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: Serine proteases are involved in a wide variety of biological reactions. Sensitive assays for these enzymes are needed because in many cases they are present in very small amounts. Also, these enzymes degrade themselves at rates that are concentration dependent. From these points of view, active-site titrants are especially useful in assaying serine proteases. Good active-site titrants react quickly with an enzyme, resulting in the formation of an enzymatically inactive, stable acyl-enzyme intermediate and the release of a stoichiometric amount of product that can be detected at low concentrations. Esters of guanidinobenzoate are excellent active-site titrants of many serine proteases. Researchers have increased the usefulness of p-guanidinobenzoate esters as active-site titrants by substituting 4-methylumbelliferone for p-nitrophenol. Another active-site titrant is fluorescein diester (FDE). FDE is nonfluorescent because the fluorescein moiety is in the lactone state. In the presence of a serine protease, however, one of the guanidinobenzoyl esters is hydrolyzed, yielding 5-(N'-4-carboxyphenyl)- thioureido- 3' - (4-guanidinobenzoyloxy) - 6' - hydroxyspiro[isobenzofuranl( 3H),9'-[9H]xanthen]-3-one, abbreviated FME for Fluorescein- MonoEster. FME should be highly fluorescent, because the fluorescein moiety is in the quinone state. By substituting chromophores, the detectability of the product in an active-site titration can be increased 5-6 orders of magnitude, from 10–6 M for p-nitrophenol.Methods in Enzymology 02/1981; 80 Pt C:414-24. DOI:10.1016/S0076-6879(81)80036-5 · 2.09 Impact Factor
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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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