Purification and characterization of a tissue plasminogen activator-inhibitor complex from human umbilical vein endothelial cell conditioned medium.
Tissue plasminogen activator-inhibitor complexes were purified from the conditioned medium of human umbilical vein endothelial cells by affinity chromatography followed by gel filtration. It was found that a single complex was isolated which can exist in two distinct interconvertible conformations. These may be separated by electrophoresis into a form with a 105,000 apparent molecular weight and a form with an 88,000 apparent molecular weight. The particular conformation which predominates may be altered by changing the pH at which preparations are incubated or by including dithiothreitol in incubation buffers. Plasminogen activator enzymatic activity may be partially recovered from purified complexes by incubation in the presence of fibrin. Incubation in 1.5 M NH4OH results in the dissociation of the complex into two major polypeptides of 67 and 40 kilodaltons (kDa). The 40-kDa protein was isolated by gel filtration high-pressure liquid chromatography. N-Terminal amino acid analysis of this protein revealed three distinct sequences. Two of these were nearly identical and matched the N-terminal sequence recently reported for the native plasminogen activator inhibitor from endothelial cells. The third sequence exactly matched an internal portion of the same protein. The results suggest that the internal sequence is located at the site where the inhibitor is cleaved by tissue plasminogen activator.
D000595:Amino Acid Sequence; D002478:Cells, Cultured; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D003470:Culture Media; D004730:Endothelium, Vascular / Q000378:metabolism*; D005260:Female; D006023:Glycoproteins / Q000302:isolation & purification* / Q000378:metabolism; D006801:Humans; D008970:Molecular Weight; D015849:Plasminogen Inactivators; D010959:Tissue Plasminogen Activator / Q000037:antagonists & inhibitors / Q000302:isolation & purification*; D014471:Umbilical Veins / Q000378:metabolism
D003470:Culture Media; D006023:Glycoproteins; D015849:Plasminogen Inactivators; D010959:Tissue Plasminogen Activator