Cescatti L, Pederzolli C, Menestrina G
Dipartimento di Fisica, Università di Trento, Italy.
J Membr Biol. 1991 Jan;119(1):53-64. doi: 10.1007/BF01868540.
Staphylococcus aureus alpha-toxin opens an ion channel in planar phospholipid bilayers, which is selective for anions over cations, supposedly because of the presence of positively charged groups along the ion pathway. To remove some positive charges of this protein toxin, we chemically modified part of its lysine residues either with diethylpyrocarbonate, followed by histidine regeneration with hydroxylamine, or with trinitrobenzenesulfonic acid. The extent of chemical modification can be followed accurately by native polyacrylamide gel electrophoresis and isoelectric focusing. Ethoxyformilation of two to three lysine residues per toxin monomer does not impair hemolysis of rabbit red blood cells nor formation of pores in model membranes. It reduces the conductance and the anion selectivity of the channel and changes the shape of its current-voltage characteristic. This indicates that positively charged lysine residues are actually important in determining the electrical properties of the pore. Ethoxyformilation of channels preassembled in planar bilayers produces the same changes as modification of toxin monomers before channel formation. Furthermore, it can be performed by adding diethylpyrocarbonate on either side of the bilayer. This suggests that the lysine residues relevant for the electrical properties of the pore are located inside its lumen where they can be reached by diethylpyrocarbonate diffusing from either entrance of the channel.
金黄色葡萄球菌α毒素可在平面磷脂双分子层中打开一个离子通道，该通道对阴离子的选择性高于阳离子，据推测这是由于离子通道沿线存在带正电荷的基团。为了去除这种蛋白质毒素的一些正电荷，我们用焦碳酸二乙酯对其部分赖氨酸残基进行化学修饰，随后用羟胺进行组氨酸再生，或者用三硝基苯磺酸进行修饰。化学修饰的程度可以通过天然聚丙烯酰胺凝胶电泳和等电聚焦精确跟踪。每个毒素单体对两到三个赖氨酸残基进行乙氧基甲酰化，既不会损害兔红细胞的溶血作用，也不会影响模型膜中孔的形成。它会降低通道的电导率和阴离子选择性，并改变其电流-电压特性的形状。这表明带正电荷的赖氨酸残基在决定孔的电学性质方面实际上很重要。对平面双分子层中预先组装的通道进行乙氧基甲酰化会产生与通道形成前毒素单体修饰相同的变化。此外，可以通过在双分子层的任一侧添加焦碳酸二乙酯来进行。这表明与孔的电学性质相关的赖氨酸残基位于孔腔内，从通道的任一入口扩散进来的焦碳酸二乙酯可以到达这些残基。
