Influência de inibidores proteicos e não-proteicos de proteases digestivas e caracterização bioquímico-cinética de cisteíno-proteases parcialmente purificadas de Anticarsia gemmatalis

• Ano de defesa: 2012
• Autor(a) principal: Mendonça, Eduardo Gomes de
• Orientador(a): Guedes, Raul Narciso Carvalho
• Banca de defesa: Visôtto, Liliane Evangelista , Silva, Camila Rocha da , Campos, Wellington Garcia
• Tipo de documento: Tese
• Tipo de acesso: Acesso aberto
• Idioma: por
• Instituição de defesa: Universidade Federal de Viçosa
• Programa de Pós-Graduação: Doutorado em Bioquímica Agrícola
• Departamento: Bioquímica e Biologia molecular de plantas; Bioquímica e Biologia molecular animal
• País: BR
• Palavras-chave em Português: Inibidores de proteases; Cisteíno-proteases; Anticarsia gemmatalis
• Palavras-chave em Inglês: Protease inhibitors; Cysteine-proteases; Anticarsia gemmatalis
• Área do conhecimento CNPq: CNPQ::CIENCIAS BIOLOGICAS::BIOQUIMICA::ENZIMOLOGIA
• Link de acesso: http://locus.ufv.br/handle/123456789/315

The protease inhibitors have protective effect of plants against damage from various types of agricultural pests, inhibiting significantly the activity of proteolytic enzymes in the gut of insects. Ingestion of protease inhibitors leads insects to use free amino acids in the synthesis of more digestive proteases, in order to compensate forms of these enzymes unavailable to catalysis, precisely because they are in the form of enzyme-inhibitor complex (EI). Consequently, instead of using bioavailable amino acids for synthesis of proteins necessary for growth, development, maintenance and breeding, insects using the amino acid reservation for biosynthesis of new proteases to perform the hydrolysis of the diet protein. The biochemical and kinetic characterization of hydrolytic enzymes in the gut of insects has been made to understand the complex system of proteases of insects from the view of the structure/function. Thus, the active centers of these enzymes can be mapped through enzyme kinetics and protein chemistry, with the goal of producing protease inhibitors specific for each. In this work we selected protease inhibitors SBBI, SKTI and E-64 in order to evaluate its effects on larval development of Anticarsia gemmatalis and on the activity of some digestive enzymes such as total proteases, trypsin-like serine proteases and cysteine proteases. These inhibitors were added individually on artificial diet at a concentration of 0.1% (w/v) and first instar larvae were fed to complete their larval cycle. The three inhibitors used caused injury in the development of larvae of A. gemmatalis, increasing the larval cycle and affecting the weight gain of the larvae to the pupal stage. The concentration of PI used was insufficient to cause the inhibitory effect of proteases present in the gut of A. gemmatalis. But the profile of proteases coincided with the profile of specific proteases trypsin-like and cysteine proteases, and the activity serine-proteases were higher than cysteine proteases. It was also done partial purification of midgut cysteine proteases of Anticarsia gemmatalis using differential precipitation with 30 - 60% (NH4)2SO4 and affinity chromatography with aprotinin-agarose column. The proteins not bound to the column, ie all the proteins present in the crude extract with the exception of serine proteases associated with aprotinin-agarose, which showed proteolytic activity were pooled enzyme and characterized using the synthetic substrate L-BApNA in the presence the serine protease inhibitor benzamidine. Observed peak activity at pH values 4.0, 5.5 and 8.0, being the best activity in pH 8.0. Regarding the influence of temperature, we observed two more discrete peaks activity at 20°C and between 35 - 45ºC, and a pronounced peak of activity 55 - 60ºC. The KM app obtained was 0.74 mM and Vmax app was 0.8 μM.min-1. Analysis of the calcium ions effect showed that partially purified cysteine proteases are calcium-dependent and showed a better activity in 30 mM CaCl2. The presence of the serine protease inhibitor benzamidine and the reducing agent DTT in the reaction medium together with the two inhibitors of serine proteases TLCK and aprotinin proved the activity of cysteine proteases tested in this study.