Conformational studies on plasminogen activator inhibitor (PAI-1) in active, latent, substrate, and cleaved forms.
Plasminogen activator inhibitor 1 (PAI-1), the primary physiological inhibitor of t-PA, is an unusual member of the serpin family of serine protease inhibitors, in that it spontaneously converts to a latent form. Latent PAI-1 has been reported to share characteristics with the cleaved form of other serpins. Here we examine the conformation of four forms of PAI-1, active and latent wild-type, together with a noninhibitory, substrate mutant that is cleavable at P1-P1', and its cleaved product. The circular dichroism spectra of active and latent PAI-1 showed differences consistent with decreased alpha-helix from 26% to 22% and increased beta-sheet from 23% to 34% as active-->latent. Active and substrate PAI-1 were less thermostable than latent PAI-1, which was 50% denatured at 70 degrees C. In contrast, cleaved PAI-1 was very stable, with little loss of structure at 100 degrees C. Cleaved PAI-1 was much more resistant to guanidinium chloride (Gdn-HCl), 50% unfolding requiring 4.5 M Gdn-HCl, while active, latent, and substrate forms of PAI-1 were 50% unfolded in 2-2.5 M Gdn-HCl. The differences in fluorescence emission maxima, latent 339 nm, active 336 nm, substrate 343 nm, and cleaved 333 nm, underline the contrast between latent and cleaved PAI-1. The conformational changes occurring on cleavage are clearly more profound than those seen on transition from active to latent PAI-1.(ABSTRACT TRUNCATED AT 250 WORDS)
Sancho
E
E
Department of Molecular & Cell Biology, University of Aberdeen, Scotland.
Declerck
P J
PJ
Price
N C
NC
Kelly
S M
SM
Booth
N A
NA
D002152:Calorimetry, Differential Scanning; D002942:Circular Dichroism; D019791:Guanidine; D006146:Guanidines / Q000494:pharmacology; D006358:Hot Temperature; D017395:Plasminogen Activator Inhibitor 1 / Q000737:chemistry*; D011489:Protein Denaturation / Q000187:drug effects; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins; D013050:Spectrometry, Fluorescence; D017550:Spectroscopy, Fourier Transform Infrared; D013329:Structure-Activity Relationship; D014568:Urokinase-Type Plasminogen Activator / Q000037:antagonists & inhibitors*
D006146:Guanidines; D017395:Plasminogen Activator Inhibitor 1; D011994:Recombinant Proteins; D014568:Urokinase-Type Plasminogen Activator; D019791:Guanidine