INTERCONVERSIONS BETWEEN ACTIVE, INERT AND SUBSTRATE FORMS OF DENATURED REFOLDED TYPE-1 PLASMINOGEN-ACTIVATOR INHIBITOR

The latent form of type-1 plasminogen activator inhibitor (PAI-1) acqu ires inhibitory activity by denaturation followed by refolding. We sho w here that the reactions of denatured/refolded PAI-1 with plasminogen activators are affected by low concentrations of SDS, which may remai n after using SDS for denaturation. Without SDS, the active fraction o f denatured/refolded PAI-1 comprised around 60%. Increasing SDS concen trations led to conversions to an inert form without inhibitory activi ty; then to a substrate form, that is being cleaved proteolytically in the reactive centre by the activators without complex formation, and finally to a second inert form. The first two conversions were associa ted with changes of the reactivity with monoclonal antibodies and of t he thermal stability, respectively. Our results define clearly differe nt interconvertible forms of denatured/refolded PAI-1, distinguish the se from the latent and the reactive-centre-cleaved forms, and provide conditions for reproducibly producing reactive-centre-cleaved PAI-1 an d PAI-1/activator complexes.