Purification and characterization of two forms of a high-molecular-weight cysteine proteinase (porphypain) from Porphyromonas gingivalis.
Porphyromonas gingivalis, and organism implicated in the etiology and pathogenesis of human periodontal diseases, produces a variety of potent proteolytic enzymes, and it has been suggested that these enzymes play a direct role in the destruction of periodontal tissues. We now report that two cell-associated cysteine proteinases of P. gingivalis W12, with molecular masses of approximately 150 kDa (porphypain-1) and 120 kDa (porphypain-2), as determined by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis, have been separated and purified to apparent homogeneity. These proteinases appear to be SDS-stable conformational variants of a 180-kDa enzyme, and they are the largest cysteine proteinases yet purified from P. gingivalis. The purified proteinases hydrolyze fibrinogen, tosyl-Gly-L-Pro-L-Arg p-nitroanilide, and tosyl-Gly-L-Pro-L-Lys p-nitroanilide. While hydrolysis of both synthetic substrates by porphypain-1 and -2 requires activation by reducing agents, is inhibited by EDTA, and is stimulated in the presence of derivatives of glycine, the Arg-amidolytic activity is sensitive to leupeptin and H-D-tyrosyl-L-prolyl-L-arginyl chloromethyl ketone, whereas the Lys-amidolytic activity is sensitive to tosyl-L-lysyl chloromethyl ketone and insensitive to leupeptin. These data suggest that porphypains contain two types of active sites. These cell-associated P. gingivalis proteinases may contribute significantly and directly to periodontal tissue destruction.
Ciborowski
P
P
Department of Periodontics, University of Pittsburgh, Pennsylvania 15261.
Nishikata
M
M
Allen
R D
RD
Lantz
M S
MS
D000595:Amino Acid Sequence; D003546:Cysteine Endopeptidases / Q000302:isolation & purification* / Q000378:metabolism; D015853:Cysteine Proteinase Inhibitors; D005340:Fibrinogen / Q000378:metabolism; D007526:Isoelectric Point; D007527:Isoenzymes / Q000302:isolation & purification* / Q000378:metabolism; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009842:Oligopeptides / Q000378:metabolism; D010449:Peptide Mapping; D016966:Porphyromonas gingivalis / Q000201:enzymology*; D011487:Protein Conformation; D017421:Sequence Analysis; D013379:Substrate Specificity
D015853:Cysteine Proteinase Inhibitors; D007527:Isoenzymes; D009842:Oligopeptides; D005340:Fibrinogen; D003546:Cysteine Endopeptidases