Purification and characterization of wtPAI-1. Panel A, Purification of wtPAI-1. The medium from High five cells was harvested 48 hours post infection and PAI-1s were purified on a heparin-Sepharose column and stained as described under Methods. Lane 1, the medium from infected High five cells, lane 2, wtPAI-1 eluted from the heparin-Sepharose column. Panel B, Treatment of fibrosarcoma- and wtPAI-1 with N-glycanase as described under Methods. Lane1 and 2 were fibrosarcoma PAI-1, lane 3, and 4 were wtPAI-1. Fibrosarcoma PAI-1 (lane 2) and wtPAI-1 (lane 4) were treated with N-glycanase. Panel C, Stability of fibrosarcoma PAI-1 or of wtPAI-1. Fibrosarcoma PAI-1 or wtPAI-1 was incubated (37°C) in the absence (empty circle and square) or in the presence of a two molar fold of vitronectin (filled circle and square) in PBS. At the indicated time periods, aliquots were removed and the inhibitory activity of the PAI-1s towards uPA was measured as described under Methods. The inhibitory activity prior to the incubation at 37°C was taken as 100%.