title: Synthesis, processing, and transport of Pseudomonas aeruginosa elastase.
abstract: Three cell-associated elastase precursors with approximate molecular weights of 60,000 (P), 56,000 (Pro I), and 36,000 (Pro II) were identified in Pseudomonas aeruginosa cells by pulse-labeling with [35S]methionine and immunoprecipitation. In the absence of inhibitors, cells of a wild-type strain as well as those of the secretion-defective mutant PAKS 18 accumulated Pro II as the only elastase-related radioactive protein. EDTA but not EGTA [ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid] inhibited the formation of Pro II, and this inhibition was accompanied by the accumulation of Pro I. P accumulated in cells labeled in the presence of ethanol (with or without EDTA), dinitrophenol plus EDTA, or carbonyl cyanide m-chlorophenyl hydrazone plus EDTA. Pro I and Pro II were localized to the periplasm, and as evident from pulse-chase experiments, Pro I was converted to the mature extracellular enzyme with Pro II as an intermediate of the reaction. P was located to the membrane fraction. Pro I but not Pro II was immunoprecipitated by antibodies specific to a protein of about 20,000 molecular weight (P20), which, as we showed before (Kessler and Safrin, J. Bacteriol. 170:1215-1219, 1988), forms a complex with an inactive periplasmic elastase precursor of about 36,000 molecular weight. Our results suggest that the elastase is made by the cells as a preproenzyme (P), containing a signal sequence of about 4,000 molecular weight and a "pro" sequence of about 20,000 molecular weight. Processing and export of the preproenzyme involve the formation of two periplasmic proenzyme species: proelastase I (56 kilodaltons [kDa]) and proelastase II (36 kDa). The former is short-lived, whereas proelastase II accumulates temporarily in the periplasm, most likely as a complex with the 20-kDa propeptide released from proelastase I upon conversion to proelastase II. The final step in elastase secretion seems to required both the proteolytic removal of a small peptide from proelastase II and dissociation of the latter from P20.
mesh_terms: D002258:Carbonyl Cyanide m-Chlorophenyl Hydrazone / Q000494:pharmacology; D004140:Dinitrophenols / Q000494:pharmacology; D004492:Edetic Acid / Q000494:pharmacology; D004533:Egtazic Acid / Q000494:pharmacology; D000431:Ethanol / Q000494:pharmacology; D015151:Immunoblotting; D007700:Kinetics; D008715:Methionine / Q000378:metabolism; D010196:Pancreatic Elastase / Q000096:biosynthesis* / Q000235:genetics; D011499:Protein Processing, Post-Translational / Q000187:drug effects; D011550:Pseudomonas aeruginosa / Q000201:enzymology* / Q000235:genetics
chemical_list: D004140:Dinitrophenols; D000431:Ethanol; D004533:Egtazic Acid; D002258:Carbonyl Cyanide m-Chlorophenyl Hydrazone; D004492:Edetic Acid; D008715:Methionine; D010196:Pancreatic Elastase