Purification and characterization of a new class of insect alpha-amylase inhibitors from barley

Barley seeds contain proteins that apparently protect them against attack by microorganisms and insects. Studies of these barley defensive proteins may lead to the development of barleys with improved natural resistance to pests. We have purified two low molecular weight proteins, designated BI alpha 1 and BI alpha 2, from barley grain, using ion-exchange chromatography and reversed-phase and gel-permeation high-performance liquid chromatography (HPLC). Both BI alpha 1 and BI alpha 2 inhibited insect (yellow meal worm, Tenebrio molitor) alpha-amylase activities. For the T. molitor alpha-amylase, the IC50 values of BI alpha 1 and BI alpha 2 were 80 mu g/mL (12.5 mu M) and 34 mu g/mL (6.8 mu M), respectively. Neither protein inhibited either human salivary alpha-amylase, barley alpha-amylase, or trypsin activities. N-terminal amino acid sequences of the inhibitors were highly homologous with those of the plant proteins called defensins. The first 20 N-terminal amino acids of BI alpha 2 were identical to those of gamma-hordothionin, but neither BI alpha 1 nor BI alpha 2 protein showed any homology with the chloroform-methanol (CM) soluble protein amino acid consensus sequence. The two inhibitors therefore apparently comprise another group of low molecular weight barley proteins that inhibit the alpha-amylase activities of some insects that attack cereal grains.