Recombinant Human u-Plasminogen Activator/Urokinase, CF Summary
Details of Functionality
Measured by its ability to cleave a peptide substrate, N-carbobenzyloxy-Gly-Gly-Arg-7-amido-4-methylcoumarin (Z-GGR-AMC). The specific activity is >2,000 pmol/min/µg, as measured under the described conditions.
Mouse myeloma cell line, NS0-derived human u-Plasminogen Activator (uPA)/Urokinase protein Met1-Leu431 with a C-terminal 10-His tag
>95%, by SDS-PAGE visualized with Silver Staining and quantitative densitometry by Coomassie® Blue Staining.
<1.0 EU per 1 μg of the protein by the LAL method.
18 kDa (long A chain), 3 kDa (short A chain), 30 kDa (B chain). Disclaimer note: The observed molecular weight of the protein may vary from the listed predicted molecular weight due to post translational modifications, post translation cleavages, relative charges, and other experimental factors.
Recombinant Human u‑Plasminogen Activator (uPA)/Urokinase (rhuPA) (Catalog # 1310-SE)
Substrate: Z-Gly-Gly-Arg-AMC (Bachem, Catalog # I-1140), 10 mM stock in DMSO
F16 Black Maxisorp Plate (Nunc, Catalog # 475515)
Fluorescent Plate Reader (Model: SpectraMax Gemini EM by Molecular Devices) or equivalent
Dilute rhuPA to 1 ng/µL in Assay Buffer.
Dilute Substrate to 200 µM in Assay Buffer.
Load 50 µL of the 1 ng/µL rhuPA into a black well plate, and start the reaction by adding 50 µL of 200 µM Substrate. Include a Substrate Blank containing 50 µL Assay Buffer and 50 µL of 200 µM Substrate without any rhuPA.
Read at excitation and emission wavelengths of 380 nm and 460 nm (top read), respectively, in kinetic mode for 5 minutes.
Calculate specific activity:
Specific Activity (pmol/min/µg) =
Adjusted Vmax* (RFU/min) x Conversion Factor** (pmol/RFU)
amount of enzyme (µg)
*Adjusted for Substrate Blank **Derived using calibration standard 7-amino, 4-Methyl Coumarin (Sigma, Catalog # A-9891)
rhuPA: 0.05 µg
Substrate: 100 µM
This product is produced by and ships from R&D Systems, Inc., a Bio-Techne brand.
Alternate Names for Recombinant Human u-Plasminogen Activator/Urokinase, CF
plasminogen activator, urokinase
urokinase-type plasminogen activator
uPA is a serine protease with an extremely limited substrate specificity, cleaving the sequence Cys-Pro-Gly-Arg560-Val561-Val-Gly-Gly-Cys in plasminogen to form plasmin (1). uPA is a potent marker of invasion and metastasis in a variety of human cancers associated with breast, stomach, colon, bladder, ovary, brain and endometrium (2). For example, the combination (both low vs. either or both high) of uPA and its inhibitor, plasminogen activator inhibitor-1 (PAI-1), outperforms the single factors as well as other traditional prognostic factors with regard to risk group assessment for breast cancer, particularly in node-negative breast cancer (3). The human uPA is initially synthesized as 431 amino acid precursor with a N-terminal signal peptide (20 residues) (4‑6). The single chain molecule is processed into a disulfide-linked two-chain molecule. The B chain starting at Ile179 corresponds to the catalytic domain. Two forms of the A chain exist, one starting at Ser21 (the long form) and the other at Lys156 (the short form). The resulting two-chain forms have different molecular weights (MW). The B chain is common for both forms whereas the long and short A chains are unique to the high and low MW forms, respectively. The long A chain contains an EGF-like domain, which is responsible for binding of the uPA receptor (uPAR). Both high and low MW forms exist in the purified recombinant human uPA.
Ellis, V. (2004) in Handbook of Proteolytic Enzymes. Barrett, A.J. et al. eds., Academic Press, San Diego, pp.1677.
Duffy, M.J. (2002) Biochem. Soc. Trans. 30:207.
Harbeck, N. et al. (2002) Clin. Breast Cancer 3:196.
Riccio, A. et al. (1985) Nucleic Acids Res. 13:2785.
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