Interaction of wheat monomeric and dimeric protein inhibitors with α-amylase from yellow mealworm (Tenebrio molitor L. larva)

The highly purified alpha-amylase from Tenebrio molitor L. larva (yellow mealworm) reversibly combines with two closely related homogeneous glycoprotein inhibitors, one dimeric (termed ‘inhibitor 0.19’) and one monomeric (termed ‘inhibitor 0.28’), from wheat flour. As established by means of difference spectroscopy and kinetic studies, molar combining ratios for the amylase–inhibitor-0.19 and amylase-inhibitor-0.28 complexes were 1:1 and 1:2 respectively. Two amylase–inhibitor-0.19 complexes with slightly different retention volumes on Bio-Gel P-300 and only one amylase–inhibitor-0.28 complex were observed. Dissociation constants of the amylase–inhibitor-0.19 and amylase–inhibitor-0.28 complexes were 0.85 nM and 0.13 nM respectively. A strong tendency of both complexes to precipitate under an ultracentrifugal field was observed; the minimum molecular weight calculated for the two complexes under such conditions was approx. 95 000. The two complexes showed difference spectra indicating involvement of structurally related or identical tryptophyl side chains in the binding of inhibitors 0.28 and 0.19 to the amylase. A model summarizing the main features of the inhibition of the insect amylase by the two wheat protein inhibitors is proposed.