FIGURE 3.
Effect of Lys129 mutation on Kgp expression and activity. Western blotting analysis of parental strain P. gingivalis W83 and mutants K129A, K129E, and K129R is shown. Late exponential/early stationary bacterial cultures (BC) were separated by centrifugation into cell-free growth medium (GM) and cell pellet. The latter was washed, giving rise to the whole cell (WC) fraction, which was further fractionated into the soluble intracellular protein fraction (CP + PP) and the cell envelope fracion (CE) by sonication and ultracentrifugation. A and B, all fractions were standardized to the initial volume of the culture subjected to centrifugation and analyzed by Western blotting to detect Kgp forms (A) and Rgps (control) (B). C and D, Kgp (C) and Rgp (D) gingipain activities determined in whole cultures, cell-free growth medium, and washed cells using specific substrates. The whole-culture activity of the wild-type strain was arbitrarily taken as 100%.