Purification and characterization of two forms of a high-molecular-weight cysteine proteinase (porphypain) from Porphyromonas gingivalis

Pawel S Ciborowski, M. Nishikata, R. D. Allen, M. S. Lantz

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Abstract

Porphyromonas gingivalis, an 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.

Original languageEnglish (US)
Pages (from-to)4549-4557
Number of pages9
JournalJournal of bacteriology
Volume176
Issue number15
DOIs
StatePublished - Jan 1 1994

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Porphyromonas gingivalis
Cysteine Proteases
Peptide Hydrolases
Molecular Weight
Ketones
Sodium Dodecyl Sulfate
Reducing Agents
Periodontal Diseases
Enzymes
Edetic Acid
Glycine
Fibrinogen
Polyacrylamide Gel Electrophoresis
Catalytic Domain
Hydrolysis
Porphyromonas gingivalis porphypain protein
leupeptin

ASJC Scopus subject areas

  • Microbiology
  • Molecular Biology

Cite this

Purification and characterization of two forms of a high-molecular-weight cysteine proteinase (porphypain) from Porphyromonas gingivalis. / Ciborowski, Pawel S; Nishikata, M.; Allen, R. D.; Lantz, M. S.

In: Journal of bacteriology, Vol. 176, No. 15, 01.01.1994, p. 4549-4557.

Research output: Contribution to journalArticle

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