MODULATION OF PLASMINOGEN-ACTIVATOR INHIBITOR-1 BY TRITON X-100 - IDENTIFICATION OF 2 CONSECUTIVE CONFORMATIONAL TRANSITIONS

Plasminogen activator inhibitor-1 (PAI-1) is a unique member of the se rpin superfamily because of its conformational and functional flexibil ity. In the present study, we have evaluated the influence of the noni onic detergent Triton X-100 (TX-100) on the functional stability and c onformational transitions of PAI-1. At 37 degrees C, TX-100 induced a concentration-dependent decrease of the functional half-life of PAI-1 resulting in half-lives of 177 +/- 54 min (mean +/- SD, n = 3), 19 +/- 2 min, 1.7 +/- 0.3 min and 0.53 +/- 0.03 min in the presence of 0.005 , 0.010, 0.020 and 0.2% TX-100, respectively, compared to a half-life of 270 +/- 146 min in the absence of TX-100. Confer mational analysis at various time points and at different temperatures (0 degrees C, 25 degrees C, 37 degrees C) revealed that this inactivation proceeds thro ugh the formation of a substrate-like intermediate followed by the for mation of the latent form. Kinetic evaluation demonstrated that this c onversion fits to two consecutive first-order transitions, i.e. active (k(1)) under right arrow substrate (k(2)) under right arrow latent. T he k(1) value was strongly dependent on the concentration of TX-100 (e .g. 0.002 +/- 0.0006 s(-1) and 0.029 +/- 0.003 s(-1) for 0.01% and 0.2 % TX-100 at 37 degrees C) whereas the conversion of substrate to laten t (k(2)) was virtually independent of the TX-100 concentration (i, e. 0.012 +/- 0.002 s(-1) and 0.011 +/- 0.001 s(-1) for 0.01 and 0.2% TX-1 00 at 37 degrees C). Experiments with a variety of other non-ionic amp hiphilic compounds revealed that the amphiphilic character of the comp ound is, at least in part, responsible for the observed effects and st rongly indicate that the currently reported mechanism of inactivation is of general importance for the conformational transitions in PAI-1. In conclusion, TX-100 changes the initial conformation of PAI-1 result ing in altered functional properties. This observation allows us to de velop a new model for the mechanism involved in the conformational fle xibility of PAI-1 and may provide new insights for the development of strategies for interference with PAI-1 activity.