Recombinant Human Cathepsin X/Z/P Protein, CF

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

Recombinant Human Cathepsin X/Z/P Protein, CF Summary

• Details of Functionality: Measured by its ability to cleave the fluorogenic peptide substrate, Mca-RPPGFSAFK(Dnp)-OH (Catalog # ES005). The specific activity is >800 pmol/min/µg, as measured under the described conditions.
• Source: Mouse myeloma cell line, NS0-derived human Cathepsin X/Z/P protein Ala21-Val303 (Gly23Val & Ser48Thr), with a C-terminal 10-His tag
• Accession #: Q9UBR2
• N-terminal Sequence: Ala21
• Structure / Form: Pro form
• Protein/Peptide Type: Recombinant Enzymes
• Gene: CTSZ
• 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: 33 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: 41 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 degreesC as supplied. 3 months, -20 to -70 degreesC under sterile conditions after opening.
• Buffer: Supplied as a 0.2 μm filtered solution in Sodium Acetate and NaCl.
• Purity: >95%, by SDS-PAGE under reducing conditions and visualized by silver stain
• Assay Procedure:
  • Activation Buffer: 25 mM Sodium Acetate, pH 3.5
  • Assay Buffer: 25 mM Sodium Acetate, 5 mM Dithiothreitol (DTT), pH 3.5
  • Dithiothreitol (DTT), 1 M stock in deionized water
  • Recombinant Human Cathepsin X/Z/P (rhCathepsin X/Z/P) (Catalog # 934-CY)
  • Substrate: MCA-Arg-Pro-Pro-Gly-Phe-Ser-Ala-Phe-Lys(DNP)-OH (Catalog # ES005)
  • F16 Black Maxisorp Plate (Nunc, Catalog # 475515)
  • Fluorescent Plate Reader (Model: SpectraMax Gemini EM by Molecular Devices) or equivalent
  1. Activate rhCathepsin X/Z/P.
  1. Dilute rhCathepsin X/Z/P to 20 µg/mL in Activation Buffer.
  2. Dilute DTT stock to 10 mM in Activation Buffer.
  3. Mix equal volumes of 20 µg/mL rhCathepsin X/Z/P and 10 mM DTT.
  4. Incubate at room temperature for 5 minutes.
  2. Dilute activated rhCathepsin X/Z/P to 0.4 ng/µL in Assay Buffer.
  3. Dilute Substrate to 20 µM in Assay Buffer.
  4. Load 50 µL of the 0.4 ng/µL rhCathepsin X/Z/P in a black well plate, and start the reaction by adding 50 µL of 20 µM Substrate. Include a Substrate Blank containing 50 µL of Assay Buffer and 50 µL of 20 µM Substrate without any rhCathepsin X/Z/P.
  5. Read at excitation and emission wavelengths of 320 nm and 405 nm (top read), respectively, in kinetic mode for 5 minutes.
  6. 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 MCA-Pro-Leu-OH (Bachem, Catalog # M-1975).

  Per Well:
  • rhCathepsin X/Z/P: 0.02 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 Human Cathepsin X/Z/P Protein, CF

• Cathepsin P
• Cathepsin X/Z/P
• Cathepsin Z
• CTSX
• CTSZ

Background

Cathepsin X (also known as Cathepsin Z and P) is a cysteine protease of the papain family (1-5). Compared to other members of the papain family, Cathepsin X has a short proregion and unique insertions. The cysteine residue in the proregion forms a covalent and reversible bond with the active site cysteine residue (6). Acting as a carboxypeptidase, Cathepsin X displays a unique specificity (7-10). It is ubiquitously expressed in human tissues and conserved in other species such as mouse, nematode and echiuran. The nematode enzyme is apparently involved in molting of third stage larvae (11).

1. Nagler, D.K. and R. Menard (1998) FEBS Lett. 434:135.
2. Santamaria, I. et al. (1998) J. Biol. Chem. 273:16816.
3. Deussing, J. et al. (2000) Biochim. Biophys. Acta 1491:93.
4. Pungercar, J. and G. Ivanovski (2000) Pflugers Arch. Eur. J. Physiol. 439:R116.
5. Pungercar, J. et al. (2000) Pflugers Arch. Eur. J. Physiol. 439:R119.
6. Sivaraman, J. et al. (2000) J. Mol. Biol. 295:935.
7. Menard, R. et al. (2001) Biol. Chem. 382:839.
8. Therrien, C. et al. (2001) Biochemistry 40:2702.
9. Klemencic, I. et al. (2000) Eur. J. Biochem. 267:5404.
10. Guncar, G. et al. (2000) Structure Fold Des. 8:305.
11. Lustigman, S. et al. (1996) J. Biol. Chem. 271:30181.

Publications for Cathepsin X/Z/P (934-CY)(3)

Publications using 934-CY

• M Buša, Z Matoušková, P Bartošová-, P Pachl, P ?ezá?ová, RM Eichenberg, P Deplazes, M Horn, S Štefani?, M Mareš An evolutionary molecular adaptation of an unusual stefin from the liver fluke Fasciola hepatica redefines the cystatin superfamily The Journal of Biological Chemistry, 2023-02-01;0(0):102970. 2023-02-01 [PMID: 36736427] (Bioassay, N/A)
• 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)
• Falgueyret JP, Black WC, Cromlish W, Desmarais S, Lamontagne S, Mellon C, Riendeau D, Rodan S, Tawa P, Wesolowski G, Bass KE, Venkatraman S, Percival MD An activity-based probe for the determination of cysteine cathepsin protease activities in whole cells. Anal. Biochem., 2004-12-15;335(2):218-27. 2004-12-15 [PMID: 15556560] (Enzyme Assay, Human)

Bioinformatics

• Gene Symbol: CTSZ
• Uniprot:
  • Q9UBR2()