Direct fluorescent assay of urokinase and plasminogen activators of normal and malignant cells: kinetics and inhibitor profiles.

Abstract

A direct rate assay for plasminogen activator has been developed using a synthetic fluorogenic peptide substrate, 7(N-Cbz-glycylglycylargininamido)4-methylcoumarin trifluoroacetate. The assay correlates well with the standard 12SI-labeled fibrin plate assay using highly purified urokinase, culture fluids from WI-38, Chinese The enhanced production of plasminogen activator (PA) activity has been shown to be a characteristic of many different cell types. The intracellular and extracellular levels of PA have been demonstrated to be substantially elevated in malignant cells in culture (1-6), cells treated with a tumor promoter (7), activated macrophages (8, 9), established cell lines (10, 11), granulosa cells during ovulation (12), embryonic cells during differentiation (13,14), and hormone-treated uteri (15). The standard system used for measuring PA in these cells is an indirect, two-step assay in which plasminogen is incubated with a source of PA and the plasmin activity generated is quantitated by using fibrin, casein, or protamine as substrates (16)(17)(18)(19). There is a need, however, for a simple, sensitive, direct assay that allows both rapid measurement and kinetic analysis of PA, independent of plasmin generation. In addition, the presence of two proteases of similar specificities in the two-step assay precludes the screening of potential PA inhibitors.A series of synthetic fluorogenic substrates, specific for a number of serine proteases, utilizing the leaving group 7-amino-4-methylcoumarin (AMC) has been described (20,21). In a continuation of this approach, we have now prepared a synthetic peptide specific for the cleavage site of PA, incorporating the same leaving group. This compound is Cbz-GlyGly-Arg-AMC. We report here the use of this substrate in the direct fluorescent assay of PA from normal and malignant cells, some kinetic parameters of these enzymes, and the effect of various low and high molecular weight protease inhibitors on the various enzymes. The various PAs were analyzed by the direct fluorescent technique in parallel with the indirect standard 25I-labeled fibrin plate assay using purified plasminogens from both canine and bovine sources. (24). When the cultures had attained high cell density (1 X 107 cells per 100-mm culture dish), the plates were washed three times with minimal medium and further incubated in serum-free minimal medium. The medium was removed from the cultures every 12 hr and was a source of extracellular PA; it is referred to as harvest fluid (HF). After it was harvested, the HF was immediately centrifuged to remove cells and cellular debris and acidified to pH 3.5 by the addition of 1 M HCl. Ammonium sulfate was added to 70% saturation, and the resulting precipitate was recovered by centrifugation and resuspended in 1/100 the volume of the original HF in 0.05 M glycine.HCI buffer, pH 3.0. MATERIALS AND METHODSWI-38, HeLa, and Chinese hamster ovary cells were grown in serum-containing medium, washed, and incubated in serum-free Higuchi medium (25). This me...

Urokinase Receptor Cleavage: A Crucial Step in Fibroblast-to-Myofibroblast Differentiation

“…Although proteases such as plasmin, uPA, and trypsin are known to cleave uPAR in the linker region between D1 and D2 (Montuori et al, 2005), the inability of leupeptin to inhibit uPAR cleavage suggests they are not involved here (Zimmerman et al, 1978). Similarly, metalloproteases cleave uPAR in the linker region, but uPAR cleavage was not inhibited by the general metalloprotease inhibitor GM6001.…”

Primary structure of N‐linked carbohydrate chains of a human chimeric plasminogen activator K2tu‐PA expressed in Chinese hamster ovary cells

“…After extensive washing with 2 M NaCl, the column was eluted I), 9005 (Kztu-PA-II), 9006 (Kztu-PA-III), and 113079 (Kztu- with 100 mM 6-aminohexanoic acid, adjusted to pH 2.0 with HC1, and collected fractions were immediately adjusted to pH 6.0 with 1 M Tris. The fractions containing K,tu-PA-I, as traced by a direct amidolytic assay with Cbz-Gly-Gly-Arg-NH-Mec as fluorogenic substrate [26], were pooled and loaded on a S-Sepharose FF cation-exchange column (Pharmacia), equilibrated in 50 mM sodium phosphate pH 6.0. The column was eluted with a linear gradient of 0-500 mM NaCl in 50 mM sodium phosphate pH 6.0.…”

Cell surface antigens in renal tumour cells: detection by immunoluminescence and enzymatic analysis

“…uPA-activity assay (1) Endogenous membrane-bound uPA activity was assayed as described (Zimmerman et al, 1978) with modifications: the reaction was started by adding 100 µl of a cell suspension (2 × 10 6 cells) to 1 ml substrate solution (Z-Gly-Gly-Arg-AMC, final concentration 0.15 mM in PBS) with or without inhibitor (D-Val-Phe-Lys-CMK, PMSF). Excitation and emission wavelengths were set at 380 nm and 450 nm, respectively.…”