Intestinal molecular physiology of Dysdercus peruvianus (Hemiptera) / Fisiologia molecular intestinal de Dysdercus Peruvianus (Hemiptera)

AUTOR(ES)
DATA DE PUBLICAÇÃO

2008

RESUMO

After identification of cathepsins L in vitro assays and in zimograms we began to purify this enzyme in insect midgut. The region V2 was selected as a source of material for purifying a cysteine proteinase because it contains most of the activity of that proteinase. After several attempts to purify this proteinase, an effective process was developed that avoid autolysis with methyl methanethiosulfonate (MMTS), a sulfhydryl-reactive and reversible sulfonating reagent for thiol-containing molecules. The purify process was made by three chromatographic steps (anion-exchange column, gel filtration column and affinity column in this order), where two cysteine proteinase were purified, cys 1 and cys 2 with 32 and 45 kDa (SDS-PAGE). The two cysteine proteinases have the same pH optimum of 6.3. Besides that, these enzymes were thermicaly inactivated following apparent first-order kinetics with a half-life of 5 min (cys1) and 4.8 min (cys2) at 40 ºC. Both Cys are inhibited by E-64 with a KD of 17.3 nM (Cys 1) and 7.11 nM (Cys2). Both Cys are more active on Z-FR-MCA than on Z-RR-MCA, suggesting they are cathepsins-L. With purpose of describe the molecular mechanisms underlying physiological phenomena in midgut of the Hemiptera Dysdercus peruvianus a cDNA library was prepared from midgut mRNA. We used ESTs from this library to identify transcripts genes related with glucose transport proteins besides digestive enzymes. Analysis of 1053 high-quality expressed sequence tags (ESTs) yielded 903 unique sequences comprised of 62 contigs and 841 singlets. Among the homologous sequences found the following are more relevant to our aim: β-glucosidase (microvillar membrane marker), α-glucosidase (perimicrovillar membrane marker), aminopeptidase (perimicrovillar space marker), cathepsin L (vesicles content) and sugar transporter protein, GLUT. These sequences had its specific transcription (or preferential) verified by semi-quantitative RT-PCR on different insect tissues (malpighian tubules, salivary gland, fat body, midgut, midgut first ventriculus, second ventriculus and third ventriculus). The glucose and water absorption across the first ventriculus of the midgut of the Hemiptera Dysdercus peruvianus were determined. The insects were fed with a 10 glucose-non-absorbable dye solution, followed by periodical dissection of insects and analysis of ventriculus contents. The transport of water and glucose can be inhibited by 0.2 mM phloretin (GLUT inhibitor) and by 0.1 mM phlorizin (SGLT inhibitor) and is activated by 50 mM K2SO4 The results suggest that D. peruvianus has a transporter uniporter like (GLUT) and K+-glucose symporter like SGLT, both co-transporting water. The transcriptome showed a GLUT homologous protein which sequence is almost complete and was analyzed by bioinformatics tools

ASSUNTO(S)

sglt dysercus peruvianus insects glut glut sglt catepsina l transcriptoma bioquímica animal dysercus peruvianus cathepsin l hemiptera hemiptera insetos transcriptome

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