Vasconcelos I M, Morais J K S, Siebra E A, Carlini C R, Sousa D O B, Beltramini L M, Melo V M M, Oliveira J T A
Laboratório de Toxinas Vegetais, Departamento de Bioquímica e Biologia Molecular, Universidade Federal do Ceará, CEP 60451-970, Fortaleza, CE, Brazil.
Toxicon. 2008 May;51(6):952-63. doi: 10.1016/j.toxicon.2007.10.005. Epub 2007 Oct 13.
SBTX, a novel toxin from soybean, was purified by ammonium sulfate fractionation followed by chromatographic steps DEAE-Cellulose, CM-Sepharose and Superdex 200 HR fast-protein liquid chromatography (FPLC). Lethality of SBTX to mice (LD(50) 5.6 mg/kg) was used as parameter in the purification steps. SBTX is a 44-kDa basic glycoprotein composed of two polypeptide chains (27 and 17 kDa) linked by a disulfide bond. The N-terminal sequences of the 44 and 27kDa chains were identical (ADPTFGFTPLGLSEKANLQIMKAYD), differing from that of 17 kDa (PNPKVFFDMTIGGQSAGRIVMEEYA). SBTX contains high levels of Glx, Ala, Asx, Gly and Lys and showed maximum absorption at 280 nm, epsilon(1cm)(1%) of 6.3, and fluorescence emission in the 290-450 nm range upon excitation at 280nm. The secondary structure content was 35% alpha-helix, 13% beta-strand and beta-sheet, 27% beta-turn, 25% unordered, and 1% aromatic residues. Immunological assays showed that SBTX was related to other toxic proteins, such as soyatoxin and canatoxin, and cross-reacted weekly with soybean trypsin inhibitor and agglutinin, but it was devoid of protease-inhibitory and hemagglutinating activities. The inhibitory effect of SBTX on growth of Cercospora sojina, fungus causing frogeye leaf spot in soybeans, was observed at 50 microg/ml, concentration 112 times lesser than that found to be lethal to mice. This effect on phytopathogenic fungus is a potential attribute for the development of transgenic plants with enhanced resistance to pathogens.
SBTX是一种来自大豆的新型毒素，通过硫酸铵分级分离，随后经过DEAE-纤维素、CM-琼脂糖凝胶和Superdex 200 HR快速蛋白质液相色谱（FPLC）等色谱步骤进行纯化。在纯化步骤中，以SBTX对小鼠的致死率（半数致死量为5.6毫克/千克）作为参数。SBTX是一种44千道尔顿的碱性糖蛋白，由两条通过二硫键连接的多肽链（27千道尔顿和17千道尔顿）组成。44千道尔顿和27千道尔顿链的N端序列相同（ADPTFGFTPLGLSEKANLQIMKAYD），与17千道尔顿链的序列不同（PNPKVFFDMTIGGQSAGRIVMEEYA）。SBTX含有高水平的谷氨酰胺、丙氨酸、天冬酰胺、甘氨酸和赖氨酸，在280纳米处有最大吸收峰，1厘米光径下的百分吸光系数为6.3，在280纳米激发下，荧光发射在290 - 450纳米范围内。二级结构含量为35%的α-螺旋、13%的β-链和β-折叠、27%的β-转角、25%的无规结构以及1%的芳香族残基。免疫分析表明，SBTX与其他有毒蛋白质有关，如大豆毒素和刀豆毒素，并且与大豆胰蛋白酶抑制剂和凝集素发生弱交叉反应，但它没有蛋白酶抑制和血凝活性。在50微克/毫升的浓度下观察到SBTX对引起大豆蛙眼叶斑病的真菌——大豆尾孢菌的生长有抑制作用，该浓度比发现对小鼠致死的浓度低112倍。这种对植物病原真菌的作用是开发具有增强病原体抗性的转基因植物的一个潜在特性。
