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A new sulfur-rich basic polypeptide. so called ω-hordothionin, has been isolated from barley endosperm by extractions with NaCl and ammonium bicarbonate followed by reverse-phase high performance liquid chromatography. Purified ω-hordothionin was found to be homogeneous by SDS/polyacrylamide gel electrophoresis, N-terminal amino-acid sequencing and electrospray-ionization mass spectrometric analysis. The complete primary structure of ω-hordothionin was determined by automatic degradation of the intact molecule and peptides obtained by proteotytic cleavage. ω-hordothionin consists of a single polypeptide chain of 48 amino acids with a molecular mass of 5508 Da deduced from its amino acid sequence, which fully coincides with the 5508.2 Da determined by elcctrospray-ionization mass spectrometry. The isolated polypeptide showed a characteristic composition with a high content of basic amino acids (five arginine residues, two lysine residues and six histidine residues) and eight cysteine residues. and has strong sequence identity (66%) with the sorghum SIα1 α-amylase inhibitor. ω-hordothionin. like γ-hordothionin. exhibited translation inhibitory activity on both cukaryotic cell-free systems from mammalian (rat liver and rabbit reticulocyte lysates) and prokaryotic cell-free systems (Escherichia coli). However, in contrast to γ-hordothiomn, ω-hordothionin did not inhibit plant systems such as Triticum aestivum, Cucumis sativus, Vicia sativa and Hordeum vulgare. ;γ-hordothionin also inhibited the α-amylase activity from human saliva, while ω-hordothionin and the other different genetic variants of thionins, α- hordothionin and β-hordothionin. failed to show any inhibitory effect. [ABSTRACT FROM AUTHOR]