Effects of proteases on the structure and activity of Pseudomonas aeruginosa exotoxin A

The effects of various proteases on the enzymatic or biological activity and structure of exotoxin A from Pseudomonas aeruginosa were systematically studied. The toxin was extremely resistant to treatment with various enzymes. The lethality of the toxin disappeared upon treatment with P. aeruginosa protease and elastase, thermolysin, and trypsin with a long incubation time (5h) in the presence of a high enzyme concentration (molar concentration of enzyme to toxin, 1:10 or 1:20), but was little altered by either alpha-chymotrypsin or subtilisin. The decrease of adenosine diphosphate ribosylation activity was moderate when the same treatment was used, regardless to the protease source, except in the case of papain, which was tested in the presence of reducing agents. The increase in activation of the treated toxin determined in the presence of a denaturant and a reducing agent was less than that of the intact toxin, except in the case of trypsin. The differences in disc and sodium dodecyl sulfate gel electropherograms of the toxins treated with these proteases, except for those treated with papain, suggested that the toxins had been nicked by the protease, which resulted in their degradation by sodium dodecyl sulfate treatment. Papain degraded the toxin into fragments and caused the disappearance of lethality or a marked decrease of adenosine diphosphate ribosylation activity.