Recombinant Human Cathepsin A/Lysosom Carboxypeptidase A, CF
Recombinant Human Cathepsin A/Lysosom Carboxypeptidase A, CF Summary
Product Specifications
Ala29-Tyr480, with a C-terminal 10-His tag
Analysis
Product Datasheets
Carrier Free
CF stands for Carrier Free (CF). We typically add Bovine Serum Albumin (BSA) as a carrier protein to our recombinant proteins. Adding a carrier protein enhances protein stability, increases shelf-life, and allows the recombinant protein to be stored at a more dilute concentration. The carrier free version does not contain BSA.
In general, we advise purchasing the recombinant protein with BSA for use in cell or tissue culture, or as an ELISA standard. In contrast, the carrier free protein is recommended for applications, in which the presence of BSA could interfere.
1049-SE
| Formulation | Supplied as a 0.2 μm filtered solution in Tris and NaCl. |
| Shipping | The product is shipped with polar packs. Upon receipt, store it immediately at the temperature recommended below. |
| Stability & Storage: | Use a manual defrost freezer and avoid repeated freeze-thaw cycles.
|
Assay Procedure
- Activation Buffer: 25 mM MES, 5 mM DTT, pH 6.0
- Assay Buffer: 25 mM MES, 5 mM DTT, pH 5.5
- Recombinant Human Cathepsin A/Lysosomal Carboxypeptidase A (rhCathepsin A) (Catalog # 1049-SE)
- Recombinant Human Cathepsin L (rhCathepsin L) (Catalog # 952-CY)
- E 64 (Tocris Catalog # 5208) , 50 mM stock in DMSO
- 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
- Dilute rhCathepsin A to 100 µg/mL in Activation Buffer.
- Dilute rhCathepsin L to 10 µg/mL in Activation Buffer.
- Combine equal volumes of rhCathepsin A and rhCathepsin L for final concentrations of 50 µg/mL and 5 µg/mL, respectively.
- Incubate reaction at 37 °C for 30 minutes.
- Stop reaction by adding E-64 to a final concentration of 10 µM.
- Dilute activated rhCathepsin A to 2.0 ng/µL in Assay Buffer.
- Dilute Substrate to 20 µM in Assay Buffer.
- Load 50 µL of 2.0 ng/µL rhCathepsin A into 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 320 nm and 405 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 MCA-Pro-Leu-OH (Bachem, Catalog # M-1975)
- rhCathepsin A: 0.1 µg
- Substrate: 10 µM
Reconstitution Calculator
Background: Cathepsin A/Lysosomal Carboxypeptidase A
Cathepsin A/lyososomal carboxypeptidase A is a member of the serine carboxypeptidase family (1). Cathepsin A is a multifunctional enzyme that expresses deaminidase and esterase activities at neutral pH and carboxypeptidase activity at acidic pH. Also known as protective protein, its association with beta -galactosidase ( beta -gal) and neuraminidase is essential for beta -gal stability and neuraminidase activation in the lysosomes. Inherited deficiency of Cathepsin A causes the lysosomal storage disorder galactosialidosis, characterized by a combined secondary deficiency of beta -gal and neuraminidase. Cathepsin A is capable of hydrolyzing a variety of bioactive peptide hormones including tachykinins, indicating that extralysosomal Cathepsin A plays a role in regulation of functions of these molecules (2). Cathepsin A is synthesized as a single-chain precursor and processed into heavy (32 kDa) and light (20 kDa) chains, which are linked by disulfide bonds.
- Pshezhetsky, A.V. (2004) in Handbook of Proteolytic Enzymes (ed. Barrett, A.J. et al.) p. 1923, Academic Press, San Diego.
- Hiraiwa, M. (1999) Cell. Mol. Life. Sci. 56:894.
Citations for Recombinant Human Cathepsin A/Lysosom Carboxypeptidase A, CF
R&D Systems personnel manually curate a database that contains references using R&D Systems products. The data collected includes not only links to publications in PubMed, but also provides information about sample types, species, and experimental conditions.
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Citations: Showing 1 - 4
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Activation of Tenofovir Alafenamide and Sofosbuvir in the Human Lung and Its Implications in the Development of Nucleoside/Nucleotide Prodrugs for Treating SARS-CoV-2 Pulmonary Infection
Authors: J Li, S Liu, J Shi, HJ Zhu
Pharmaceutics, 2021-10-11;13(10):.
Species: Human
Sample Types: Whole Tissue
Applications: Enzyme Activity -
Lysosome-targeted beta-galactosidase negatively regulates neuraminidase 1 (NEU1) and promotes NEU1 deficiency in GM1 gangliosidosis
Authors: AR Luu, H Wong, V Agrawal, N Wise, B Handyside, MJ Lo, G Pacheco, JB Felix, A Giaramita, A d'Azzo, J Vincelette, S Bullens, S Bunting, TM Christians, C Hague, JH Lebowitz, G Yogalingam
J. Biol. Chem., 2020-07-28;0(0):.
Species: Human
Sample Types: Whole Cells
Applications: Bioassay -
Mechanism of activation of PSI-7851 and its diastereoisomer PSI-7977.
Authors: Murakami E, Tolstykh T, Bao H, Niu C, Steuer HM, Bao D, Chang W, Espiritu C, Bansal S, Lam AM, Otto MJ, Sofia MJ, Furman PA
J. Biol. Chem., 2010-08-26;285(45):34337-47.
Species: Human
Sample Types: Small Molecule
Applications: Enzyme Assay -
Cathepsin A is expressed in primary human antigen-presenting cells.
Authors: Reich M, Spindler KD, Burret M, Kalbacher H, Boehm BO, Burster T
Immunol. Lett., 2009-11-30;128(2):143-7.
Species: Human
Sample Types: Buffer, N/A
Applications: Enzyme Assay, Western Blot
FAQs
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Where does Cathepsin A cleave Mca-RPPGFSAFK(Dnp)-OH Fluorogenic Peptide Substrate, Catalog # ES005?
Although the QC assay conditions provided on our recombinant Cathepsin A datasheets should favor carboxypeptidase activity, it is possible that there is more than one site in the ES005 peptide recognized and cleaved by Cathepsin A. We have not performed verification experiments to confirm the cleavage site preferred in our reaction conditions.
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