Kreger A S, Griffin O K
Infect Immun. 1974 May;9(5):828-34. doi: 10.1128/iai.9.5.828-834.1974.
Fractionation of the culture supernatant fluids of a cornea-virulent strain of Pseudomonas aeruginosa by ammonium sulfate precipitation, diafiltration, isoelectric focusing, ion-exchange chromatography, gel filtration, and sucrose density gradient centrifugation failed to separate the rabbit cornea-damaging activity and the in vitro protease activity of the preparations. Three proteases having similar molecular weights (approximately 20,000) and isoelectric points of approximately 4.6, 5.8, and 8.8 were obtained free of detectable amounts of other known extracellular pseudomonal enzymes. Heating a mixture of the three proteases for 15 min at 80 C resulted in complete loss of protease and cornea-damaging activities. The sterile culture filtrate of a nonproteolytic but lethal toxin-producing strain of P. aeruginosa did not contain cornea-damaging activity. Cultivation of the proteolytic strain in broth containing 4.7% ammonium sulfate yielded a culture supernatant fluid free of protease and cornea-damaging activities. The results obtained support the conclusion that a cornea-virulent strain of P. aeruginosa can produce, in vitro, at least three different extracellular proteases capable of eliciting rapid and extensive damage to rabbit corneas.
通过硫酸铵沉淀、透析过滤、等电聚焦、离子交换色谱、凝胶过滤和蔗糖密度梯度离心对铜绿假单胞菌角膜毒力菌株的培养上清液进行分级分离，未能将制剂的兔角膜损伤活性和体外蛋白酶活性分开。获得了三种分子量相似（约20,000）且等电点分别约为4.6、5.8和8.8的蛋白酶，未检测到其他已知的细胞外假单胞菌酶。将这三种蛋白酶的混合物在80℃加热15分钟会导致蛋白酶活性和角膜损伤活性完全丧失。铜绿假单胞菌的一种非蛋白水解但产生致死毒素的菌株的无菌培养滤液不具有角膜损伤活性。在含有4.7%硫酸铵的肉汤中培养蛋白水解菌株，得到的培养上清液没有蛋白酶活性和角膜损伤活性。所得结果支持以下结论：铜绿假单胞菌的角膜毒力菌株在体外可产生至少三种不同的细胞外蛋白酶，这些蛋白酶能够对兔角膜造成快速且广泛的损伤。
