Purification and identification of two structural variants of porcine tissue plasminogen activator by affinity adsorption on fibrin.
Porcine tissue plasminogen activator has been purified from delipidized heart tissue by affinity adsorption to fibrin. A crude fraction is prepared from an acid tissue extract by precipitation with ammonium sulphate. The tissue activator of this fraction is isolated by adsorption on fibrin and elution with KSCN. The procedure also includes chromatography on arginine-Sepharose and two gel-filtration steps. The final product has a specific activity of 250 000 IU/mg (+/- 16 000) as compared to an international urokinase reference preparation. The yield calculated from the active ammonium sulphate precipitate is about 28%. An approx. 7 000-fold increase of specific activity is obtained, most of which is achieved in the fibrin step. The native tissue plasminogen activator consists of a single chain molecule with a molecular weight of 64 000 as measured by SDS-polyacrylamide gel electrophoresis. In a previous report, it was claimed that the activator is composed of two disulphide-connected polypeptide chains. These results were due to a preparation artefact, caused by proteolytic activity present in the tissue extracts. The introduction of the protease inhibitor aprotinin and 6-amino-hexanoic acid in the purification procedure has abolished the effect of the protease contaminant, leading to the production of a one-chain activator. Treatment with plasmin transforms the native, one-chain tissue activator into a variant composed of two chains of about equal size (Mr 32 000) connected by disulphide bonding. This modified activator is indistinguishable from the one obtained at insufficient protection against proteolytic enzymes. The cleavage by plasmin causes about an 8-fold increase of amidolytic activity as measured on H-D-Val-Gly-Arg-p-nitroanilide. The fibrinolytic activity as measured by clot lysis in only slightly increased. The physiological significance of the cleavage is discussed.
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D002846:Chromatography, Affinity; D005337:Fibrin; D005340:Fibrinogen / Q000378:metabolism; D014644:Genetic Variation; D006801:Humans; D007700:Kinetics; D009206:Myocardium / Q000378:metabolism; D010960:Plasminogen Activators / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D013552:Swine
D000596:Amino Acids; D005337:Fibrin; D005340:Fibrinogen; D010960:Plasminogen Activators