Mendez E, Moreno A, Colilla F, Pelaez F, Limas G G, Mendez R, Soriano F, Salinas M, de Haro C
Servicio de Endocrinologia, Hospital Ramon y Cajal, Madrid, Spain.
Eur J Biochem. 1990 Dec 12;194(2):533-9. doi: 10.1111/j.1432-1033.1990.tb15649.x.
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.
一种新的富含硫且呈碱性的多肽，命名为γ-大麦硫堇蛋白，已通过半制备纯化法从大麦胚乳中分离出来，该纯化法包括用挥发性盐溶液萃取，随后使用反相C4柱进行高效液相色谱分析。通过在十二烷基硫酸钠存在下的微量二维凝胶电泳发现，分离出的多肽是均一的。γ-大麦硫堇蛋白的完整一级结构是通过对完整的、S-羧甲基化和S-吡啶基乙基化的γ-大麦硫堇蛋白以及通过蛋白水解裂解获得的片段进行自动降解来确定的。γ-大麦硫堇蛋白由一条47个氨基酸的单多肽链组成，计算分子量为5250 Da，含有四个二硫键。γ-大麦硫堇蛋白在多个水平上抑制源自哺乳动物（兔网织红细胞、小鼠肝脏）以及非哺乳动物（卤虫胚胎）细胞的无细胞系统中的翻译。在低浓度（1 - 10 microM）时，该蛋白似乎主要影响多肽链起始过程，尽管它也可能在延伸水平起作用。在较高浓度（20 - 80 microM）时，这种抑制剂会诱导在添加血红素的网织红细胞裂解物中真核多肽链起始因子2α亚基（eIF - 2α）激酶的激活，血红素缺乏时也是如此。γ-大麦硫堇蛋白中二硫键的存在似乎是eIF - 2α激酶激活所必需的。基于其在结构和功能水平上与硫堇蛋白（来自小麦和大麦的α和β硫堇蛋白）的不同遗传变体的相似性，γ-大麦硫堇蛋白是硫堇蛋白家族的一个推定成员。
