Propriedades bioquímicas e cinético-enzimáticas de cisteíno- proteases do intestino médio da lagarta da soja

• Autor(a) principal: Mendonça, Eduardo Gomes de
• Data de Publicação: 2008
• Tipo de documento: Dissertação
• Idioma: por
• Título da fonte: LOCUS Repositório Institucional da UFV
• Texto Completo: http://locus.ufv.br/handle/123456789/2394
• topic: Soja; Lagarta da soja; Cisteíno-proteases; Soybean; Velvetbean caterpillar; Cysteine-proteases; CNPQ::CIENCIAS BIOLOGICAS::BIOQUIMICA::ENZIMOLOGIA

Protease inhibitors that react with insect specific proteases are candidates to have their genetic code inserted in genetically modified plants. The first step to achieve this is the characterization of proteolytic enzymes of these insects intestines. Cysteine-protease of the soluble and insoluble fractions from the midgut of Anticarsia gemmatalis were characterized using the substrate LBApNA. The Soluble Fraction, called Fraction I, was obtained from the suspension medium after nine cycles of freezing and thawing of the intestine of A. gemmatalis, and the Insoluble Fraction, called Fraction II, was obtained from the maceration with detergent Brij 35 of the resulting pellet of the Fraction I. Two pH values were found with high activity in both Fractions they were pH 3.6 and 8.0 for the Fraction I and 4.6 and 8.0 for the Fraction II. And the peak of activity when tested the influence of temperature was at 35oC for the Fraction I and 60°C for the Fraction II. The KM app found for the Fractions I and II were 2.28 mM and 0.44 mM, respectively, and the Vmax app for the respective Fractions were 297.68 nM.s-1 and 122.95 nM.s-1. Four protease inhibitors were tested, which one from one class of protease: TLCK (inhibitor of serine-protease), E-64 (inhibitor for cysteine-protease), EDTA (inhibitor of metallo-protease) and Pepstatin A (inhibitor of aspartyl-protease). The higher inhibition was observed when E-64 was added to the medium for both Fractions, once that all concentrations tested decreased significantly the activity of cysteine- protease. An increase of TLCK into the medium made a gradual decrease in the activity of cysteine-protease, due to the reaction with the amino acid histidine residue in the catalytic triad, which is also part of the triad of cisteine-protease. The effect of EDTA in the activity of cysteine-protease of A. gemmatalis on L-BApNA shows the difference between the two Fractions analyzed. The Fraction I do not depend on the divalent ions as Ca2 + for its activity. However, the Fraction II is a calcium- dependent, since EDTA reduced significantly its activity. The Pespstatin A did not influenced the cysteine-proteases of the Fraction I, exerting little influence on the Fraction II.