2 research outputs found
Characterization of a membrane-bound aminopeptidase purified from Acyrthosiphon pisum midgut cells: a major binding site for toxic mannose lectins
1-ACL (articles avec comitĂ© de lecture)A single membraneâbound aminopeptidaseâN (APN) occurs in the pea aphid (Acyrthosiphonâpisum Harris) midgut, with a pH optimum of 7.0, pI of 8.1 and molecular mass of 130âkDa. This enzyme accounts for more than 15.6% of the total gut proteins. After being solubilized in detergent, APN was purified to homogeneity. The enzyme is a glycoprotein rich in mannose residues, which binds the entomotoxic lectins of the concanavalin family. The internal sequence of APN is homologous with a conservative domain in APNs, and degenerated primers of highly conserved APN motifs were used to screen a gut cDNA library. The complete sequence of APN has standard residues involved in zinc coâordination and catalysis and a glycosylâphosphatidylinositol anchor, as in APNs from Lepidoptera. APN has a broad specificity towards Nâterminal amino acids, but does not hydrolyze acidic aminoacylâpeptides, thus resembling the mammalian enzyme (EC 3.4.11.2). The kcat/Km ratios for different diâ, triâ, tetraâ, and pentaâpeptides suggest a preference for tripeptides, and that subsites S1, S2âČ and S3âČ are pockets able to bind bulky aminoacyl residues. Bestatin and amastatin bound APN in a rapidly reversible mode, with Ki values of 1.8â”m and 0.6â”m, respectively. EDTA inactivates this APN (kobs 0.14âmâ1·sâ1, reaction order of 0.44) at a rate that is reduced by competitive inhibitors. In addition to oligopeptide digestion, APN is proposed to be associated with aminoâacidâabsorption processes which, in contrast with aminopeptidase activity, may be hampered on lectin binding