THE PROPEPTIDE OF PSEUDOMONAS-AERUGINOSA ELASTASE ACTS AS AN ELASTASEINHIBITOR

Elastase, an extracellular protease of Pseudomonas aeruginosa, is synt hesized as a preproenzyme containing a large amino-terminal propeptide . The propeptide is cleaved within the periplasm to form a noncovalent complex with the elastase moiety. The propeptide-elastase complex was purified from the cell extract of P. aeruginosa by affinity chromatog raphy on Gly(3)-D-Phe-Sepharose. The purified fraction was proteolytic ally inactive and contained the propeptide-elastase complex as the maj or protein component. Activation by Limited proteolysis with trypsin w as associated with the disappearance of the propeptide. To correlate i ndividual proteins in the preparation with proteolytic activity, the p urified fraction was subjected to polyacrylamide gel electrophoresis u nder nondenaturing conditions and subsequent incubation of the separat ion gel over a skim milk-agarose-indicator gel. Clearing zones due to proteolysis were produced either by mature elastase (control) or the f ree processed periplasmic enzyme, a low level of which was present in the purified propeptide-elastase complex preparation. No clearing was evident with the propeptide-elastase complex, indicating inhibition by the bound propeptide. Proteolytic activity of mature elastase was inh ibited by various Pseudomonas cell fractions. This inhibition was abol ished by antipropeptide antibodies, and, as evident from immunoblottin g analysis, was consistent with propeptide presence in the effective f raction, whole cell extract, cytosol, and one of the two periplasmic f ractions obtained upon conversion of P. aeruginosa cells to spheroplas ts. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and elec troblotting of the various cell fi actions onto nitrocellulose membran es followed by incubation of the membranes with elastase and subsequen t probing with antielastase antibodies revealed elastase propeptide bi nding. This binding of mature elastase to the propeptide was prevented by antibodies to the propeptide but not by inhibitors of elastase act ivity. Thermolysin, a neutral metalloprotease homologous to elastase, was not recognized by the elastase propeptide. In addition, propeptide containing P. aeruginosa fractions that were inhibitory to elastase h ad no effect on thermolysin activity. We conclude that elastase propep tide functions as an elastase inhibitor. Inhibition is specific, effec tive against both periplasmic and mature elastase, and depends on enzy me propeptide binding.