Submitted to: American Society of Plant Physiologists Meeting
Publication Type: Abstract Only
Publication Acceptance Date: 7/26/1999
Publication Date: N/A
Technical Abstract: Properties of proteolytic enzymes used by the alfalfa weevil, Hypera postica, for nutrient assimilation are being determined. The effect on natural substrate hydrolysis of pH, thiol activators, low-molecular weight (low-Mr) inhibitors, and protein inhibitors (PIs) with transgenic potential is being evaluated using crude extracts prepared from excised larval midguts. Optimal hydrolysis occurs at pH 3.5 and pH 4.5-5.0 with hemoglobi or azocasein as the substrate, respectively. Much less hydrolysis (<20% of maximum) occurs at pH 7 and higher. The effect of thiol activator on hydrolysis varies with each assay depending on the choice of activator, substrate, and pH, but generally results in a two to three-fold enhancement. Maximal inhibition by class-specific inhibitors also varies with assay composition, but the order of effectiveness is E-64 (50-80% inhibition), pepstatin A (25-50%), and then PMSF (3-20%). This corresponds to a relative importance among major insect proteinase classes of cysteine > aspartyl > serine. The activity assigned to the cysteine class results primarily from the action of cathepsin L, and to a lesser extent cathepsin B, as suggested by treatment with the diazomethylketone inhibitors Z-Phe-Phe-CHN2 and Z-Phe-Ala-CHN2. The aspartyl activity is consistent with a cathepsin D-like enzyme. Likewise, plant-derived PIs with specificities toward cathepsins L and D, namely oryzacystatin I and potato cathepsin D inhibitor, most effectively inhibit midgut activity. Work is continuing to identify PIs with transgenic potential that can be combined to inhibit the full spectrum of alfalfa weevil digestive enzymes.