Recombinant Mouse Cathepsin A/Lysosom Carboxypeptidase A, CF

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

Recombinant Mouse Cathepsin A/Lysosom Carboxypeptidase A, 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 >160 pmol/min/μg, as measured under the described conditions.
• Source: Mouse myeloma cell line, NS0-derived mouse Cathepsin A/Lysosomal Carboxypeptidase A protein
  Ala24-Tyr474, with a C-terminal 10-His tag
• Accession #: P16675
• N-terminal Sequence: Ala24
• Protein/Peptide Type: Recombinant Enzymes
• Purity: >95%, by SDS-PAGE visualized with Silver Staining and quantitative densitometry by Coomassie® Blue Staining.
• Endotoxin Note: <0.10 EU per 1 μg of the protein by the LAL method.

Applications/Dilutions

• Dilutions:
  • Enzyme Activity
• Theoretical MW: 53 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: 52-60 kDa, reducing conditions

Packaging, Storage & Formulations

• Storage: Use a manual defrost freezer and avoid repeated freeze-thaw cycles.
  • 6 months from date of receipt, -70 °C as supplied.
  • 3 months, -70 °C under sterile conditions after opening.
• Buffer: Supplied as a 0.2 μm filtered solution in Tris and NaCl.
• Purity: >95%, by SDS-PAGE visualized with Silver Staining and quantitative densitometry by Coomassie® Blue Staining.
• Assay Procedure:
  • Assay Buffer: 25 mM MES, 0.5 mM TCEP, pH 5.5
  • Recombinant Mouse Cathepsin A/Lysosomal Carboxypeptidase A (rmCathepsin A) (Catalog # 9789-SE)
  • Activator: Recombinant Human Cathepsin L (rhCathepsin L) (Catalog # 952-CY)
  • E 64 (Tocris, Catalog # 5208), 50 mM stock in DMSO
  • Fluorogenic Peptide Substrate V: MCA-RPPGFSAFK-(DNP)-OH (Catalog # ES005), 2 mM stock in DMSO
  • F16 Black Maxisorp Plate (Nunc, Catalog # 475515)
  • Plate Reader (Model: SpectraMax Gemini EM by Molecular Devices) or equivalent
  1. Dilute rmCathepsin A to 100 μg/mL in Assay Buffer.
  2. Dilute rhCathepsin L to 10 μg/mL in Assay Buffer.
  3. Combine equal volumes of rmCathepsin A and rhCathepsin L for final concentrations of 50 μg/mL and 5 μg/mL, respectively. Include a control containing Assay Buffer in place of rmCathepsin A (Cathepsin L Control).
  4. Incubate at room temperature for 30 minutes.
  5. Dilute E 64 to 20 μM in Assay Buffer.
  6. Combine equal volumes of activated rmCathepsin A and 20 μM E 64 to stop the reaction. Combine equal volumes of Cathepsin L Control and 20 μM E 64.
  7. Dilute activated rmCathepsin A to 3 μg/mL in Assay Buffer. Perform equivalent dilution on Cathepsin L Control.
  8. Dilute Substrate to 40 μM in Assay Buffer.
  9. Load into a plate 50 μL of 3 μg/mL rmCathepsin A and Cathepsin L Control, and start the reaction by adding 50 μL of 40 μM Substrate.
  10. Read plate at excitation and emission wavelengths of 320 nm and 405 nm, respectively, (top read) in kinetic mode for 5 minutes.
  11. Calculate specific activity:

  Specific Activity (pmol/min/µg) = (Adjusted Vmax* (RFU/min) x Conversion Factor** (pmol/RFU)) / (amount of enzyme (µg))

  *Adjusted for Cathepsin L Control.
  **Derived using calibration standard Mca-PL-OH (Bachem, Catalog # M-1975).

  Per Well:
  • rmCathepsin A: 0.15 μg
  • Substrate: 20 μM

Notes

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

Alternate Names for Recombinant Mouse Cathepsin A/Lysosom Carboxypeptidase A, CF

• beta-galactosidase 2
• beta-galactosidase protective protein
• Carboxypeptidase C
• Carboxypeptidase L
• Cathepsin A
• cathepsin ANGBE
• CTSA
• EC 3.4.16
• EC 3.4.16.5
• GLB2
• GSL
• Lysosomal Carboxypeptidase A
• lysosomal protective protein
• PPCA
• PPGBprotective protein for beta-galactosidase (galactosialidosis)
• Protective protein cathepsin A
• Protective protein for beta-galactosidase

Background

Cathepsin A/lyososomal carboxypeptidase A is a member of the serine carboxypeptidase family (1). Cathepsin A, also known as protective protein, is synthesized as a single-chain precursor and processed into heavy (32 kDa) and light (20 kDa) chains, which are linked by disulfide bonds. Cathepsin A is a multifunctional enzyme that expresses deaminidase and esterase activities at neutral pH and carboxypeptidase activity at acidic pH (2, 3). Cathepsin A is capable of hydrolyzing a variety of bioactive peptide hormones including endothelin and bradykinin making it a promising target in heart failure (2-5). A hereditary variant of cathepsin A results in cathepsin A-related arteriopathy with strokes and leukoencephalapathy (CARASAL) (6). In addition, Cathepsin A's association with beta -galactosidase ( beta -gal) and neuraminidase in a complex is essential for beta -gal stability and neuraminidase activation in the lysosomes. Inherited deficiency of Cathepsin A causes Galactosialidosis, a lysosomal storage disorder, characterized by a combined secondary deficiency of beta -gal and neuraminidase (7, 8).

1. Pshezhetsky, A.V. (2004) in Handbook of Proteolytic Enzymes (ed. Barrett, A.J. et al.) p. 1923, Academic Press, San Diego.
2. Hiraiwa, M. (1999) Cell. Mol. Life. Sci. 56:894.
3. Schreuder, H.A. et al. (2014) Biochem. Biophys. Res. Commun. 445:451.
4. Ruf, S. et al. (2013) Future Med. Chem. 5:399.
5. Timur, Z.K. et al. (2016) Front. Mol. Biosci. 3:68.
6. Bugiani, M. et al. (2016) Neurology 84:1777.
7. Caciotti, A. et al. (2013) Ophanet. J. Rare Dis. 8:114.
8. Annunziata, I. et al. (2017) Expert Opin. Orphan Drugs 5:131.

Bioinformatics

• Uniprot:
  • P16675()