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 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,...

Isolation and Properties of Floral Defensins from Ornamental Tobacco and Petunia

“…NaD1, PhD1, and PhD2 and the floral defensins, FST from cultivated tobacco (Gu et al, 1992) and TPP3 from tomato (Milligan and Gasser, 1995) all have a predicted C-terminal domain of 27 to 33 amino acids in addition to the defensin domain. In contrast, the floral defensins PPT from petunia (Karunanandaa et al, 1994) and TGAS118 from tomato (van den Heuvel et al, 2001) lack this C-terminal domain, as do the seed defensins from radish (Rs-AFP2; [Terras et al, 1995], alfalfa [alfAFP; Gao et al, 2000]), and wheat (␥1-P; Colilla et al, 1990;Fig. 1A).…”

“…The first plant defensins were isolated from wheat (Triticum aestivum) and barley (Hordeum vulgare), and were initially classified as a subgroup of the thionin family called the ␥-thionins (Colilla et al, 1990;Mendez et al, 1990). Subsequent identification of other ␥-thionin-like proteins in other plant families, together with structural information, revealed striking differences between ␥-thionins and classical thionins (Bruix et al, 1993;Terras et al, 1995).…”

“…Thevissen andcolleagues (1996, 2000) have suggested that the defensin initiates this response by interaction with a membrane-bound receptor rather than by permeabilizing the membrane by direct defensin-lipid interaction. Certain members within the plant defensin family also display other biological activities, including proteinase (Wijaya et al, 2000;Melo et al, 2002) and ␣-amylase (Bloch and Richardson, 1991;Zhang et al, 1997) inhibitory activity and inhibition of protein translation (Colilla et al, 1990;Mendez et al, 1990; that may contribute to their role in defense.…”

A Review: Biological and Technological Functions of Barley Seed Pathogenesis-Related Proteins (PRs)

“…The first plant defensins were isolated from wheat and barley seeds 214 . Most plant defensins are seed specific, but are also expressed in vegetative tissues.…”

“…Loop 2 has been shown to be important for interaction and enzyme inhibition; it may prevent the entrance of the substrate by insertion into the active site of TMA. Moreover, it was found that positively charged residues in loops 1 and 2 may be important for interaction with α-amylases 192,214 . The ability of defensins to chelate calcium 51 may cause enzyme inhibition by destabilizing the enzyme molecule 258 .…”