The propeptide of Pseudomonas aeruginosa elastase acts an elastase inhibitor.
Elastase, an extracellular protease of Pseudomonas aeruginosa, is synthesized 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 chromatography on Gly3-D-Phe-Sepharose. The purified fraction was proteolytically inactive and contained the propeptide-elastase complex as the major protein component. Activation by limited proteolysis with trypsin was associated with the disappearance of the propeptide. To correlate individual proteins in the preparation with proteolytic activity, the purified fraction was subjected to polyacrylamide gel electrophoresis under nondenaturing conditions and subsequent incubation of the separation gel over a skim milk-agarose-indicator gel. Clearing zones due to proteolysis were produced either by mature elastase (control) or the free 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 inhibited by various Pseudomonas cell fractions. This inhibition was abolished by antipropeptide antibodies, and, as evident from immunoblotting analysis, was consistent with propeptide presence in the effective fraction, whole cell extract, cytosol, and one of the two periplasmic fractions obtained upon conversion of P. aeruginosa cells to spheroplasts. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electro-blotting of the various cell fractions onto nitrocellulose membranes followed by incubation of the membranes with elastase and subsequent probing with antielastase antibodies revealed elastase propeptide binding. This binding of mature elastase to the propeptide was prevented by antibodies to the propeptide but not by inhibitors of elastase activity. 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 had no effect on thermolysin activity. We conclude that elastase propeptide functions as an elastase inhibitor. Inhibition is specific, effective against both periplasmic and mature elastase, and depends on enzyme propeptide binding.
Kessler
E
E
Maurice and Gabriela Goldschleger Eye Research Institute, Tel-Aviv University Sackler Faculty of Medicine, Sheba Medical Center, Tel-Hashomer, Israel.
Safrin
M
M
D000595:Amino Acid Sequence; D002846:Chromatography, Affinity; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D004792:Enzyme Precursors / Q000302:isolation & purification / Q000378:metabolism* / Q000494:pharmacology; D015151:Immunoblotting; D007700:Kinetics; D008969:Molecular Sequence Data; D010196:Pancreatic Elastase / Q000037:antagonists & inhibitors* / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism* / Q000494:pharmacology; D011485:Protein Binding; D011550:Pseudomonas aeruginosa / Q000201:enzymology*
D004792:Enzyme Precursors; C022443:proelastase; D010196:Pancreatic Elastase