Purification and characterization of an extracellular protease of Legionella pneumophila.

An extracellular proteolytic enzyme of Legionella pneumophila was purified by sequential batch separation with DEAE-cellulose, hydrophobic interaction chromatography with octyl-Sepharose, and ion-exchange chromatography with DEAE-Bio-Gel A (Bio-Rad Laboratories, Richmond, Calif.). The resulting protease preparation was determined to be homogeneous by polyacrylamide gel electrophoresis in the presence and absence of sodium dodecyl sulfate. Although free of contaminating proteins, the purified protease separated into two antigenically indistinguishable proteins both of which possessed proteolytic activity. The apparent masses of the proteins were 38 and 40 kilodaltons (kDa) as determined by polyacrylamide gel electrophoresis in sodium dodecyl sulfate, whereas gel filtration chromatography revealed a single mass of 34 kDa. Immunoblot analysis indicated that the 38-kDa protein probably originated from the 40-kDa protein during purification. The isoelectric points of the two protease species were 4.20 and 4.42. Enzyme activity, which was optimum between pH 5.5 and 7.5, was inhibited by various metal chelators; however, no effect was observed after treatment with phenylmethylsulfonyl fluoride, chymostatin, trypsin inhibitor, or dithiothreitol. Enzyme activity inhibited by metal chelators was restored upon the addition of various metal ions, including Zn2+, Fe2+, Mn2+, Cu2+, and Fe3+, but was not restored by Mg2+ or Ca2+. Atomic absorption analysis of the purified protease revealed a single gram-atom of zinc per mole of enzyme. Our findings indicate that the L. pneumophila protease resembles neutral zinc-containing metalloproteases similar to those found in other bacterial species.