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The amino acid sequence of a 20 kDa bifunctional subtilisin/α‐amylase inhibitor from brain of rice ( Oryza saliva L.) seeds
Author(s) -
Ohtsubo Ken-Ichi,
Richardson Michael
Publication year - 1992
Publication title -
febs letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.593
H-Index - 257
eISSN - 1873-3468
pISSN - 0014-5793
DOI - 10.1016/0014-5793(92)80741-x
Subject(s) - subtilisin , biochemistry , protease , trypsin , glutenin , chemistry , ammonium sulfate precipitation , chymotrypsin , trypsin inhibitor , amylase , bran , biology , chromatography , enzyme , size exclusion chromatography , protein subunit , gene , raw material , organic chemistry
A 20 kDa bifunctional inhibitor of the microbial proteinase, subtilisin, and the α‐amylase from the larvae of the red flour beetle ( Tribolium castaneum ) was purified from bran of rice seeds by saline extraction, precipitation with ammonium sulphate, ion‐exchange chromatography on DEAE‐Cellulose and Toyopearl CM‐650, and preparative HPLC on Vydac C 18 . The complete primary structure was determined by automatic degradation of the intact, reduced and S‐alkylated protein, and by manual DABITC/PITC micro‐sequencing of peptides obtained from the protein following separate enzymic digestions with trypsin, pepsin, chymotrypsin, clastase and the protease from S. aureus V8. The protein sequence, which contained 176 residues, showed strong homology with similar bifunctional inhibitors previously isolated from wheat and barley which are related to the Kunitz family of proteinase inhibitors from legume seeds.