A quantitative assay for the activation of plasminogen by transformed cells in situ and by urokinase
An in situ quantitative assay for the plasminogen activator of transformed cells is described in which as few as 3500 transformed cells are incubated with purified plasminogen for various time intervals and the amount of plasmin formed is then quickly titrated with the new fluorogenic, active-site titrant 3′,6′-bis(4-guanidinobenzoyloxy)-5-[N′-(4-carboxyphenyl) thioureido]spiro[isobenzofuran-1(3H),9′-[9H]xanthen]-3-one. The kinetics of plasminogen activation are linear and obey the Michaelis-Menten rate equation. The Km(app) and Vmax for the plasminogen activator of a rat neurotumor cell line with the two forms of dog plasminogen fractionated by lysine-Sepharose column chromatography are determined. The Km(app) for fraction I plasminogen is 6.6 μM and for fraction II plasminogen 3.4 μM. No secreted plasminogen activator is observed during the assay, indicating that under these conditions the plasminogen activator is cell associated. The assay is also used to show that lysine is a competitive inhibitor of transformed cell plasminogen activator. As a result of the ability to measure the plasminogen activator activity of cells quantitatively, the role of this enzyme in a variety of physiological processes can be more easily elucidated. The kinetics of activation of two forms of dog plasminogen by human urokinase, a soluble as opposed to cell-associated plasminogen activator, were also analyzed in a two-step assay. The rate of plasmin formation is linear and shown to obey the Michaelis-Menten rate equation. For the activation of fraction I plasminogen, the Km(app) is 31.7 μM and the kcat is 1.98 s-1; for the activation of fraction II plasminogen, the Km(app) is 19.2 μM and the Kcat is 1.86 s-1. The assay conditions are flexible so that factors which influence the rate and extent of activation, e.g., fibrin and lysine, can now be characterized quantitatively. The apparent Michaelis constants for the transformed cell-associated plasminogen activator are 5-fold less than those for urokinase and are close to the in vivo concentration of plasminogen.
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