Partial purification and characterization of serine and cysteine proteases of Sitophilus zeamais (Coleoptera: Curculionidae) / Purificação e caracterização parciais de serino e cisteíno proteases de Sitophilus zeamais (Coleoptera: Curculionidae)

AUTOR(ES)
DATA DE PUBLICAÇÃO

2009

RESUMO

Serine and cysteine proteases of a susceptible and two resistant strain (one with a cost associated with resistance and other without cost) of the maize weevil Sitophilus zeamais were partially purified using an affinity column of aprotinin-agarose . The highest purification factor was obtained for the resistant no-cost strain, 51.20x, with a yield of 14.8% and specific activity of 875μM∕min∕mg protein. In the SDS-PAGE a band of 56kDa was visible in the three strains, and the resistant no-cost strain also exhibited another band of 70kDa. The enzymes partially purified from the three strains were inhibited by PMSF, TLCK, aprotinin, benzamidine and SBTI, indicating the presence of serine and histidine in the enzyme active center, which is characteristic of trypsin-like serine proteases. The enzyme purified of the resistant strains have higher affinity for the substrate (lower value of KM app), and the catalytic efficiency for the resistant strain nocost was approximately 2-fold higher relative of the susceptible strain. A thiolsepharose affinity column was used to purify the cysteine protease of the three strains. Cysteine proteases partially purified from the three strains exhibited a different behavior in casein degradation and its inhibition by specied inhibitors. The enzyme partially purified from the resistant cost strain was less sensitive to the inhibitor E-64 exhibiting higher I50 compared to the other strains studied. The affinity (i.e. KM app) of the enzyme for the substrate was similar among the strains, but the Vmax app for the enzyme partially purified from the resistant cost strain was 5x greater and of resistant no-cost was 3x greater than the Vmax app of the susceptible strain. The results of this investigation confirm that the pyrethroid-resistant strains have elevated activity of trypsin-like proteases, which has an important role in mitigating the physiological cost associated with maintaining the mechanism of resistance to insecticides in some strains of S. zeamais. The role of cysteine in mitigating the cost associated with the resistance appears to be of secondary relative to that of serine proteases.

ASSUNTO(S)

entomologia agricola enzymes kinetics resistance to insecticides gorgulho do milho enzimas digestivas maize weevil custo fisiológico cinética de enzima digestive enzymes resistência a inseticidas physiological cost

ACESSO AO ARTIGO

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