PURIFICATION AND CHARACTERIZATION OF 2 FORMS OF A HIGH-MOLECULAR-WEIGHT CYSTEINE PROTEINASE (PORPHYPAIN) FROM PORPHYROMONAS-GINGIVALIS

Porphyromonas gingivalis, an organism implicated in the etiology and p athogenesis of human periodontal diseases, produces a variety of poten t proteolytic enzymes, and it has been suggested that these enzymes pl ay a direct role in the destruction of periodontal tissues. We now rep ort that two cell-associated cysteine proteinases of P. gingivalis W12 , with molecular masses of approximately 150 kDa (porphypain-1) and 12 0 kDa (porphypain-2), as determined by sodium dodecyl sulfate (SDS) po lyacrylamide gel electrophoresis, have been separated and purified to apparent homogeneity. These proteinases appear to be SDS-stable confor mational variants of a 180-kDa enzyme, and they are the largest cystei ne proteinases yet purified from P. gingivalis. The purified proteinas es hydrolyze fibrinogen, tosyl-Gly-L-Pro-L-Arg p-nitroanilide, and tos yl-Gly-L-Pro-L-Lys p-nitroanilide. While hydrolysis of both synthetic substrates by porphypain-1 and -2 requires activation by reducing agen ts, is inhibited by EDTA, and is stimulated in the presence of derivat ives of glycine, the Arg-amidolytic activity is sensitive to leupeptin and H-D-tyrosyl-L-prolyl-L-arginyl chloromethyl ketone, whereas the L ys-amidolytic activity is sensitive to tosyl-L-lysyl chloromethyl keto ne and insensitive to leupeptin. These data suggest that porphypains c ontain two types of active sites. These cell-associated P. gingivalis proteinases may contribute significantly and directly to periodontal t issue destruction.