The purification and characterization of an 88-kDa Porphyromonas endodontalis 35406 protease

A Porphyromonas endodontalis ATCC 35406 protease was purified from Triton X -114 cell extracts by preparative SDS-PAGE followed by electroelution. The purified enzyme exhibits a molecular size of 88 kDa and was dissociated int o two polypeptides of 43 and 41 kDa upon heating in the presence of sodium dodecyl sulfate with or without a reducing agent. The protease (pH optimum 7.5-8.0) degraded the extracellular matrix proteins fibrinogen and fibronec tin. Collagen IV was also degraded at 37 degreesC but not at 28 degreesC. T he protease also cleaved the bioactive peptide angiotensin at amino acid re sidue phenylalanine-8 and tyrosine-4 but failed to hydrolyze bradykinin, va sopressin and synthetic chromogenic substrates with phenylalanine or tyrosi ne at the P1 position. In addition, two peptidases were detected in P. endo dontalis cells: a proline aminopeptidase that remained associated with the cell pellet after detergent extraction and peptidase/s that partitioned int o the Triton X-114 phase after phase separation and degraded the bioactive peptides bradykinin and vasopressin. These P. endodontalis peptidases and p roteases may play an important role in both the nutrition and pathogenicity of these assacharolytic microorganisms. The inactivation of bioactive pept ides and degradation of extracellular matrix proteins by bacterial enzymes may contribute to the damage of host tissues accompanied with endodontic in fections.