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) acquires inhibitory activity by denaturation followed by refolding. We show here that the reactions of denatured/refolded PAI-1 with plasminogen activators are affected by low concentrations of SDS, which may remain after using SDS for denaturation. Without SDS, the active fraction of denatured/refolded PAI-1 comprised around 60%. Increasing SDS concentrations led to conversions to an inert form without inhibitory activity; 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 associated with changes of the reactivity with monoclonal antibodies and of the thermal stability, respectively. Our results define clearly different interconvertible forms of denatured/refolded PAI-1, distinguish these from the latent and the reactive-centre-cleaved forms, and provide conditions for reproducibly producing reactive-centre-cleaved PAI-1 and PAI-1/activator complexes.
Department of Molecular Biology, University of Aarhus, Denmark.
M
M
Munch
C W
CW
Heegaard
P A
PA
Andreasen
D000818:Animals; D000906:Antibodies / Q000276:immunology; D000915:Antibody Affinity; D002460:Cell Line / Q000201:enzymology; D002522:Chlorocebus aethiops; D004795:Enzyme Stability; D006801:Humans; D017395:Plasminogen Activator Inhibitor 1 / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism*; D010960:Plasminogen Activators / Q000378:metabolism; D011487:Protein Conformation; D011489:Protein Denaturation; D012967:Sodium Dodecyl Sulfate
D000906:Antibodies; D017395:Plasminogen Activator Inhibitor 1; D012967:Sodium Dodecyl Sulfate; D010960:Plasminogen Activators