Twining S S, Kirschner S E, Mahnke L A, Frank D W
Department of Biochemistry, Medical College of Wisconsin, Milwaukee 53226.
Invest Ophthalmol Vis Sci. 1993 Aug;34(9):2699-712.
To determine the effects of exoproducts from the corneal pathogen Pseudomonas aeruginosa on corneal proteinases and proteins.
Whole rabbit corneas were cultured in the presence or absence of broths conditioned with Pseudomonas aeruginosa, elastase, alkaline protease, and exotoxin A. Protein synthesis was assayed by adding 35S-methionine during the last 6 hours of culture. Caseinolytic assays and zymography on sodium dodecyl sulfate polyacrylamide gels containing casein and gelatin were used in the presence and absence of inhibitors to quantify and identify corneal proteinases.
The major proteinases released by the corneas were 92/89 kD (MMP9) and 65 kD (72 kD gelatinase, MMP2) gelatinases and a 97 kD caseinase. Minor proteinases observed included 184, 166, 156, 153, 126, 111, 102, 60, 57, and 43 kD gelatinases and 170, 136, 85, and 54 kD caseinases. P. aeruginosa elastase at 1 microgram/ml cleaved the 92 kD gelatinase to yield a 77 kD active form and cleaved the 65 kD gelatinase to yield a 57 kD active form. At 25 micrograms/ml elastase, the gelatinases were degraded. P. aeruginosa alkaline protease had no effect on the 92 or 65 kD gelatinases. Both elastase and alkaline protease degraded the 97 kD caseinase. Proteinases other than elastase and alkaline protease in P. aeruginosa103- and P. aeruginosa01-conditioned broths also activated and/or degraded corneal proteinases. Exotoxin A inhibited the synthesis of the 92 kD gelatinase and most other proteins. The 72 kD gelatinase and the 97 kD caseinase were released in the presence of exotoxin A.
Pseudomonas aeruginosa exoproducts can contribute directly to keratitis caused by Pseudomonas organisms through toxic effects on corneal cells and degradation of corneal proteins and indirectly through the activation of corneal proteinases.
确定角膜病原体铜绿假单胞菌的外产物对角膜蛋白酶和蛋白质的影响。
将完整的兔角膜在有或无经铜绿假单胞菌、弹性蛋白酶、碱性蛋白酶和外毒素A处理的培养液的条件下培养。在培养的最后6小时加入35S-甲硫氨酸来测定蛋白质合成。在有或无抑制剂的情况下，对含有酪蛋白和明胶的十二烷基硫酸钠聚丙烯酰胺凝胶进行酪蛋白溶解测定和酶谱分析，以定量和鉴定角膜蛋白酶。
角膜释放的主要蛋白酶是92/89 kD（基质金属蛋白酶9，MMP9）和65 kD（72 kD明胶酶，MMP2）明胶酶以及一种97 kD酪蛋白酶。观察到的次要蛋白酶包括184、166、156、153、126、111、102、60、57和43 kD明胶酶以及170、136、85和54 kD酪蛋白酶。1微克/毫升的铜绿假单胞菌弹性蛋白酶可切割92 kD明胶酶产生77 kD活性形式，并切割65 kD明胶酶产生57 kD活性形式。在25微克/毫升弹性蛋白酶作用下，明胶酶被降解。铜绿假单胞菌碱性蛋白酶对92或65 kD明胶酶无影响。弹性蛋白酶和碱性蛋白酶均能降解97 kD酪蛋白酶。铜绿假单胞菌103和铜绿假单胞菌01处理的培养液中除弹性蛋白酶和碱性蛋白酶之外的蛋白酶也能激活和/或降解角膜蛋白酶。外毒素A抑制92 kD明胶酶和大多数其他蛋白质的合成。72 kD明胶酶和97 kD酪蛋白酶在外毒素A存在的情况下被释放。
铜绿假单胞菌外产物可通过对角膜细胞的毒性作用和角膜蛋白质的降解直接导致铜绿假单胞菌引起的角膜炎，也可通过激活角膜蛋白酶间接导致角膜炎。
