title: Localization of the binding sites of porcine tissue-type plasminogen activator and plasminogen to heparin.
abstract: To localize the binding region of porcine tissue-type plasminogen activator (EC 3.4.21.31) (t-plasminogen activator) to heparin, functionally active A and B chains (molecular mass of each 33 kDa) were separated from the two-chain t-plasminogen activator after mild reduction and alkylation. The A chain bound to fibrin-Sepharose, but not to heparin-Sepharose. In contrast, the B chain showed amidase activity toward HD-Ile-Pro-Arg-p-nitroanilide (S-2288) and a high affinity for heparin-Sepharose, but no affinity for fibrin-Sepharose. Plasminogen activator activity of the B chain was stimulated by heparin (about 3-fold), but not by fibrin. On the other hand, the elastase digestion fragments of plasminogen, kringle 1-3 and kringle 4, had no affinity for a heparin-Sepharose column, whereas the other fragment, Val442-plasminogen, efficiently bound to the column and was eluted with 1.6 M KSCN-containing buffer. The stimulatory effect of fibrin on two-chain t-plasminogen activator-catalyzed Val442-plasminogen activation was clearly diminished by heparin. These results suggest that heparin can form a complex with both t-plasminogen activator and plasminogen molecules through their catalytic regions located in each B chain, and that the heparin connection between t-plasminogen activator and plasminogen may improve the plasminogen activation kinetics by making a situation in which t-plasminogen activator is easily approachable to plasminogen.
mesh_terms: D000818:Animals; D001665:Binding Sites; D002846:Chromatography, Affinity; D004591:Electrophoresis, Polyacrylamide Gel; D005337:Fibrin / Q000378:metabolism; D006493:Heparin / Q000378:metabolism*; D009842:Oligopeptides / Q000378:metabolism; D010958:Plasminogen / Q000378:metabolism*; D013552:Swine; D010959:Tissue Plasminogen Activator / Q000378:metabolism*
chemical_list: D009842:Oligopeptides; C041319:isoleucyl-prolyl-arginine-4-nitroanilide; D005337:Fibrin; D010958:Plasminogen; D006493:Heparin; D010959:Tissue Plasminogen Activator