Characterization of two Acanthoscelides obtectus alpha-amylases and their inactivation by wheat inhibitors.
Wheat alpha-amylase inhibitors represent an important tool in engineering crop plants against bean bruchids. Because Acanthoscelides obtectus is a devastating storage bean insect-pest, we attempted to purify and characterize its gut alpha-amylases, to study their interaction with active proteinaceous inhibitors. Two digestives alpha-amylases (AoA1 and AoA2) were purified from gut larvae, showing molecular masses of 30 and 45 kDa for each one, respectively. The stoichiometry interaction between these alpha-amylases with two wheat inhibitors (0.19 and 0.53) showed a binding complex of 1:1 enzyme:inhibitor. In vivo activities of these inhibitors against A. obtectus were also evaluated using a rich ammonium sulfate inhibitor fraction (F(20)(-)(40)) and purified inhibitors after reversed phase high-performance liquid chromatography columns. Incorporation of three different inhibitor concentrations (0.25, 0.5, and 1.0% w/w) into artificial seeds showed that addition of the purified 0.19 inhibitor at the highest concentration (1.0%) reduced the larval weight by 80%. Similar data were observed when 0.53 inhibitor was incorporated at 0.5%. When the concentration of purified 0.53 was enhanced to 1.0%, no larvae or adult emergence were observed. Our data suggest that these alpha-amylase inhibitors present great potential for use in Phaseolus genetic improvement programs.
Franco
Octávio L
OL
EMBRAPA/Recursos Genéticos e Biotecnologia, Brasília-DF, Brazil, Universidade Católica de Brasília, Brasília-DF, Brazil. ocfranco@pos.ucb.br
Melo
Francislete R
FR
Mendes
Paulo A
PA
Paes
Norma S
NS
Yokoyama
Massaru
M
Coutinho
Marise V
MV
Bloch
Carlos
C
Grossi-de-Sá
Maria F
MF
D000818:Animals; D002851:Chromatography, High Pressure Liquid; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D007814:Larva / Q000201:enzymology; D027805:Phaseolus; D014908:Triticum / Q000737:chemistry*; D048889:Weevils / Q000201:enzymology*; D000516:alpha-Amylases / Q000037:antagonists & inhibitors* / Q000302:isolation & purification
D004791:Enzyme Inhibitors; D000516:alpha-Amylases