Primary structure of omega-hordothionin, a member of a novel family of thionins from barley endosperm, and its inhibition of protein synthesis in eukaryotic and prokaryotic cell-free systems.
A new sulfur-rich basic polypeptide, so called omega-hordothionin, has been isolated from barley endosperm by extractions with NaCl and ammonium bicarbonate followed by reverse-phase high performance liquid chromatography. Purified omega-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 omega-hordothionin was determined by automatic degradation of the intact molecule and peptides obtained by proteolytic cleavage. Omega-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 electrospray-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 SI alpha 1 alpha-amylase inhibitor. Omega-hordothionin, like gamma-hordothionin, exhibited translation inhibitory activity on both eukaryotic cell-free systems from mammalian (rat liver and rabbit reticulocyte lysates) and prokaryotic cell-free systems (Escherichia coli). However, in contrast to gamma-hordothionin, omega-hordothionin did not inhibit plant systems such as Triticum aestivum, Cucumis sativus, Vicia sativa and Hordeum vulgare. Gamma-hordothionin also inhibited the alpha-amylase activity from human saliva, while omega-hordothionin and the other different genetic variants of thionins, alpha-hordothionin and beta-hordothionin, failed to show any inhibitory effect.
Méndez
E
E
Unidad de Análisis Estructural de Proteínas, Centro Nacional de Biotecnología, Madrid, Spain.
Rocher
A
A
Calero
M
M
Girbés
T
T
Citores
L
L
Soriano
F
F
D000595:Amino Acid Sequence; D000818:Animals; D023181:Antimicrobial Cationic Peptides; D002474:Cell-Free System; D002851:Chromatography, High Pressure Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism; D001467:Hordeum / Q000737:chemistry*; D006801:Humans; D006868:Hydrolysis; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D010940:Plant Proteins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D014176:Protein Biosynthesis; D011500:Protein Synthesis Inhibitors / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D011817:Rabbits; D051381:Rats; D012463:Saliva / Q000201:enzymology; D017386:Sequence Homology, Amino Acid; D000516:alpha-Amylases / Q000037:antagonists & inhibitors
D023181:Antimicrobial Cationic Peptides; D010940:Plant Proteins; D011500:Protein Synthesis Inhibitors; C036092:hordothionin protein, Hordeum vulgare; D000516:alpha-Amylases