Identificação de atividades ATPásica e Amilásica em larvas de Pachymerus nucleorum (Fabricius, 1792) (Coleoptera: Chrysomelidae: Bruchinae)

Abstract

ABSTRACT - CHAPTER I - Pachymerus nucleorum (Fabricius, 1792) is a beetle specie of the Bruchinae sub-family, family Chysomelidae, that principal host of this insect is a palm babassu (Orbinya spp). The objective of this work was identify and to characterize the ATPase activity in P. Nucleorum larvae. The P. nucleorum larvae guts were discartated and the greasy body was homogenized in 50 mM phosphate buffer, pH 7,5 containing protease inhibitor (0,2 ug/mL Aprotinine, 1 mM Benzamidine, 250 mM sucrose and 1 mM EDTA), after that the samples were centrifugated at 15000xg for 30 minutes, 4 º C. Amonium sulfate was added to the supernatant fraction (S1) and centrifugated at 45000xg for 30 minutes, 4 º C. The precipitated fraction (P2) was ressuspended in Imidazol buffer, pH 8,0 containing 1 mM EDTA and 0,2 mM β-mercaptoetanol and centrifugated at 45000xg for 30 minutes, 4 º C. Final concentrations of 10 mM ATP and 5 mM MgCl2 was added to the P3 fraction and centrifugated. The fraction P4 presented Mg-ATPase e Co- ATPase activity and main polipeptides with 180, 54, 43 and 17 kDa. The Mg- ATPase activity was not estimulated by calcium or calcium/calmoduline, and KEDTA ATPase activity was absent. The Mg-ATPase was not estimulated by other cátions and was slightly inhibited by vanadate. The Mg-GTPase activity was about 50% of Mg-ATPase activity. ABSTRACT - CHAPTER II - Pachymerus nucleorum (Fabricius, 1792) is a beetle specie of the Bruchinae sub-family, family Chysomelidae and principal host is coconut of the palm babassu (Orbinya spp), typical of the varzea zones and river valleys. The objective in this work was identify and to characterize the amylolytic activity in P. nucleorum larvae guts. The guts were immediately homogenized in phosphatate buffer 50 mM pH 7,5 containing protease inhibitor (Aprotinine 0,2 ug/mL and Benzamidine 1 mM), sucrose 250 mM and EDTA 1 mM, after that the samples were centrifugated at 15000xg for 30 minutes, 4 º C. The supernatant fraction (S1) presented higher amylolytic activity in relation to the precipitated fraction (P1). Analyses in SDS-PAGE revealed polipeptides of 62 kDa, in fraction P1 and in fraction S1 polipetides of 12 and 40 kDa. The fraction S1 presented maximum amylolytic activity in 50°C, and estimulation of the activity in 20% and 45% in presence of calcium 0,5 mM or sodium chloride 14 mM, respectively. The preparation containing sucrose and EDTA presented higher amylolytic activity in relation to the preparation without sucrose and EDTA. An increase in the amylolytic activity was observed in both preparations when stored in refrigerator, however the preparation without sucrose and EDTA presented higher increase in this activity.