Primary structure and inhibition of protein synthesis in eukaryotic cell‐free system of a novel thionin, γ‐hordothionin, from barley endosperm

Abstract

A new sulfur‐rich and basic polypeptide, designated as γ‐hordothionin, has been isolated from barley endosperm by a semi‐preparative purification consisting of extraction with a volatile salt solution followed by high‐performance liquid chromatography using a reversed‐phase C 4 column. The isolated polypeptide was found to be homogeneous by micro‐two‐dimensional gel electrophoresis in the presence of sodium dodecyl sulfate. The complete primary structure of γ‐hordothionin was determined by automatic degradation of the intact, S‐carboxymethylated and S‐pyridylethylated γ‐hordothionin and fragments obtained by proteolytic cleavage. γ‐Hordothionin consists of a single polypeptide chain of 47 amino acids with a calculated molecular mass of 5250 Da and contains four disulfide bridges. γ‐Hordothionin inhibits translation in cell‐free systems derived from mammalian (rabbit reticulocyte, mouse liver) as well as non‐mammalian (Artemia embryo) cells, at several levels. At low concentrations (1–10 μM) the protein seems to affect mainly the polypeptide‐chain‐initiation process, although it might also act at the elongation level. At higher concentrations (20–80 μM) this inhibitor induces activation of an eukaryotic polypeptide‐chain initiation factor 2 α‐subunit (eIF‐2α) kinase in hemin‐supplemented reticulocyte lysates, as does hemin deficiency. The presence of the disulfide bridges in γ‐hordothionin appears to be essential for the eIF‐2α kinase activation. Based on its similarity at both the structural and functional level with the different genetic variants of thionins (α and β‐thionins, from wheat and barley), γ‐hordothionin is a putative member of the thionin family.