The complete assignment of the proton NMR spectra of the homologous gamma 1-hordothionin and gamma 1-purothionin (47 amino acids, 4 disulfide bridges) from barley and wheat, respectively, has been performed by two-dimensional sequence-specific methods. A total of 299 proton-proton distance constraints for gamma 1-H and 285 for gamma 1-P derived from NOESY spectra have been used to calculate the three-dimensional solution structures. Initial structures have been generated by distance geometry methods and further refined by dynamical simulated annealing calculations. Both proteins show identical secondary and tertiary structure with a well-defined triple-stranded antiparallel beta-sheet (residues 1-6, 31-34, and 39-47), an alpha-helix (residues 16-28), and the corresponding connecting loops. Three disulfide bridges are located in the hydrophobic core holding together the alpha-helix and the beta-sheet and forming a cysteine-stabilized alpha-helical (CSH) motif. Moreover, a clustering of positive charges is observed on the face of the beta-sheet opposite to the helix. The three-dimensional structures of the gamma-thionins differ remarkably from plant alpha- and beta-thionins and crambin. However, they show a higher structural analogy with scorpion toxins and insect defensins which also present the CSH motif.

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A new sulfur-rich and basic polypeptide, designated as y-hordothionin, has been isolated from barley endosperm by a semi-preparative purification consisting of extraction with a volatile salt solution followed by highperformance liquid chromatography using a reversed-phase C4 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 y-hordothionin was determined by automatic degradation of the intact, Scarboxymethylated and S-pyridylethylated y-hordothionin and fragments obtained by proteolytic cleavage. yHordothionin consists of a single polypeptide chain of 47 amino acids with a calculated molecular mass of 5250 Da and contains four disulfide bridges. y-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 pM) 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 pM) this inhibitor induces activation of an eukaryotic polypeptide-chain initiation factor 2 a-subunit (eIF-2a) kinase in hemin-supplemented reticulocyte lysates, as does hemin deficiency. The presence of the disulfide bridges in y-hordothionin appears to be essential for the eIF-2a kinase activation. Based on its similarity at both the structural and functional level with the different genetic variants of thionins (a and p-thionins, from wheat and barley), y-hordothionin is a putative member of the thionin family.Two groups of protein synthesis inhibitors have been described from the endosperm of several Gramineae. Type 1 ribosome-inactivating proteins, which are single-chain basic polypeptides with molecular mass of around 30 kDa and are N-terminally blocked [l], belong to the first group. They have been found in wheat germ and in wheat, barley, rye and corn grains [l -61, as well as in a number of other plant species from different sources [2, 5, 71. An inhibitory effect has been shown with in vitro cell-free systems of protein synthesis from rabbit reticulocyte lysates, Ehrlich ascites cell lysates [2 -51 and with in vivo fungal growths [8]. These molecules inhibit protein synthesis in eucaryotic cells by interfering with the ability of the 60s subunit to bind the elongation factor 2 [2,The second group comprises a family of low-molecularmass proteins of around 5 kDa, rich in basic amino acids and cysteines and named thionins [9-lo]. Thionins are toxic to bacteria [11 -121, certain strains of yeasts [ l l , 131, insect larvae [14] and cultured cells [15-161 and they modify membraneCorrespondence to E. Mendez, Servicio de Endocrinologia, Hospital Ramon y Cajal, E-28034 Madrid, SpainAbbreviations. cIF-24 the a subunit of eukaryotic polypeptidechain initiation factor 2; HCI, heme-controlled translational inhibitor (an eIF-2cr kinase); GSSG, oxidized glutathione; Pth,...

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We present a new generation of a robust sandwich R5-ELISA with good reproducibility (8.7%) and repeatability (7.7%). Its gluten-detection limit of 3.2 ppm is lower than the existing threshold of 20-200 ppm. The ELISA, which is equally sensitive to barley, wheat and rye prolamins, is compatible with the quantitative cocktail extraction procedure for heat-processed foods. Along with the cocktail procedure, the Working Group on Prolamin Analysis and Toxicity is currently evaluating an R5-ELISA system as proposed by the Codex Alimentarius Commission.