Inhibition of digestive proteinases of stored grain Coleoptera by oryzacystatin, a cysteine proteinase inhibitor from rice seed

Electrophoresis of midgut extracts from the rice weevil, Sitophilus oryzae, and the red flour beetle, Tribolium castaneum, in polyacrylamide gels containing sodium dodecyl sulfate and gelatin revealed there was one major proteinase (apparent molecular mass = 40,000) in the rice weevil and two major proteinases (apparent molecular masses = 20,000 and 17,000) in the red flour beetle. The pH optima using 3H]casein as substrate were about pH 6.8 for the rice weevil and pH 5.2 for the red flour beetle. Use of specific inhibitors, including L-trans-epoxysuccinyl-leucylamino-(4- guanidino)-butane (E-64), p-chloromercuriphenylsulfonic acid (PCMS), and oryzacystatin, indicated that nearly all of the proteinase activity against casein was contributed by cysteine proteinases. The estimated IC50 values for oryzacystatin were 2 x 10(-6) M and 4 x 10(-7) M when tested against midgut extracts from T. castaneum and S. oryzae, respectively.