Midgut juice components affect pore formation by the Bacillus thuringiensis insecticidal toxin Cry9Ca
Midgut juice components affect pore formation by the Bacillus thuringiensis insecticidal toxin Cry9Ca
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DOI：
10.1016/j.jip.2010.04.007
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发表时间：
2010-07-01
影响因子：
3.4
通讯作者：
Laprade, Raynald
中科院分区：
文献类型：
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作者：
Brunet, Jean-Frederic;Vachon, Vincent;Laprade, Raynald
苏云金芽孢杆菌毒素Cry 9 Ca，它的两个单位点突变体R164 A和R164 K，和55-kDa的片段产生的蛋白水解切割在R164的孔形成能力进行了评估，在各种实验条件下，使用电生理测定。所有四种毒素制剂去极化的顶端膜新鲜分离的第三龄天蛾sexta midguts沐浴在含有122 mM KCl的溶液中，pH值为10.5，但55-kDa的片段是相当多的活性比Cry 9 Ca及其突变体。然而，当实验在五龄幼虫存在下进行时，后一种毒素的活性大大增强。六中肠液在中肠液蛋白质通过在95摄氏度下加热变性后，或者在无机离子和小分子通过广泛透析从中肠液中去除后，也观察到这种效果。当在从等体积的中肠汁液中提取的脂质存在下进行实验时，也观察到毒素活性的类似刺激。细胞膜的去极化也大大增强，在没有中肠汁，通过添加水溶性蛋白酶抑制剂的鸡尾酒。这些结果表明，取决于切割位点和所用的实验条件，活化的Cry 9 Ca毒素的进一步蛋白水解可以刺激其活性或对其活性有害，并且M. sexta中肠汁液可能含有蛋白酶抑制剂，其可能在B的活性中起主要作用。苏云金杆菌毒素在昆虫中肠中的作用。(C)2010年爱思唯尔公司All rights reserved.
The pore-forming ability of the Bacillus thuringiensis toxin Cry9Ca, its two single-site mutants R164A and R164K, and the 55-kDa fragment resulting from its proteolytic cleavage at R164 was evaluated under a variety of experimental conditions using an electrophysiological assay. All four toxin preparations depolarized the apical membrane of freshly isolated third-instar Manduca sexta midguts bathing in a solution containing 122 mM KCl at pH 10.5, but the 55-kDa fragment was considerably more active than Cry9Ca and its mutants. The activity of the latter toxins was greatly enhanced, however, when the experiments were conducted in the presence of fifth-instar M. sexta midgut juice. This effect was also observed after midgut juice proteins had been denatured by heating at 95 degrees C or after inorganic ions and small molecules had been removed from the midgut juice by extensive dialysis. A similar stimulation of toxin activity was also observed when the experiments were carried out in the presence of the lipids extracted from an equivalent volume of midgut juice. Depolarization of the cell membrane was also greatly enhanced, in the absence of midgut juice, by the addition of a cocktail of water-soluble protease inhibitors. These results indicate that, depending on the cleavage site and on the experimental conditions used, further proteolysis of the activated Cry9Ca toxin can either stimulate or be detrimental to its activity and that M. sexta midgut juice probably contains protease inhibitors that could play a major role in the activity of B. thuringiensis toxins in the insect midgut. (C) 2010 Elsevier Inc. All rights reserved.