Biological and antigenic characterization of three BApNA-hydrolyzing proteases from the culture supernatant of Porphyromonas gingivalis.

Biological and antigenic distinction of 3-N-alpha-benzoyl-DL-arginine p-nitroanilide (BApNA)-hydrolyzing proteases (Pase-B, Pase-C and Pase-S) isolated from the culture supernatant of Porphyromonas gingivalis were determined. Immunoblotting analysis of these enzymes using a polyclonal antibody against Pase-S, which is a soluble, clostripain-like protease, revealed immunological distinction from Pase-C, a vesicle-associated thiol-protease. Pase-B, a vesicle-associated clostripain-like protease, reacted with the antibody and was also found to contain a considerable amount of carbohydrates in its structure, as compared with the others. Analysis of N-terminal amino acids of Pase-B provided a sequence not found in the SwissProt data bank or previously reported as N-terminal sequences of proteases from P. gingivalis. Pase-S, resembling Pase-B in its hydrolytic specificity, cleaved only arginine residues of peptides and degraded type IV and denatured type I collagen. Pase-C hydrolyzed N-alpha-benzoyl-DL-lysine p-nitroanilide and showed the strongest capacity of degrading native type I collagen. This enzyme was also the only one to possess hemagglutinating activity. Our findings suggest that Pase-S from P. gingivalis is less active than Pase-C and that the enzyme may be an isozyme of Pase-B.