A quantitative assay for the activation of plasminogen by transformed cells in situ and by urokinase
University of Essex, Colchester, England, United KingdomBiochemistry (Impact Factor: 3.02). 08/1981; 20(15):4307-14. DOI: 10.1021/bi00518a011
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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ABSTRACT: The molecule 3',6'-bis(4-guanidinobenzoyloxy)-5-[N'-(4-carboxyphenyl)thioureido[spirop]isobenzofuran-1-(3H),9'-[9H]xanthen]-3-one, abbreviated FDE, was designed and synthesized as a fluorogenic active-site titrant for serine proteases. It is an analogue of p-nitrophenyl p-guanidino-benzoate (NPGB) in which a fluorescein derivative is substituted for p-nitrophenol. FDE and NPGB exhibit similar kinetic characteristics in an active-site titration of trypsin in phosphate-buffered saline, pH 7.2. The rate of acylation with FDE is extremely fast (k2 = 1.05 s-1) and the rate of deacylation extremely slow (k3 = 1.66 X 10(-5) s-1). The Ks is 3.06 X 10(-6) M, and the Km(app) is 4.85 X 10(-11) M. With two of the serine proteases involved in fibrinolysis, the rate of acylation with FDE is also fast, K2 = 0.112 s-1 for urokinase and 0.799 s-1 for plasmin, and the rate of deacylation is slow, k3 = 3.64 X 10(-4) s-1 for urokinase and 6.27 X 10(-6) s-1 for plasmin. The solubility limit of FDE in phosphate-buffered saline is 1.3 X 10(-5) M, and the first-order rate constant for spontaneous hydrolysis is 5.1 X 10(-6) s-1. The major difference between FDE and NPGB is the detectability of the product in an active-site titration. p-Nitrophenol can be detected at concentrations no lower than 10(-6) M whereas fluorescein can be detected at concentrations as low as 10(-12) M. Thus, FDE should be useful in quantitatively assaying serine proteases as very low concentrations.Biochemistry 08/1981; 20(15):4298-306. DOI:10.1021/bi00518a010 · 3.02 Impact Factor
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ABSTRACT: Most determinations of tissue plasminogen activator (TPA) activity have been done with qualitative or semiquantitative variations of the fibrin-overlay technique [1–4]. However, to meaningfully compare renal TPA activities between animal species or in same kidneys under different conditions, a quantitative and sensitive assay is required. We are reporting here the development of such an assay based on a two-step reaction: (1) activation of plasminogen (Plg) to plasmin by renal TPA, and (2) quantitation of plasmin by using S2251, a chromogenic substrate highly specific for this enzyme.Kidney International 08/1982; 22(1):80-3. DOI:10.1038/ki.1982.136 · 8.56 Impact Factor
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ABSTRACT: The effects of variations in cell density on the expression of the plasminogen activator activity of a tumorigenic rat cell line were analyzed. At low cell densities, the plasminogen activator activity per cell was high and independent of cell density. As the cell density increased, the plasminogen activator activity per cell decreased until it eventually became inversely proportional to cell density. Inhibition of the plasminogen activator activity per cell by increases in cell density was not the result of the presence of a soluble inhibitor but seemed to require cell-to-cell contact. The V(max) per cell for the activation of plasminogen changed at high cell densities, but the K(m) did not change. This change in the V(max) per cell was in part the result of a change in the catalytic rate constant for the conversion of plasminogen to plasmin. This was inferred from studies on the kinetics of inhibition of plasminogen activator activity by diisopropyl fluorophosphate as a function of cell density. For cells growing at high densities, the rate of inhibition was constant, exhibiting a second-order rate constant of 2.6 x 10(-2)M(-1) s(-1). For cells growing at low densities, the plasminogen activator activity was inhibited at two different rates, one exhibiting a second-order rate constant of 2.6 x 10(-2)M(-1) s(-1) and the other exhibiting a second-order rate constant of 9.4 x 10(-2)M(-1) s(-1). We discuss the importance of cell density in assays of the plasminogen activator activity of cells, the use of this cell line to study the biochemical basis of the density dependence of plasminogen activator activity, and the density-dependent role of plasminogen activator activity in tumor formation and metastasis.Molecular and Cellular Biology 12/1982; 2(11):1410-6. DOI:10.1128/MCB.2.11.1410 · 4.78 Impact Factor
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