Characterization of a membrane-bound aminopeptidase purified from Acyrthosiphon pisum midgut cells. A major binding site for toxic mannose lectins.
A single membrane-bound aminopeptidase N (APN) occurs in the pea aphid (Acyrthosiphon pisum Harris) midgut, with a pH optimum of 7.0, pI of 8.1 and molecular mass of 130 kDa. 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 co-ordination and catalysis and a glycosyl-phosphatidylinositol anchor, as in APNs from Lepidoptera. APN has a broad specificity towards N-terminal amino acids, but does not hydrolyze acidic aminoacyl-peptides, thus resembling the mammalian enzyme (EC 3.4.11.2). The kcat/Km ratios for different di-, tri-, tetra-, and penta-peptides suggest a preference for tripeptides, and that subsites S1, S2' and S3' are pockets able to bind bulky aminoacyl residues. Bestatin and amastatin bound APN in a rapidly reversible mode, with Ki values of 1.8 microM and 0.6 microM, respectively. EDTA inactivates this APN (k(obs) 0.14 M(-1) x s(-1), 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 amino-acid-absorption processes which, in contrast with aminopeptidase activity, may be hampered on lectin binding.
Cristofoletti
Plinio T
PT
Departamento de Bioquímica, Instituto de Química, Universidade de São Paulo, Brazil.
de Sousa
Flavia A Mendonça
FA
Rahbé
Yvan
Y
Terra
Walter R
WR
D000595:Amino Acid Sequence; D000626:Aminopeptidases / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D000818:Animals; D001042:Aphids / Q000166:cytology / Q000201:enzymology*; D001483:Base Sequence; D001665:Binding Sites; D001667:Binding, Competitive; D018076:DNA, Complementary / Q000096:biosynthesis / Q000235:genetics; D004064:Digestive System / Q000201:enzymology*; D007700:Kinetics; D007915:Lepidoptera / Q000201:enzymology; D037241:Mannose-Binding Lectins / Q000378:metabolism* / Q000494:pharmacology; D008969:Molecular Sequence Data; D010802:Phylogeny; D016415:Sequence Alignment; D013379:Substrate Specificity
D018076:DNA, Complementary; D037241:Mannose-Binding Lectins; D000626:Aminopeptidases