Purification and characterization of three types of proteases from culture supernatants of Porphyromonas gingivalis

Abstract

Three types of caseinolytic proteases (Pase-A, Pase-B, and Pase-C) were isolated and purified from culture supernatants of Porphyromonas gingivalis 381 by the combined procedures of acetone precipitation, gel filtration, solubilization with octylthioglucoside followed by affinity chromatography on arginine-Sepharose 4B, high-performance liquid chromatography (HPLC) on Biofine IEC-DEAE, and HPLC on TSK-G4000SW. By sodium dodecyl sulfate-polyacrylamide gel electrophoresis, Pase-A and -B showed diffuse protein bands of 105 to 110 and 72 to 80 kDa, respectively, and Pase-C showed a clear band of about 44 kDa. Pase-B and -C hydrolyzed some synthetic substrates for trypsin, but Pase-B did not act on the carboxyl side of lysine in insulin chain B or on a synthetic substrate which trypsin and Pase-C acted on. Pase-A did not act on the synthetic substrates but cleaved the peptide bonds Glu-Ala and Ala-Leu of insulin. Leupeptin inhibition of the caseinolytic activity of both Pase-A and -B was similar to its inhibition of Pase-C. Phenylmethylsulfonyl fluoride and diisopropyl fluorophosphate strongly inhibited Pase-A, but no significant effect on the other enzymes was observed, suggesting that only Pase-A is a serine protease. The inhibitory characteristics of Pase-B and -C were very similar. Pase-A was not thiol dependent for enzyme activity, but Pase-B was strongly dependent, i.e., even more so than Pase-C. Pase-A inactivated the inhibitory activity of plasma alpha-1-antitrypsin, but the other two did not. These results show that P. gingivalis produces different types of proteases other than the trypsinlike protease generally reported.