Studies of enzyme inhibitors and endochitinase in seeds of job’s tears (Coix lachryna-jobi)

Abstract

Studies of the purification, characterization and primary structure of protein inhibitors of trypsin and -amylase from seeds of Job's Tears (Coix lachryma-jobi) were undertaken. The major trypsin inhibitor from seeds of Coix was purified by heat treatment, fractional precipitation with ammonium sulphate, ion-exchange chromatography, gel filtration and preparative reversed-phase HPLC. The complete amino acid sequence was determined by analysis of peptides derived from the reduced and S- carboxymethylated protein by digestion with trypsin, chymotrypsin and the S.aureus V8 protease. The polypeptide contained 64 amino acids with a high content of cysteine. The sequence exhibited strong similarity with a number of Bowman-Birk inhibitors from legume and cereal seeds. A protein inhibitor of locust gut ζ-amylase was purified from seeds of Coix using ammonium sulphate precipitation, affinity chromatography on Red Sepharose and reversed-phase HPLC. It consisted of two major isomers, each a dimer of two identical or closely similar subunits of M(_r) about 26 400. These two isomers also had very similar amino acid compositions. The major isomer showed no inhibitory activity against amylases from other sources: human saliva, porcine pancreas, B. subtilis. A. oryzae and barley malt. The manual DABITC/PITC method was used to determine about half of the amino acid sequence of the major isoform. This showed a high degree of similarity with previously reported sequences of endochitinase enzymes from several species (tobacco, potato, barley, bean). Endochitinase activity was demonstrated by following the release of radioactivity from [(^3)H] chitin. As far as can be ascertained from the literature this is the first characterization of a plant protein with activity as an enzyme and as an enzyme inhibitor. Preliminary molecular studies were also carried out, including the isolation and in vitro translation of mRNA fractions from developing seeds of Coix.