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. 2003 Jul 8;4:5. doi: 10.1186/1471-2091-4-5

Figure 4.

Figure 4

Specific activity, functional stability an complex formation of E351A and E351R Panel A, Specific activities of wtPAI-1 and mutants were measured by a direct chromogenic assay using uPA and substrate S-2444 in microtiter plate as described under Methods. One unit of PAI-1 activity is defined as the amount that completely neutralizes 1 U of u-PA. Panel B, wtPAI-1, or mutants in assay buffer were incubated at 37°C. At times indicated, aliquots were taken out and determined their activity immediately as described above. The specific activity obtained at time zero was assigned a value of 100%. Panel C, complex formation of PAI-1s with tPA: certain amount of wtPAI-1, E351A and E351R were incubated with equal amount of tPA at 37°C for 30 minutes before put on ice, at same time these PAI-1s were incubated in PBS at 37°C for 3 hrs, respectively, before they were incubated with same amount of tPA at 37°C for 30 minutes, then the protein samples prepared at two time points were subjected to SDS-PAGE, and probed the membrane blot with anti-PAI-1 monoclonal antibody. Lane1, 2 and 3 for wtPAI-1, E351A and E351R at zero time point, lane 4, 5, 6 for wtPAI-1, E351A and E351R after 3 hours' incubation at 37°C.