Solvent effects on activity and conformation of plasminogen activator inhibitor-1

We have studied effects of the solvent composition on the activity and the conformation of human plasminogen activator inhibitor-1 (PAI-1) from HT-108 0 fibrosarcoma cells. Non-ionic detergents, including Triton X-100, reduced the inhibitory activity of PAI-1 more than 20-fold at 0 degrees C, but les s than 2-fold at 37 degrees C, while glycerol partly prevented the detergen t-induced activity-loss at 0 degrees C. The activity-loss was associated wi th an increase in PAI-1 substrate behaviour. Evaluating the PAI-1 conformat ion by proteolytic susceptibility of specific peptide bonds, we found that the V8-proteinase susceptibility of the Glu332-Ser333 (P-17-P-16) bond, par t of the hinge between the reactive centre loop (RCL) and beta-strand 5A, a nd the endoproteinase Asp-N susceptibility of several bonds in the beta-str and 2A-alpha-helix E region were increased by detergents at both 0 and 37 d egrees C. The susceptibility of the Gln321-Ala322 and the Lys325-Va1326 bon ds in beta-strand 5A to papain and trypsin, respectively, was increased by detergents at 0 degrees C, but not at 37 degrees C, showing a strict correl ation between proteinase susceptibility of beta-strand 5A and activity-loss at 0 degrees C. Since the beta-strand 2A-alpha-helix E region also showed differential susceptibility to endoproteinase Asp-N in latent, active, and reactive centre-cleaved PAI-1, we propose that a detergent-induced conforma tional change of the beta-strand 2A-alpha-helix E region influences the mov ements of beta-sheet A, resulting in a cold-induced conformational change o f beta-strand 5A and thereby an increased substrate behaviour at low temper atures. These results provide new information about the structural basis fo r serpin substrate behaviour.