Characterization of the trypsin-like enzymes of Porphyromonas gingivalis W83 using a radiolabelled active-site-directed inhibitor.
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.
MRC Dental Research Unit, London Hospital Medical College, Whitechapel, UK.
M A
MA
Curtis
M
M
Ramakrishnan
J M
JM
Slaney
D000345:Affinity Labels / Q000378:metabolism*; D000590:Amino Acid Chloromethyl Ketones / Q000378:metabolism*; D000595:Amino Acid Sequence; D001422:Bacterial Adhesion; D001665:Binding Sites; D006384:Hemagglutination / Q000187:drug effects; D007527:Isoenzymes; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016966:Porphyromonas gingivalis / Q000201:enzymology*; D014357:Trypsin / Q000302:isolation & purification / Q000378:metabolism*; D014361:Trypsin Inhibitors / Q000378:metabolism*
D000345:Affinity Labels; D000590:Amino Acid Chloromethyl Ketones; D007527:Isoenzymes; D014361:Trypsin Inhibitors; C082167:tyrosyl-alanyl-lysyl-arginine chloromethyl ketone; D014357:Trypsin