Aminopeptidase-N from the Helicoverpa armigera (Hubner) brush border membrane vesicles as a receptor of Bacillus thuringiensis Cry1Ac δ-endotoxin

Abstract

Brush border membrane vesicles (BBMVs) were prepared from the 2nd instar larvae of Helicoverpa armigera. Binding of the activated CrylAc of Bacillus thuringiensis (Bt) toxin was shown by immunoblot. A 120-kDa protein was identified as a receptor for the CrylAc type δ-endotoxin. The aminopeptidase-N activity of BBMVs was measured as the hydrolysis of L-leucine p-nitroanilide. The specific activity was 35 units/mg protein. The BBMV preparation also showed low level of alkaline phosphatase activity. Zn++ chelating agents 2,2′-dipyridyl and 1,10-phenanthroline inhibited aminopeptidase activity at 10 mM concentration, indicating the presence of zinc-dependent aminopeptidase in the brush border of H. armigera. The aminopeptidase activity was increased with increasing concentration of δ-endotoxin. The purified 120-kDa binding protein was N-terminally sequenced. The first 10-aminoacid sequence showed 60-77% similarity with human cysteine-rich secretory protein-1 precursor, inhibin alpha chain precursor. Salmonella flagellar hook protein and yeast carboxypeptidase S.