Geoffroy C, Alouf J E
J Biol Chem. 1983 Aug 25;258(16):9968-72.
Alveolysin has been purified in milligram quantities to apparent immunochemical and electrophoretic homogeneity from the supernatant culture fluid of Bacillus alvei. Purification involved ultrafiltration with concomitant molecular sieving on Amicon hollow fibers, thiol-disulfide interchange chromatography on thiopropyl-Sepharose 6B, and gel filtration on Sephacryl S-200 and Bio-Gel P-100. The purified toxin was found to be a single polypeptide chain of 582 amino acids (Mr = 63,000) free of carbohydrate, with alanine as NH2-terminal amino acid. Nonpolar amino acids accounted for 40% of the total residues. Immunochemical analysis of the precipitation curve of the toxin with anti-alveolysin immune sera indicated the presence of approximately 13 epitopes. The toxin exhibited a specific activity of 10(6) hemolytic units/mg of protein and behaved as an amphiphilic protein as inferred from charge-shift electrophoresis in a three-detergent system, suggesting the presence of substantial hydrophobic region(s). The inhibitory effects of various thiol reagents and that of cholesterol and related 3 beta-hydroxysterols with aliphatic side chains at carbon 17 indicated that alveolysin is a typical member of the group of bacterial--SH-activated toxins. The stoichiometry of cholesterol-toxin interaction was apparently equimolar. The complexes between alveolysin and [3H]cholesterol were characterized by sucrose gradient ultracentrifugation. Cholesterol-containing liposomes were disrupted by the toxin.
肺泡溶素已从蜂房芽孢杆菌的上清培养液中纯化出毫克量，达到明显的免疫化学和电泳均一性。纯化过程包括用Amicon中空纤维进行超滤并同时进行分子筛分，在硫丙基-琼脂糖6B上进行硫醇-二硫键交换色谱，以及在Sephacryl S-200和Bio-Gel P-100上进行凝胶过滤。发现纯化后的毒素是一条由582个氨基酸组成的单多肽链（Mr = 63,000），不含碳水化合物，以丙氨酸作为氨基末端氨基酸。非极性氨基酸占总残基的40%。用抗肺泡溶素免疫血清对毒素沉淀曲线进行免疫化学分析表明存在约13个表位。该毒素表现出10(6)溶血单位/毫克蛋白质的比活性，并且从在三去污剂系统中的电荷转移电泳推断其为两亲性蛋白质，这表明存在大量疏水区域。各种硫醇试剂以及胆固醇和相关的在17位带有脂肪族侧链的3β-羟基甾醇的抑制作用表明肺泡溶素是细菌-SH激活毒素组中的典型成员。胆固醇与毒素相互作用的化学计量比显然是等摩尔的。肺泡溶素与[3H]胆固醇之间的复合物通过蔗糖梯度超速离心进行表征。含胆固醇的脂质体被该毒素破坏。
