Butko P, Huang F, Pusztai-Carey M, Surewicz W K
Institute for Biological Sciences, National Research Council of Canada, Ottawa, Ontario, Canada.
Biochemistry. 1996 Sep 3;35(35):11355-60. doi: 10.1021/bi960970s.
CytA is a member of a functionally defined family of insecticidal delta-endotoxins occurring in parasporal crystals of Bacillus thuringiensis var. israelensis. We investigated the ability of CytA to permeabilize the membrane and release fluorescence marker molecules from unilamellar lipid vesicles. Both protoxin (27 kDa) and proteolytically activated toxin (24 kDa) were very effective in permeabilization of unilamellar lipid vesicles: concentrations as low as several nanomolar produced a significant effect. The toxin was about 2-3 times more effective than the protoxin. The concentration of CytA required for the same extent of calcein release in large unilamellar vesicles (LUV) was 5-10 times lower than that in small unilamellar vesicles (SUV). Both small (calcein) and large (fluorescein-dextrans, MW 3000 and 10 000) molecules were released from the vesicles by CytA with comparable single-exponential kinetics. The release was an all-or-none event, i.e., each vesicle either released all of its contents or remained completely intact. Binding of CytA to lipid membranes did not show appreciable cooperativity, the apparent binding constant (Kapp) being on the order of 10(5) M-1. The plots of kinetics of release vs bound protein/ lipid ratio and the differential effects of CytA on LUV vs SUV indicate that at least 140 toxin molecules or 311 protoxin molecules must bind to an LUV before the latter starts losing its integrity. The necessity of adsorption of this relatively large number of toxin molecules to trigger permeabilization, together with the lack of discrimination in the size of the released marker molecules, suggests that the effect of CytA is a general, detergent-like, perturbation of the membrane rather than creation of small, well-defined, proteinaceous channels.
CytA是苏云金芽孢杆菌以色列变种伴孢晶体中出现的具有杀虫活性的δ-内毒素功能家族的一员。我们研究了CytA使单层脂质体膜通透并释放荧光标记分子的能力。原毒素（27 kDa）和经蛋白水解激活的毒素（24 kDa）在使单层脂质体通透方面都非常有效：低至几纳摩尔的浓度就能产生显著效果。毒素的效果比原毒素约高2至3倍。在大单层脂质体（LUV）中实现相同程度的钙黄绿素释放所需的CytA浓度比小单层脂质体（SUV）低5至10倍。小（钙黄绿素）和大（分子量为3000和10000的荧光素 - 葡聚糖）分子都以类似的单指数动力学从脂质体中被CytA释放出来。释放是一个全或无的事件，即每个脂质体要么释放其所有内容物，要么保持完全完整。CytA与脂质膜的结合未显示出明显的协同性，表观结合常数（Kapp）约为10⁵ M⁻¹。释放动力学与结合的蛋白质/脂质比的关系图以及CytA对LUV和SUV的不同影响表明，在大单层脂质体开始失去其完整性之前，至少必须有140个毒素分子或311个原毒素分子与其结合。需要吸附相对大量的毒素分子来触发通透作用，以及对释放的标记分子大小缺乏区分，这表明CytA的作用是对膜进行一般性的、类似去污剂的扰动，而不是形成小的、明确的蛋白质通道。
