Characterization of the Trypsin-Like Enzymes of Porphyromonas Gingivalis W83 Using a Radiolabelled Active-Site-Directed Inhibitor

J Gen Microbiol. 1993 May;139(5):949-55. doi: 10.1099/00221287-139-5-949.

Abstract

The trypsin-like enzyme activity of Porphyromonas gingivalis is an important virulence determinant of this organism in destructive periodontitis. An active-site-directed inhibitor, tyrosyl-alanyl-lysyl-arginine chloromethyl ketone (YAKR-CK) was radio-iodinated and used with SDS-PAGE and autoradiography to determine the number and molecular masses of enzymes with trypsin-like specificity produced by P. gingivalis W83. Two forms (I + II) were detected in both crude culture supernatant and whole cell sonicates. Protease I was a sharp band (47 kDa) on reducing SDS-PAGE; Protease II electrophoresed as a diffuse band in the range 70-90 kDa. The specificity with which the inhibitor bound to Protease I was established in competition experiments using other active-site-directed agents. YAKR-CK inhibited P. gingivalis whole cell haemagglutination, supporting the possible role of trypsin-like proteases of this organism in adhesion mechanisms.

MeSH terms

  • Affinity Labels / metabolism*
  • Amino Acid Chloromethyl Ketones / metabolism*
  • Amino Acid Sequence
  • Bacterial Adhesion
  • Binding Sites
  • Hemagglutination / drug effects
  • Isoenzymes
  • Molecular Sequence Data
  • Molecular Weight
  • Porphyromonas gingivalis / enzymology*
  • Trypsin / isolation & purification
  • Trypsin / metabolism*
  • Trypsin Inhibitors / metabolism*

Substances

  • Affinity Labels
  • Amino Acid Chloromethyl Ketones
  • Isoenzymes
  • Trypsin Inhibitors
  • tyrosyl-alanyl-lysyl-arginine chloromethyl ketone
  • Trypsin