Characterization of sodium dodecyl sulfate-stable Bacteroides gingivalis proteases by polyacrylamide gel electrophoresis.
Profiles of the proteolytic activities found in Bacteroides gingivalis culture supernatants, outer membranes, vesicles, and cell extracts were analyzed in sodium dodecyl sulfate-polyacrylamide gels containing covalently bound bovine serum albumin. A total of eight distinct bands of proteolytic activity could be detected. Four of these were found in the culture supernatant (P1, P2, P3, and P4). The outer membranes, vesicles, and the cell extract each contained seven major proteolytic bands (P1, P3, P4, P5, P6, P7, and P8). No activity was found in the membrane-free extract, suggesting that the proteases were associated with the cell envelope. With the exception of P7 and P8, all the proteolytic bands were dependent on reducing agents for activity. The eight proteolytic bands were distributed in an identical manner in all four strains of B. gingivalis studied. The effects of protease inhibitors, pH, and heat were determined. Sulfhydryl group reagents and N-alpha-p-tosyl-L-lysine chloromethyl ketone reduced proteolytic activity. The optimum pH was found to be between 7 and 8. A 30-min preincubation at 50 degrees C inactivated the P6, P7, and P8 proteolytic bands. All proteolytic activity was lost after the samples were heated at 75 degrees C for 30 min.
Grenier
D
D
Department of Microbiology, University of British Columbia, Vancouver, Canada.
Chao
G
G
McBride
B C
BC
D001439:Bacteroides / Q000201:enzymology* / Q000254:growth & development; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D004795:Enzyme Stability; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D010447:Peptide Hydrolases / Q000302:isolation & purification* / Q000378:metabolism; D011480:Protease Inhibitors / Q000494:pharmacology; D012835:Silver Nitrate; D012967:Sodium Dodecyl Sulfate; D013194:Staining and Labeling
D011480:Protease Inhibitors; D012967:Sodium Dodecyl Sulfate; D012835:Silver Nitrate; D010447:Peptide Hydrolases