ISOLATION AND PARTIAL CHARACTERIZATION OF A SOYBEAN CYSTATIN CYSTEINE PROTEINASE-INHIBITOR OF COLEOPTERAN DIGESTIVE PROTEOLYTIC ACTIVITY

A 12 000-Da protein with cysteine proteinase inhibitory activity characteristic of cystatins was isolated from soybean seeds. The inhibitor was homologous in N-terminal amino acid sequence to rice oryz-acystatin and chicken egg white cystatin. It inhibited papain and ficin but did not inhibit bromelain, bovine trypsin, bovine alpha-chymotrypsin, and porcine pepsin. The soybean cystatin also inhibited, to varying degrees, the proteolytic activity of crude extracts of larval midguts of several Coleopteran insects with known digestive cysteine proteinase activity. Of the insects tested, the inhibitor was most effective against extracts of the cowpea weevil, Callosobruchus maculatus, and the red flour beetle, Tribolium castaneum. The soybean cystatin inhibitor was similar in potency on a molar basis to that of the cysteine proteinase inhibitors E-64 and chicken egg white cystatin. The isoelectric point of the soybean cystatin was pH 5.3, and a glycoprotein stain was negative. The inhibitory activity of the protein was lost after heating at 100-degrees-C for 30 min.