Effect of p97 on pro-uPA, tPA, and plasminogen. The serine activity of 90 nM pro-uPA (A) and 75 nM tPA (B) were measured in the absence (○) or presence (•) of 70 nM p97 without plasminogen. The reaction was performed in a final volume of 200 μL as described in “Materials and methods.” In both panels A and B, controls were also performed with p97 (▪) but without pro-uPA or tPA (n = 9 for pro-uPA; n = 6 for tPA). (C) The effect of p97 on pro-uPA was evaluated after incubation in the presence or absence of plasminogen. 2 μg p97 (lane 1), 1 μg pro-uPA (lane 2), and 2 μg plasminogen (lane 3) were incubated for 5 minutes at 37°C alone as controls. Pro-uPA (2 μg) was incubated at 37°C for 5 minutes with 2 μg p97 (lane 4). Plasminogen and pro-uPA were added without incubation (lane 5) and with 5 minutes' incubation at 37°C (lane 6). Pro-uPA with 2 μg of both p97 and plasminogen was added without incubation (lane 7) or with 5 minutes' incubation at 37°C (lane 8). Tc-uPA (2 μg) was also loaded as a control (lane 9). Proteins were then separated by SDS-PAGE, using a 12.5% acrylamide gel under reducing conditions. After electrophoresis, proteins were visualized by staining the gel with Coomassie blue. (D) Effect of p97 on plasminogen degradation by pro-uPA. Quantities (3 μg) of p97 (lane 1), Glu-plasminogen (lane 2), and Lys-plasminogen (lane 3) were incubated alone for 6 hours at 37°C. In lane 4, 3 μg of both glu-plasminogen and p97 was also incubated for 6 hours at 37°C. Pro-uPA (20 ng) was added to plasminogen for the same period of incubation at 37°C (lane 5). p97 was added to pro-uPA and plasminogen for 6 hours at 37°C (lane 6) or 4°C (lane 7). In lane 8, 3 μg angiostatin was also added as a control. Proteins were then separated by SDS-PAGE, using a 7.5% acrylamide gel under nonreducing conditions. After electrophoresis, proteins were visualized by staining the gel with Coomassie blue.