Isolation And Characterization Of A Plasminogen Activator (PA) From Human Myocardial Tissue
Pig heart is one of the major sources for isolation of PAs. However, no attempt has been made to isolate PA from human heart. It was the aim of the study to isolate a PA from human myocardial tissue and to compare it with the vascular plasminogen activator (VPA) derived from cadaver vessel eluates.Human heart was obtained from cadavers and myocardial tissue was prepared, homogenized, and after extraction with a neutral buffer, a pH=4.2 extract was obtained containing about 60-70% of PA activity originally present in the homogenate. The latter extract was made 2 M with (NH4)2SO4 at pH=7.0 precipitating all of the activator activity. PA activity could be purified to apparent homogeneity by reverse ammonium sulfate gradient solubilization followed by hydrophobic interaction chromatography on octly- Sepharose, affinity chromatography on arginin-Sepharose and gel filtration on Sephadex G-150. The material obtained showed a single band in SDS polyacrylamide gel electrophoresis corresponding to Mr=70.000 and the region of the gel where PA activities could be eluted.Kinetic analysis of the purified activator with synthetic paranitroanilide substrates revealed a KM of 0.8 mM,4 mM, and 0.4 mM for H-D-Ile-Pro-Arg-paranitroanilide, H-D-Val- Gly-Arg-paranitroanilide, and H-D-Phe-Aze-Arg-paranitro- anilide, respectively, which were almost identic to those obtained with the VPA using the same subtrates. Plasminogen activation with the myocardial PA showed in a purified system a strong dependence on the presence of fibrin as it could be observed with VPA.From the similar isolation characteristics, the molecular weight, the amidolytic activities, and the fibrin effect on plasmin formation it can be concluded that the PA isolated from human myocardial tissue is similar or identic to the VPA.