Primary structure and inhibition of protein synthesis in eukaryotic cell-free system of a novel thionin, gamma-hordothionin, from barley endosperm.
A new sulfur-rich and basic polypeptide, designated as gamma-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 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 gamma-hordothionin was determined by automatic degradation of the intact, S-carboxymethylated and S-pyridylethylated gamma-hordothionin and fragments obtained by proteolytic cleavage. gamma-Hordothionin consists of a single polypeptide chain of 47 amino acids with a calculated molecular mass of 5250 Da and contains four disulfide bridges. gamma-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 microM) 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 microM) this inhibitor induces activation of an eukaryotic polypeptide-chain initiation factor 2 alpha-subunit (eIF-2 alpha) kinase in hemin-supplemented reticulocyte lysates, as does hemin deficiency. The presence of the disulfide bridges in gamma-hordothionin appears to be essential for the eIF-2 alpha kinase activation. Based on its similarity at both the structural and functional level with the different genetic variants of thionins (alpha and beta-thionins, from wheat and barley), gamma-hordothionin is a putative member of the thionin family.
Mendez
E
E
Servicio de Endocrinologia, Hospital Ramon y Cajal, Madrid, Spain.
Moreno
A
A
Colilla
F
F
Pelaez
F
F
Limas
G G
GG
Mendez
R
R
Soriano
F
F
Salinas
M
M
de Haro
C
C
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000737:chemistry; D023181:Antimicrobial Cationic Peptides; D002474:Cell-Free System; D002851:Chromatography, High Pressure Liquid; D015180:Electrophoresis, Gel, Two-Dimensional; D001467:Hordeum / Q000235:genetics / Q000378:metabolism*; D006868:Hydrolysis; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D010770:Phosphotransferases / Q000378:metabolism; D010940:Plant Proteins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D014176:Protein Biosynthesis; D011500:Protein Synthesis Inhibitors; D012639:Seeds; D013438:Sulfhydryl Compounds / Q000378:metabolism; D014357:Trypsin
D000596:Amino Acids; D023181:Antimicrobial Cationic Peptides; D010940:Plant Proteins; D011500:Protein Synthesis Inhibitors; D013438:Sulfhydryl Compounds; C036092:hordothionin protein, Hordeum vulgare; D010770:Phosphotransferases; D014357:Trypsin