Figure 5

Activation of plasminogen bound to LppA. A, B Binding ability of M. bovis LppA to plasminogen and tPA was detected by dot blot, with 6 × His peptides acting as the negative control. C Determination of kinetic curves of LppA-promoted plasminogen activation by tPA. Plasminogen and rLppA were incubated on a microtiter plate for 1 h, and then tPA and the chromogenic substrate d-Val-Leu-Lys p-nitroaniline dihydrochloride were added. Plasmin activity was detected photometrically at 405 nm for every 15 min for 135 min. Wells without plasminogen or tPA were used as negative controls. rLppA-bound plasminogen was converted to plasmin, which cleaved the substrate d-Val-Leu-Lys p-nitroaniline dihydrochloride in a time-dependent manner. ε-ACA inhibited d-Val-Leu-Lys p-nitroaniline dihydrochloride cleavage. The absorbance at 405 nm of the 6 × His peptide control was significantly lower than that of the rLppA-added wells.