An inhibitor affecting subtilisin [EC 3.4.21.14] was isolated from barley (Hordeum vulgare L cv. Kikaihadaka) by extraction with 1% sodium chloride, fractionation with ammonium sulfate, chromatography on CM- and DEAE-cellulose columns, and gel filtration on Sephadex G-100. The final preparation appeared to be homogeneous on the basis of polyacrylamide gel electrophoresis; the inhibitory activity against subtilisin was increased about 140-fold during purification. This inhibitor was protein having a molecular weight of about 20,000, and containing 177 amino acid residues. Both the amino- and carboxyl-terminal residues were alaine. The inhibitor inactivated subtilisin, probably for formation of an enzyme-inhibitor complex in a molar ratio of 1: 1, but had little or no effect on the activities of other enzymes tested. The dissociation constant of the subtilisin-inhibitor complex was 1.5 X 10(-10) M. The inhibitor appears to be distinct from the barley microbial proteinase isoinhibitors reported by Mikola and Suolinna, in respect of most of its physiochemical and inhibitory properties.
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.
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