Shai Y, Bach D, Yanovsky A
Department of Membrane Research, Weizmann Institute of Science, Rehovot, Israel.
J Biol Chem. 1990 Nov 25;265(33):20202-9.
Six analogues of teh 33-residue shark repellent neurotoxin pardaxin were synthesized by the solid phase method: [Ala13]pardaxin, [Gly14,Gly15]pardaxin, des[1----9]pardaxin, [N1-succinamido]pardaxin, C33-dihydroxyethylamido]pardaxin, and C33-[diaminoethylamido]pardaxin. The spectroscopic and functional characterizations of the analogues are described. The peptides were characterized spectroscopically by circular dichroism (CD) before and after binding to soybean vesicles. They were characterized functionally by measuring their potential to evoke the dissipation of diffusion potential and calcein release from sonicated unilamellar soybean liposomes, by determining their ability to create single channels in planar bilayers, and by measuring their cytolytic activity on human erythrocytes. The behavior of the analogues modified at the C terminus is similar to that of pardaxin. [N'-succinamido]Pardaxin, however, reveals an increase in alpha-helicity both alone and in the presence of liposomes. It has the same potency as pardaxin to dissipate diffusion potential, to evoke calcein release and to produce single channels in lipid bilayers, but at a slower rate than that of pardaxin. It has more than 70-fold less cytolytic activity than pardaxin. [Ala13] Pardaxin has twice the alpha-helical content than pardaxin, both alone and in the presence of vesicles, yet it has less effect on the diffusion potential and calcein release, and it does not have cytolytic activity on human erythrocytes. Both [Gly14,Gly15]pardaxin and des[1----9]pardaxin are much less potent than pardaxin in all effects. However des[1----9]pardaxin exhibits a slight change in alpha-helicity upon binding to vesicles, whereas [Gly14,Gly15]pardaxin does not. The results support a model in which pardaxin is composed of two putative alpha-helices separated by proline. The N-terminal alpha-helix is important for the insertion of the peptide to the lipid bilayer, and the C-terminal amphiphilic alpha-helix is the ion channel lining segment of pardaxin.
采用固相法合成了33个氨基酸残基的鲨鱼驱避神经毒素pardaxin的6种类似物：[Ala13]pardaxin、[Gly14,Gly15]pardaxin、des[1----9]pardaxin、[N1-琥珀酰氨基]pardaxin、C33-二羟乙酰胺基]pardaxin和C33-[二氨基乙酰胺基]pardaxin。描述了这些类似物的光谱和功能特性。通过圆二色性（CD）对肽与大豆囊泡结合前后进行光谱表征。通过测量它们引起扩散电位消散和从超声处理的单层大豆脂质体中释放钙黄绿素的潜力、确定它们在平面双层中形成单通道的能力以及测量它们对人红细胞的细胞溶解活性来对其进行功能表征。在C末端修饰的类似物的行为与pardaxin相似。然而，[N'-琥珀酰氨基]pardaxin无论是单独存在还是在脂质体存在的情况下，α-螺旋度都有所增加。它在消散扩散电位、引起钙黄绿素释放以及在脂质双层中产生单通道方面与pardaxin具有相同的效力，但速度比pardaxin慢。它的细胞溶解活性比pardaxin低70多倍。[Ala13]pardaxin无论是单独存在还是在囊泡存在的情况下，其α-螺旋含量都是pardaxin的两倍，但它对扩散电位和钙黄绿素释放的影响较小，并且对人红细胞没有细胞溶解活性。[Gly14,Gly15]pardaxin和des[1----9]pardaxin在所有效应方面都比pardaxin的效力低得多。然而，des[1----9]pardaxin在与囊泡结合后α-螺旋度有轻微变化而[Gly14,Gly15]pardaxin则没有。这些结果支持了一个模型，其中pardaxin由两个被脯氨酸隔开且假定的α-螺旋组成。N末端的α-螺旋对于肽插入脂质双层很重要，而C末端的两亲性α-螺旋是pardaxin的离子通道内衬片段。
