Several proteins which strongly inhibit trypsin have been found in adzuki bean seeds. Two of them, designated as adzuki proteinase inhibitors (API) I-A and I-A', were analyzed for their amino acid sequences by conventional methods. Inhibitors I-A and I-A' exhibited strong homology with other Bowman-Birk type proteinase inhibitors from leguminous seeds in spite of belonging to different genera. Inhibitors I-A and I-A' consisted of 78 and 72 amino acid residues and their molecular weights were 9,100 and 8,300, respectively. Inhibitor I-A' lacked the six amino acid residues of the amino terminus of inhibitor I-A and had an asparagine residue in place of the aspartic acid residue at position 40 of inhibitor I-A. The results showed the occurrence of some genetic variants of proteinase inhibitors in adzuki bean seeds. Inhibitor I-A was a double-headed one, and the reactive sites for trypsin were Lys-Ser and Arg-Ser bonds. Therefore, inhibitor I-A' was also assumed to be a double-headed one having Lys-Ser and Arg-Ser bonds as the reactive sites for the enzyme.
A subtilisin inhibitor was isolated from adzuki beans (Phaseolus angularis) by chromatography on CM-cellulose, Sephadex G-75, DEAE-cellulose, and SP-Sephadex C-25. The final preparation was confirmed to be homogeneous on polyacrylamide gel electrophoreses, and its pi value was 3.7. The preparation was a powerful inhibitor of microbial serine-proteinases but its activity was destroyed by trypsin and chymotrypsin. Dissociation constant of the complex of the inhibitor with subtilisin was 0.16 nM. The inhibitor was heat-stable over the pH range examined in spite of a lack of intramolecular disulfide bonds: little or no subtilisin-inhibitory activity was lost at 80°C for lOmin, though heating to 100°C at.pH 12 caused a decrease of about 50% in the activity. The inhibitor molecule consisted of 97 amino acid residues, containing relatively large amounts of glutamic acid and valine residues and no half-cystine residues, unlike adzuki-bean proteinase inhibitors I and II. The molecular weight of the inhibitor was determined to be 12,300 by gelfiltration and calculated to be approximately 1 1,000 on the basis of the amino acid composition.
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