Purification and pore-forming activity of two hydrophobic polypeptides from the secretion of the Red Sea Moses sole (Pardachirus marmoratus).
Purification and pore-forming activity of two hydrophobic polypeptides from the secretion of the Red Sea Moses sole (Pardachirus marmoratus).
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DOI：
10.1016/s0021-9258(18)66622-0
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发表时间：
1986-12
期刊：
影响因子：
--
通讯作者：
P. Lazarovici;N. Primor;L. Loew
中科院分区：
文献类型：
--
作者：
P. Lazarovici;N. Primor;L. Loew
一个新的柱层析程序，离子交换，色谱聚焦，和反相高压液相色谱的基础上，采用分离的两个主要的蛋白质，有毒，细胞溶解，孔形成因子的分泌物的红海摩西唯一Pardachirus marmoratus。包含10%的腺体分泌蛋白的帕达辛I显示出比帕达辛II（8%的腺体分泌蛋白）更高的毒性、细胞溶解性和膜孔形成活性5-10倍。凝胶电泳，氨基酸分析，和NH 2-末端氨基酸序列揭示了高度的同质性和相似性之间的两种毒素。富含天冬氨酸、丝氨酸、甘氨酸和丙氨酸，缺乏精氨酸、酪氨酸和色氨酸。它们的氨基末端残基序列为NH_2-Gly-Phe-Phe。它们的疏水性从疏水基质上的色谱行为、在NH 2末端存在9个连续疏水残基以及在色谱纯化期间活性级分洗脱期间液滴尺寸减小中是明显的。通过十二烷基硫酸钠凝胶电泳和氨基酸分析，测得帕达辛I的最小分子量约为3500。它由35个氨基酸组成，不含碳水化合物和唾液酸残基。腺体分泌物和纯化毒素的乙酸乙酯提取物的质谱分析揭示了分泌物中存在甾醇，但纯化毒素中不存在甾醇。碘化的Pardaxin I不影响其化学和成孔性能。它与不同磷脂组成的脂质体结合。在超极化单层脂质体中，pardaxin I产生快速的、非特异性的透化作用，而在多层脂质体中，产生缓慢的、阳离子特异性的孔。这表明，pardaxins发挥其作用，由于其疏水性和成孔性能。
A new column chromatography procedure, based on ion exchange, chromatofocusing, and reverse phase high pressure liquid chromatography was employed to isolate the two main proteinaceous, toxic, cytolytic, pore-forming factors from the secretion of the Red Sea Moses sole Pardachirus marmoratus. Pardaxin I, comprising 10% of the gland secretion proteins, was shown to be 5-10 times more toxic, cytolytic, and active in membrane pore formation than pardaxin II (8% of gland secretion proteins). Gel electrophoresis, amino acid analysis, and NH2-terminal amino acid sequence reveals a high degree of homogeneity and resemblance between the two toxins. They are rich in aspartic acid, serine, glycine, and alanine and devoid of arginine, tyrosine, and tryptophan. Their NH2-terminal residue sequence was found to be NH2-Gly-Phe-Phe. Their hydrophobicity is evident from chromatographic behavior on a hydrophobic matrix, presence of 9 successive hydrophobic residues at the NH2 terminus, and a decrease in drop size during elution of active fractions during chromatographic purification. The minimal molecular weight of pardaxin I is about 3500 as determined by sodium dodecyl sulfate gel electrophoresis and amino acid analyses. It is composed of 35 amino acids and is free of carbohydrate and sialic acid residues. Mass spectrometry of the ethyl acetate extract of the gland secretion and purified toxin reveals the presence of sterols in the secretion but their absence in the purified toxins. Pardaxin I was iodinated without affecting its chemical and pore-forming properties. It binds to liposomes of different phospholipid compositions. In hyperpolarized unilamellar liposomes, pardaxin I produced a fast, nonspecific permeabilization and in multilamellar liposomes, a slow, cation-specific pore. It is suggested that pardaxins exert their effects due to their hydrophobic and pore-formation properties.