Analysis of binding protein for tissue-type plasminogen activator in human endothelial cells.
The specific binding sites for tissue-type plasminogen activator (t-PA) were investigated in human umbilical vein endothelial cells. After adding 125I-t-PA (M.W. 70 kDa) to endothelial cells in suspension culture, the ligand was recovered from the cell extract after disuccinimidyl suberate treatment as a high molecular complex with M.W. of 90 kDa on SDS-PAGE. The complex reacted to only anti-t-PA IgG but not to anti-PAI-1 IgG immunoblot analysis, indicating a t-PA specific binding protein. 125I-t-PA ligand blotting of the cell extract revealed that the binding protein had M.W. 20 kDa. The binding of 125I-t-PA to endothelial cells was reduced in the presence of an excess amount of t-PA, plasminogen and 6-aminohexanoic acid, indicating that the binding sites were also recognized by plasminogen, and that t-PA and plasminogen were bound via lysine binding sites in the molecule. These findings suggest that human endothelial cells have specific t-PA binding molecules which may be expressed on the cell surface as t-PA receptors.
Fukao
H
H
Department of Physiology, Kinki University School of Medicine, Osakasayama, Japan.
Hagiya
Y
Y
Nonaka
T
T
Okada
K
K
Matsuo
O
O
D015119:Aminocaproic Acid / Q000494:pharmacology; D002478:Cells, Cultured; D004591:Electrophoresis, Polyacrylamide Gel; D004730:Endothelium, Vascular / Q000378:metabolism*; D006801:Humans; D015151:Immunoblotting; D007074:Immunoglobulin G; D007457:Iodine Radioisotopes; D008970:Molecular Weight; D010751:Phosphopyruvate Hydratase / Q000276:immunology; D010958:Plasminogen / Q000378:metabolism / Q000494:pharmacology; D011956:Receptors, Cell Surface / Q000378:metabolism*; D013388:Succinimides / Q000494:pharmacology; D010959:Tissue Plasminogen Activator / Q000378:metabolism; D014471:Umbilical Veins
D007074:Immunoglobulin G; D007457:Iodine Radioisotopes; D011956:Receptors, Cell Surface; D013388:Succinimides; D010958:Plasminogen; C067262:PLAT protein, human; D010959:Tissue Plasminogen Activator; D010751:Phosphopyruvate Hydratase; D015119:Aminocaproic Acid; C019358:disuccinimidyl suberate