojirimicina e o outro é a swainsonina. O Ki encontrado para o primeiro inibidor foi de 0,12 mM para Man 1 e 0,15 mM para Man 2 e o Ki para o segundo inibidor foi de 67,8 nM para Man 1 e 63 nM para Man 2, sendo ambos inibidores competitivos. O fato destas enzimas serem inibidas apenas por Swainsonina em concentrações razoáveis, permite a sua classificação como tipo II. Isso sugere que elas são derivadas da forma lisossômica, embora apresente pH ótimo alterado. / Studies of intestinal function were prompted after noticing that the gut is a huge and relatively unprotected interface between the insect and the environment and can thus be used as a target for pest control. In this context, our work involves the purification and characterization of an soluble alpha-mannosidase and detection of a membrane α-mannosidase. α-Mannosidases are a family of exoglycosidases which hydrolyse α-D-mannosyl residues from terminal non-reducing end of oligossacharides. These enzymes are implicated in the catabolism of carbohydrates and N-linked protein glycosylations in insects, but little is known on this biochemistry. T.molitor is a Coleoptera studied in our laboratory because of its relevance as agricultural pest and its position at a strategic point in the phylogenetic tree of insects. α-Mannosidase is more active in the anterior and middle midgut content of T.molitor larvae, although there is a significant activity in the membrane fraction. To confirm the existence of this membrane enzyme, microvilli were purified by differential precipitation with calcium. Aminopeptidase was used as a marker, since it is known that it is a typical microvilar membrane enzyme. Most α-mannosidase activity is soluble. This led us to purify this enzyme for further characterization. The purification of T. molitor α-mannosidase was attained by using a combination of four chromatographic steps: an anion-exchange chromatography in Hitrap Q XL (Amersham/Bioscience), two gel filtration chromatographies, one in Superdex 200 and another in Superdex 75 (Amersham/Bioscience) using an AKTA system, and the last step is a Hydrophobic cromatography in Phenyl Superose. Two peaks of activity were resolved: Man 1 and Man 2, suggesting the existence of two soluble α-mannosidases, differing only in hydrophobicity. The optimum pH of the α- mannosidases is 5.6 and the molecular mass is 123 KDa determined by gel filtration and 70 KDa in the case of SDS PAGE. This suggests that the holoenzyme has two subunits. In a native gel revealed with the fluorescent substrate (methylumbelliferyl-α-D-mannopyranoside) only one band of activity is seen. Man 2 has pI 3.38. T. molitor α-mannosidases followed Michaelis-Menten kinetics with a Km value of 0.84 mM for Man 1 and 0.62 mM for Man 2 using p-nitrophenyl-α-D-mannopyranoside as substrate. Inhibition tests were made with typical inhibitors of α-mannosidases: one is the 1-deoxymannojirimycin and the other is the Swainsonine. The Ki for the first was of 0.12 mM for Man 1 and 0.15 mM for Man 2 and for the second was 67.8 nM for Man 1 and 63 nM for Man 2. Both were competitive inhibitors. The fact that the enzymes are inhibited only by swainsonine in reasonable concentrations, allows us to classify them as type II. This suggests that they are derived from the lysosomal form, although they have an altered optimum pH.
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α-Manosidases intestinais da larva de Tenebrio molitor (Coleoptera) / α-Mannosidases intestinal from Tenebrio molitor (Coleoptera) larvaeMoreira, Nathália Ramalho 19 September 2008 (has links)
Os estudos da função intestinal foram particularmente estimulados após a conscientização de que o tubo digestivo é uma enorme interface relativamente pouco protegida entre o inseto e o meio ambiente e pode ser usado como alvo para controle de pragas. Neste contexto, nosso trabalho envolve a purificação e caracterização de uma α-manosidase solúvel e a detecção de uma α-manosidase de membrana. As α-manosidases pertencem a uma família de exoglicosidases as quais hidrolisam resíduos de α-D-manosil a partir de terminais não redutores de oligossacarídeos. Estas enzimas são implicadas no catabolismo de carboidratos e na via de N-glicosilação protéica em insetos, mas pouco se sabe sobre a bioquímica destas glicosidases. O Tenebrio molitor é um Coleoptera bastante estudado pelo nosso laboratório devido a sua relevância como praga agrícola e o seu posicionamento em um ponto estratégico da árvore filogenética de insetos. O estudo de distribuição desta enzima mostrou que a α-manosidase encontra-se, principalmente, como uma enzima solúvel no conteúdo anterior e médio do intestino médio, mas também existe uma atividade significante na fração de membrana. Para confirmar a existência desta enzima de membrana, microvilosidades foram purificadas por precipitação diferencial com cálcio. A enzima aminopeptidase foi utilizada como marcadora, uma vez que sabe-se que esta enzima é uma típica de membrana microvilar. Como a α-manosidase solúvel é majoritária demos início a sua purificação e posterior caracterização. A sua purificação foi realizada utilizando uma combinação de quatro passos de cromatografia: Uma de troca iônica em Hitrap Q XL (Amersham/Bioscience), duas filtrações em gel, uma em Superdex 75 e outra em Superdex 200 (Amersham/Bioscience) usando o sistema AKTA, e o último passo é uma hidrofóbica em Phenyl Superose. Nós observamos a presença de dois picos de atividade nomeados de Man 1 e Man 2, sugerindo a existência de duas α- manosidases solúveis, que se diferem quanto a hidrofobicidade. O pH ótimo das α-manosidases é de 5,6 e sua massa molecular, determinada por cromatografia de 8 filtração em gel, é de 123 kDa, e no SDS-PAGE observamos uma única banda de 70 KDa, indicando a existência de duas subunidades. Em um gel nativo revelado com o substrato fluorescente (metilumbelliferil-α-D-manopiranosídeo) nota-se somente uma banda de atividade. A Man 2 possui pI de 3,38. α-manosidases de T. molitor seguem a cinética de Michaelis-Menten com Km para o substrato p-nitrofenil-α-D-manopiranosídeo de 0,84 mM para Man 1 e 0,62 mM para Man 2. Também foram feitos ensaios de inibição com dois inibidores que sabidamente inibem carboidrases, um é o deoximanojirimicina e o outro é a swainsonina. O Ki encontrado para o primeiro inibidor foi de 0,12 mM para Man 1 e 0,15 mM para Man 2 e o Ki para o segundo inibidor foi de 67,8 nM para Man 1 e 63 nM para Man 2, sendo ambos inibidores competitivos. O fato destas enzimas serem inibidas apenas por Swainsonina em concentrações razoáveis, permite a sua classificação como tipo II. Isso sugere que elas são derivadas da forma lisossômica, embora apresente pH ótimo alterado. / Studies of intestinal function were prompted after noticing that the gut is a huge and relatively unprotected interface between the insect and the environment and can thus be used as a target for pest control. In this context, our work involves the purification and characterization of an soluble alpha-mannosidase and detection of a membrane α-mannosidase. α-Mannosidases are a family of exoglycosidases which hydrolyse α-D-mannosyl residues from terminal non-reducing end of oligossacharides. These enzymes are implicated in the catabolism of carbohydrates and N-linked protein glycosylations in insects, but little is known on this biochemistry. T.molitor is a Coleoptera studied in our laboratory because of its relevance as agricultural pest and its position at a strategic point in the phylogenetic tree of insects. α-Mannosidase is more active in the anterior and middle midgut content of T.molitor larvae, although there is a significant activity in the membrane fraction. To confirm the existence of this membrane enzyme, microvilli were purified by differential precipitation with calcium. Aminopeptidase was used as a marker, since it is known that it is a typical microvilar membrane enzyme. Most α-mannosidase activity is soluble. This led us to purify this enzyme for further characterization. The purification of T. molitor α-mannosidase was attained by using a combination of four chromatographic steps: an anion-exchange chromatography in Hitrap Q XL (Amersham/Bioscience), two gel filtration chromatographies, one in Superdex 200 and another in Superdex 75 (Amersham/Bioscience) using an AKTA system, and the last step is a Hydrophobic cromatography in Phenyl Superose. Two peaks of activity were resolved: Man 1 and Man 2, suggesting the existence of two soluble α-mannosidases, differing only in hydrophobicity. The optimum pH of the α- mannosidases is 5.6 and the molecular mass is 123 KDa determined by gel filtration and 70 KDa in the case of SDS PAGE. This suggests that the holoenzyme has two subunits. In a native gel revealed with the fluorescent substrate (methylumbelliferyl-α-D-mannopyranoside) only one band of activity is seen. Man 2 has pI 3.38. T. molitor α-mannosidases followed Michaelis-Menten kinetics with a Km value of 0.84 mM for Man 1 and 0.62 mM for Man 2 using p-nitrophenyl-α-D-mannopyranoside as substrate. Inhibition tests were made with typical inhibitors of α-mannosidases: one is the 1-deoxymannojirimycin and the other is the Swainsonine. The Ki for the first was of 0.12 mM for Man 1 and 0.15 mM for Man 2 and for the second was 67.8 nM for Man 1 and 63 nM for Man 2. Both were competitive inhibitors. The fact that the enzymes are inhibited only by swainsonine in reasonable concentrations, allows us to classify them as type II. This suggests that they are derived from the lysosomal form, although they have an altered optimum pH.
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