Devido aos problemas, ambientais e à população humana, causados pelos inseticidas químicos, novas investigações para o controle de insetos tornaram-se necessárias. Para isto, um maior conhecimento sobre a fisiologia digestiva dos insetos torna-se essencial, visto que o intestino é uma interface, grande e relativamente desprotegida, entre o inseto e o seu ambiente. Neste contexto, nosso trabalho envolve a purificação e caracterização de uma dipeptidase e de uma carboxipeptidase digestivas de T. molitor. Estas enzimas, compreendem as classes de enzimas digestivas de insetos menos estudadas. O estudo de distribuição das atividades de dipeptidase e carboxipeptidase nas diferentes regiões do intestino médio de larvas de T. molitor mostrou que essas enzimas encontram-se majoritariamente no conteúdo luminal. Para a purificação da dipeptidase e carboxipeptidase foram utilizadas cromatografias de troca iônica e filtração em gel. A carboxipeptidase purificada era muito instável para estudos mais detalhados. O estudo dos parâmetros cinéticos mostrou que a dipeptidase digestiva de T. molitor possui massa molecular de 38,6 kDa, pH ótimo 7,4, baixa solubilidade a fenantrolina e parece preferir dipeptídeos com cadeia lateral volumosa na posição P1. Devido a essas propriedades, mesmo sendo solúvel, assemelha-se a dipeptidase de membrana (EC 3.4.13.19). / Because of adverse effects caused by insecticides in environment and in animals and humans, new methods for insect control are necessary. Knowledge on insect digestive physiology may be instrumental in this direction, as the gut is the major interface between insect and its environment. Our work involves the purification and characterization of a digestive carboxypeptidase and a digestive dipeptidase from Tenebrio molitor. Those enzymes comprise the class of insect digestive enzymes less studied. Distribution studies showed that T. molitor dipeptidase and carboxypeptidase are more active in the midgut content. The purification of T. molitor dipeptidase and carboxypeptidase was attained using a combination of anion-exchange chromatographies and gel filtration. The optimum pH of dipeptidase is 7.6 and the carboxypeptidase is 7.4. The kinetic parameters showed that the T. molitor digestive dipeptidase prefers as substrates dipeptides having a large lateral chain in P1 position.

Devido aos problemas, ambientais e à população humana, causados pelos inseticidas químicos, novas investigações para o controle de insetos tornaram-se necessárias. Para isto, um maior conhecimento sobre a fisiologia digestiva dos insetos torna-se essencial, visto que o intestino é uma interface, grande e relativamente desprotegida, entre o inseto e o seu ambiente. Neste contexto, nosso trabalho envolve a purificação e caracterização de uma dipeptidase e de uma carboxipeptidase digestivas de T. molitor. Estas enzimas, compreendem as classes de enzimas digestivas de insetos menos estudadas. O estudo de distribuição das atividades de dipeptidase e carboxipeptidase nas diferentes regiões do intestino médio de larvas de T. molitor mostrou que essas enzimas encontram-se majoritariamente no conteúdo luminal. Para a purificação da dipeptidase e carboxipeptidase foram utilizadas cromatografias de troca iônica e filtração em gel. A carboxipeptidase purificada era muito instável para estudos mais detalhados. O estudo dos parâmetros cinéticos mostrou que a dipeptidase digestiva de T. molitor possui massa molecular de 38,6 kDa, pH ótimo 7,4, baixa solubilidade a fenantrolina e parece preferir dipeptídeos com cadeia lateral volumosa na posição P1. Devido a essas propriedades, mesmo sendo solúvel, assemelha-se a dipeptidase de membrana (EC 3.4.13.19). / Because of adverse effects caused by insecticides in environment and in animals and humans, new methods for insect control are necessary. Knowledge on insect digestive physiology may be instrumental in this direction, as the gut is the major interface between insect and its environment. Our work involves the purification and characterization of a digestive carboxypeptidase and a digestive dipeptidase from Tenebrio molitor. Those enzymes comprise the class of insect digestive enzymes less studied. Distribution studies showed that T. molitor dipeptidase and carboxypeptidase are more active in the midgut content. The purification of T. molitor dipeptidase and carboxypeptidase was attained using a combination of anion-exchange chromatographies and gel filtration. The optimum pH of dipeptidase is 7.6 and the carboxypeptidase is 7.4. The kinetic parameters showed that the T. molitor digestive dipeptidase prefers as substrates dipeptides having a large lateral chain in P1 position.

<p>Enzymes are useful tools as specific analytical reagents. Two different analysis methods were developed for use in the separate fields of protein science and organic synthesis. Both methods rely on the substrate specificity of enzymes. Enzyme catalysis and substrate specificity is described and put in context with each of the two developed methods.</p><p>In <strong>paper I </strong>a method for C-terminal peptide sequencing was developed based on conventional Carboxypeptidase Y digestion combined with matrix assisted laser desorption/ionization mass spectrometry. An alternative nucleophile was used to obtain a stable peptide ladder and improve sequence coverage.</p><p>In paper<strong> II </strong>and <strong>III</strong>, three different enzymes were used for rapid analysis of enantiomeric excess and conversion of O-acylated cyanohydrins synthesized by a defined protocol. Horse liver alcohol dehydrogenase,<em> Candida antarctica</em> lipase<strong> </strong>B<strong> </strong>and pig liver esterase were sequentially added to a solution containing the O-acylated cyanohydrin. Each enzyme caused a drop in absorbance from oxidation of NADH to NAD⁺. The conversion and enantiomeric excess of the sample could be calculated from the relative differences in absorbance.</p>

Enzymes are useful tools as specific analytical reagents. Two different analysis methods were developed for use in the separate fields of protein science and organic synthesis. Both methods rely on the substrate specificity of enzymes. Enzyme catalysis and substrate specificity is described and put in context with each of the two developed methods. In paper I a method for C-terminal peptide sequencing was developed based on conventional Carboxypeptidase Y digestion combined with matrix assisted laser desorption/ionization mass spectrometry. An alternative nucleophile was used to obtain a stable peptide ladder and improve sequence coverage. In paper II and III, three different enzymes were used for rapid analysis of enantiomeric excess and conversion of O-acylated cyanohydrins synthesized by a defined protocol. Horse liver alcohol dehydrogenase, Candida antarctica lipase B and pig liver esterase were sequentially added to a solution containing the O-acylated cyanohydrin. Each enzyme caused a drop in absorbance from oxidation of NADH to NAD+. The conversion and enantiomeric excess of the sample could be calculated from the relative differences in absorbance. / QC 20101108

Glutamate carboxypeptidase II (GCPII), also known as prostate-specific membrane antigen (PSMA), is a membrane metallopeptidase overexpressed on most prostate cancer cells. Additionally, GCPII also attracted neurologists' attention because it cleaves neurotransmitter N-acetyl-L-aspartyl-L-glutamate (NAAG). Since NAAG exhibits neuroprotective effects, GCPII may participate in a number of brain disorders, which were shown to be ameliorated by GCPII selective inhibitors. Therefore, GCPII has become a promising target for imaging and prostate cancer targeted therapy as well as therapy of neuronal disorders. Globally, prostate cancer represents the second most prevalent cancer in men. With the age, most men will develop prostate cancer. However, prostate tumors are life threatening only if they escape from the prostate itself and start to spread to other tissues. Therefore, considerable efforts have been made to discover tumors earlier at more curable stages as well as to target aggressive metastatic cancers that have already invaded other tissues and become resistant to the standard treatment. Since patients undergoing a conventional therapy (a combination of chemotherapy and surgery) suffer from severe side effects, more effective ways of treatment are being searched for. Novel approaches include selective...