Recombinant Mouse Cathepsin H Protein, CF

• Reactivity: Mu
• Applications: Enzyme Activity
• Format: Carrier-Free

Recombinant Mouse Cathepsin H Protein, CF Summary

• Details of Functionality: Measured by its ability to cleave the fluorogenic peptide substrate, Arg-7-amido-4-methylcoumarin (R-AMC). The specific activity is >400 pmol/min/µg, as measured under the described conditions. See Activity Assay Protocol on www.RnDSystems.com
• Source: Mouse myeloma cell line, NS0-derived mouse Cathepsin H protein
  Glu22-Val333, with a C-terminal 10-His tag
• Accession #: Q3UCD6
• N-terminal Sequence: Glu22
• Structure / Form: Pro form
• Protein/Peptide Type: Recombinant Enzymes
• Gene: Ctsh
• Purity: >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: 36 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: 40 kDa, reducing conditions

Packaging, Storage & Formulations

• Storage: 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: 50 mM MES, 10 mM CaCl₂, 150 mM NaCl, pH 6.5
  • Assay Buffer: 50 mM MES, pH 6.5
  • Dithiothreitol (DTT) (Sigma, Catalog # D-0632), 1 M stock in deionized water
  • Recombinant Mouse Cathepsin H (rmCathepsin H) (Catalog # 1013-CY)
  • Bacterial Thermolysin (Catalog # 3097-ZN)
  • Phosphoramidon (Catalog # EI006), 20 mM in methanol
  • Substrate: Arg-7-amido-4-methylcoumarin (R-AMC) (Chem-Impex, Catalog # 5859), 10 mM stock in DMSO
  • F16 Black Maxisorp Plate (Nunc, Catalog # 475515)
  • Fluorescent Plate Reader (Model: SpectraMax Gemini EM by Molecular Devices) or equivalent
  1. Dilute rmCathepsin H to 200 µg/mL in Activation Buffer.
  2. Dilute Thermolysin to 100 µg/mL in Activation Buffer.
  3. Mix equal volumes of 100 µg/mL Thermolysin and 200 µg/mL rmCathepsin H.
  4. Incubate at 37 °C for 1 hour.
  5. Stop reaction by adding an equal volume of 2 mM Phosphoramidon in Assay Buffer to reaction mixture.
  6. Incubate at room temperature for 10 minutes.
  7. Then add an equal volume of Assay Buffer containing 20 mM DTT to reaction mixture. The concentration of rmCathepsin H is now 25 ng/µL.
  8. Incubate reaction at room temperature for 30 minutes.
  9. Dilute activated rmCathepsin H to 0.5 ng/µL in Assay Buffer.
  10. Dilute Substrate to 200 µM in Assay Buffer.
  11. In a plate load 50 µL of 0.5 ng/µL rmCathepsin H to wells, 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.
  12. Read at excitation and emission wavelengths of 380 nm and 460 nm (top read), respectively, in kinetic mode for 5 minutes.
  13. 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 (AMC) (Sigma, Catalog # A-9891)

  Per Well:
  • rmCathepsin H: 0.025 µg
  • Substrate: 100 µM

Notes

This product is produced by and ships from R&D Systems, Inc., a Bio-Techne brand.

Alternate Names for Recombinant Mouse Cathepsin H Protein, CF

• ACC-4
• ACC-5
• aleurain
• cathepsin B3
• cathepsin BA
• Cathepsin H
• CPSB
• CTSH
• DKFZp686B24257
• EC 3.4.22
• EC 3.4.22.16
• MGC1519
• minichain
• N-benzoylarginine-beta-naphthylamide hydrolase

Background

Cathepsin H is a lysosomal cysteine protease of the papain family (1). It is synthesized as a precursor protein, consisting of a signal peptide (residues 1‑20), a propeptide (residues 21‑95), a mini chain (residues 96‑103), a heavy chain (residues 114‑290) and a light chain (residues 291‑333) (2, 3). A truncated form with a 12 amino acid deletion in the signal peptide region is secreted (4). Cathepsin H is the only known mono-aminopeptidase in the papain family (5). Cathepsin H expression is significantly increased in disease states such as in prostate tumors, sera of asthmatic patients, and mucosa of colorectal cancer patients (4, 6, 7).

1. Kirschke, H. (2004) in Handbook of Proteolytic Enzymes (ed. Barrett, et al.) p. 1089, Academic Press, San Diego. 
   
2. Lafuse, W.P. et al. (1995) J. Leukoc. Biol. 57:663.
3. Soderstrom, M. et al. (1999) Biochim. Biophys. Acta 1446:35.
4. Waghray, A. et al. (2002) J. Biol. Chem. 277:11533.
5. Guncar, G. et al. (1998) Structure 6:51.
6. Cimerman, N. et al. (2001) Clin. Chim. Acta 310:113.
7. del Re, E.C. et al. (2000) Br. J. Cancer. 82:1317.

Publications for Cathepsin H (1013-CY)(1)

Publication using 1013-CY

• Burster T, Marin-Esteban V, Boehm BO, Dunn S, Rotzschke O, Falk K, Weber E, Verhelst SH, Kalbacher H, Driessen C Design of protease-resistant myelin basic protein-derived peptides by cleavage site directed amino acid substitutions. Biochem. Pharmacol., 2007-08-02;74(10):1514-23. 2007-08-02 [PMID: 17803968] (Enzyme Assay, Human)

Bioinformatics

• Gene Symbol: Ctsh
• Uniprot:
  • Q3UCD6()