Bovine endothelial cell plasminogen activator inhibitor

Abstract

A plasminogen activator inhibitor (PAI) was purified from bovine endothelial cell conditioned medium by a simple procedure in the absence of protein denaturant The yield was 2.2 mg from 1.6 1 conditioned medium in a typical experiment. The purified inhibitor showed a single band on sodium dodecyl sulfate/polyacrylamide gel electrophoresis and reverse fibrin autography with an apparent molecular mass of 45 kDa. The amino‐terminal 40‐amino‐acid sequence was determined and found to be 70% similar to the reported corresponding sequence of human PAI‐1. The amino acid composition also revealed a close relationship between bovine PAI and human PAI‐1. The purified PAI was substantially inactive (570 U/mg) but it could be activated by treatment with protein denaturants such as 1% SDS (1.8 × 10⁵ U/mg) and 4 M guanidine‐HCl (l.5 × 10⁵ U/mg). A more effective activation of this latent PAI was achieved by heat treatment at 100°C for 2.S min, generating the specific activity of 1.0 × 10⁶ U/mg. The heat‐activated PAI lost its activity during incubation at 56°C for 30 min. but repeated heat at 100°C for 2.5 min could regenerate about 70% of the initial activity. Treatment at 37°C, 56°C and 80°C. however, failed to activate the latent PAI at all. These findings suggest that the buried reactive site of the latent PAI is exposed as a result of a heat‐induced, specific conformational change, but tends to be masked again during renaturalion under mild conditions, i.e. the PAI protein takes on preferentially a latent form.