Comparative study of four proteases from spent culture media of Porphyromonas gingivalis (FAY-19M-1).

Four gelatin cleaving proteases were partially purified from culture media of Porphyromonas gingivalis (FAY-19M-1) by sequential chromatography on columns of DEAE-Sepharose, Sephadex G-100 and chromatofocusing on PBE-94. The molecular mass of each of these proteases, estimated by relative mobility on gelatin-containing SDS-PAGE, was 50 kDa (Pool D1b), 120 kDa (Pool E1a), approximately 160 kDa (Pool E1b) and > 300 kDa (Pool A1a), respectively. These proteases also differed with respect to charge characteristics, inhibition profile and cleavage specificity. Protease pools A1a and E1a were inhibited by thiol modifying reagents. Protease pool A1a was also inhibited by N-tosyl-L-lysine chloromethyl ketone, and E1a was inhibited by antipain. Protease pool D1b was inhibited by E-64, leupeptin and antipain, and protease E1b was not inhibited by either of these inhibitors. The detailed substrate specificity of these proteases was checked by using chromogenic substrates, synthetic peptides and native proteins. Protease E1b was very active in degrading collagen, fibrinogen, fibronectin, IgG, IgA, third component of complement (C3), serum albumin, transferrin and varies; is directly proportional to 1-acid glycoprotein as substrates. Fibrinogen, fibronectin and complement C3 component were also cleaved by A1a, D1b and E1a. Synthetic peptides insulin B chain, cecropin P-1 and magainin were cleaved by E1b. Based on FAB analysis E1b showed preferential cleavage at hydrophobic or neutral residues. Protease A1a was active towards chromogenic substrates with either lys or arg in P1 position. Protease D1b cleaved chromogenic substrates with arg in P1 position and cleaved synthetic peptides magainin and (KIAGKIA)3-NH2 at lys residues also. Protease E1a showed glycyl-prolyl peptidase activity.