Measured by its ability to cleave the fluorogenic peptide substrate, Gly-Arg-7-amido-4-methylcoumarin (GR-AMC). The specific activity is >8,000 pmol/min/µg, as measured under the described conditions.
Source
Mouse myeloma cell line, NS0-derived mouse Cathepsin C/DPPI protein Asp25-Leu462, with a C-terminal 10-His tag Accession # P97821
>95%, by SDS-PAGE under reducing conditions and visualized by silver stain
Endotoxin Note
<1.0 EU per 1 μg of the protein by the LAL method.
Applications/Dilutions
Dilutions
Enzyme Activity
Theoretical MW
51 kDa. 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.
SDS-PAGE
52 kDa, reducing conditions
Publications
Read Publication using 1034-CY in the following applications:
Use a manual defrost freezer and avoid repeated freeze-thaw cycles.
6 months from date of receipt, -20 to -70 °C as supplied.
3 months, -20 to -70 °C under sterile conditions after opening.
Buffer
Supplied as a 0.2 μm filtered solution in MES and NaCl.
Purity
>95%, by SDS-PAGE under reducing conditions and visualized by silver stain
Assay Procedure
Activation Buffer: 25 mM MES, 5 mM DTT, pH 5.5
Assay Buffer: 25 mM MES, 50 mM NaCl, 5 mM DTT, pH 6.0
Recombinant Mouse Cathepsin C/DPPI (rmCathepsin C) (Catalog # 1034-CY)
Recombinant Human Cathepsin L (rhCathepsin L) (Catalog # 952-CY)
Substrate: Gly-Arg-AMC (Bachem, Catalog # I-1215)
F16 Black Maxisorp Plate (Nunc, Catalog # 475515)
Fluorescent Plate Reader (Model: SpectraMax Gemini EM by Molecular Devices) or equivalent
Dilute rmCathepsin C to 20 µg/mL in Activation Buffer.
Dilute rhCathepsin L to 2 µg/mL in Activation Buffer.
Combine equal volumes of diluted rmCathepsin C and diluted rhCathepsin L for final concentrations of 10 µg/mL rmCathepsin C and 1 µg/mL for rhCathepsin L.
Incubate at room temperature for 20 minutes to activate rmCathepsin C.
Dilute activated rmCathepsin C to 0.04 ng/µL in Assay Buffer.
Dilute Substrate to 20 µM in Assay Buffer.
Load 50 µL of the 0.04 ng/µL rmCathepsin C in a plate, and start the reaction by adding 50 µL of 20 µM Substrate. Include a Substrate Blank containing 50 µL Assay Buffer and 50 µL of 20 µM Substrate.
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 # A9891).
Per Well:
rmCathepsin C: 0.002 µg
Substrate: 10 µM
Notes
This product is produced by and ships from R&D Systems, Inc., a Bio-Techne brand.
Alternate Names for Recombinant Mouse Cathepsin C/DPPI Protein, CF
Cathepsin C
cathepsin CEC 3.4.14.1
Cathepsin J
CPPIHMS
CTSC
dipeptidyl peptidase 1
Dipeptidyl peptidase I
Dipeptidyl transferase
dipeptidyl-peptidase I
DPP1
DPPI
DPP-I
JP
JPD
PALS
PLS
Background
Cathepsin C is a cysteine protease of the papain family (1). Cathepsin C sequentially removes dipeptides from the free N-termini of proteins and peptides. It has broad specificity except that it does not cleave a basic amino acid (Arg or Lys) in the N-terminal position or Pro on either side of the scissle bond. It requires halide ions for activity. The pro form contains a pro peptide and a catalytic region, which can be further processed into heavy/ alpha and light/ beta chains that are linked by a disulfide bond. It is broadly distributed. Cathepsin C plays a role in the lysosomal degradation. It also functions as a key enzyme in the activation of granule serine proteases in cytotoxic T lymphocytes and natural killer cells (granzymes A and B), mast cells (tryptase and chymase), and neutrophils (Cathepsin G and elastase) by removing their N-terminal activation dipeptides (2).
Turk, B. et al. (2004) in Handbook of Proteolytic Enzymes. Barrett, et al. eds. p. 1192, Academic Press, San Diego.
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