title: Purification and partial characterization of an aminopeptidase from the midgut tissue of Dysdercus peruvianus.
abstract: The surface of midgut cells in Hemiptera is ensheathed by a lipoprotein membrane (the perimicrovillar membrane), which delimits a closed compartment with the microvillar membrane, the so-called perimicrovillar space. In Dysdercus peruvianus midgut perimicrovillar space a soluble aminopeptidase maybe involved in the digestion of oligopeptides and proteins ingested in the diet. This D. peruvianus aminopeptidase was purified to homogeneity by ion-exchange chromatography on an Econo-Q column, hydrophobic interaction chromatography on phenyl-agarose column and preparative polyacrylamide gel electrophoresis. The results suggested that there is a single molecular species of aminopeptidase in D. peruvianus midgut. Molecular mass values for the aminopeptidase were estimated to be 106kDa (gel filtration) and 55kDa (SDS-PAGE), suggesting that the enzyme occurs as a dimer under native conditions. Kinetic data showed that D. peruvianus aminopeptidase hydrolyzes the synthetic substrates LpNA, RpNA, AβNA and AsnMCA (K(m)s 0.65, 0.14, 0.68 and 0.74mM, respectively). The aminopeptidase activity upon LpNA was inhibited by EDTA and 1,10-phenanthroline, indicating the importance of metal ions in enzyme catalysis. One partial sequence of BLAST-identified aminopeptidase was found by random sequencing of the D. peruvianus midgut cDNA library. Semi-quantitative RT-PCR analysis showed that the aminopeptidase genes were expressed throughout the midgut epithelium, in the epithelia of V1, V2 and V3, Malphigian tubules and fat body, but it was not expressed in the salivary glands. These results are important in furthering our understanding of the digestive process in this pest species.
authors.affiliation: Laboratório de Química e Função de Proteínas e Peptídeos, Universidade Estadual do Norte Fluminense Darcy Ribeiro, 28030-600, Campos dos Goytacazes, RJ, Brazil.
authors.forename: Inês A
authors.initials: IA
authors.lastname: Costa
authors.forename: Richard I
authors.initials: RI
authors.lastname: Samuels
authors.forename: Thaís D
authors.initials: TD
authors.lastname: Bifano
authors.forename: Walter R
authors.initials: WR
authors.lastname: Terra
authors.forename: Carlos P
authors.initials: CP
authors.lastname: Silva
mesh_terms: D000595:Amino Acid Sequence; D000626:Aminopeptidases / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D000818:Animals; D005260:Female; D020062:Heteroptera / Q000201:enzymology*; D019476:Insect Proteins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D007413:Intestinal Mucosa / Q000201:enzymology; D008969:Molecular Sequence Data; D017386:Sequence Homology, Amino Acid
chemical_list: D019476:Insect Proteins; D000626:Aminopeptidases