Bovine endothelial cell plasminogen activator inhibitor. Purification and heat activation.
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 1.61 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 X 10(5) U/mg) and 4 M guanidine-HCl (1.5 X 10(5) U/mg). A more effective activation of this latent PAI was achieved by heat treatment at 100 degrees C for 2.5 min, generating the specific activity of 1.0 X 10(6) U/mg. The heat-activated PAI lost its activity during incubation at 56 degrees C for 30 min, but repeated heat at 100 degrees C for 2.5 min could regenerate about 70% of the initial activity. Treatment at 37 degrees C, 56 degrees C and 80 degrees 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 renaturation under mild conditions, i.e. the PAI protein takes on preferentially a latent form.
Katagiri
K
K
Central Research Laboratories, Banyu Pharmaceutical Co. Ltd, Tokyo, Japan.
Okada
K
K
Hattori
H
H
Yano
M
M
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D002339:Carotid Arteries / Q000032:analysis; D002417:Cattle; D002478:Cells, Cultured; D004591:Electrophoresis, Polyacrylamide Gel; D004730:Endothelium, Vascular / Q000032:analysis*; D006023:Glycoproteins / Q000302:isolation & purification*; D006358:Hot Temperature; D006801:Humans; D007525:Isoelectric Focusing; D015849:Plasminogen Inactivators
D000596:Amino Acids; D006023:Glycoproteins; D015849:Plasminogen Inactivators