• Purity

  >95%, by SDS-PAGE under reducing conditions and visualized by silver stain

• Endotoxin Level

  <1.0 EU per 1 μg of the protein by the LAL method.  

• Activity

  Measured by its ability to cleave the fluorogenic peptide substrate, Mca-RPPGFSAFK(Dnp)-OH (Catalog # ). The specific activity is >75 pmol/min/µg, as measured under the described conditions. See Activity Assay Protocol on www.RnDSystems.com

• Source

  Mouse myeloma cell line, NS0-derived Ala29-Tyr480, with a C-terminal 10-His tag

• Accession #

• N-terminal Sequence    
  Analysis

  Ala29

• Structure / Form

  Pro form

• Predicted Molecular Mass

  53 kDa

• SDS-PAGE

  55 kDa, reducing conditions

Assay Procedure

Materials

• 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 # )

• E-64 (trans-epoxysuccinyl-L-leucylamido-(4-Guanidino)butane) (V-Biognostics, Catalog # E640), 1 mM stock in DMSO

• Substrate: MCA-Arg-Pro-Pro-Gly-Phe-Ser-Ala-Phe-Lys(DNP)-OH (Catalog # )

• F16 Black Maxisorp Plate (Nunc, Catalog # 475515)

• Fluorescent Plate Reader (Model: SpectraMax Gemini EM by Molecular Devices) or equivalent

1. Dilute rhCathepsin A to 100 µg/mL in Activation Buffer.

2. Dilute rhCathepsin L to 10 µg/mL in Activation Buffer.

3. Combine equal volumes of rhCathepsin A and rhCathepsin L for final concentrations of 50 µg/mL and 5 µg/mL, respectively.

4. Incubate reaction at 37 °C for 30 minutes.

5. Stop reaction by adding E-64 to a final concentration of 10 µM.

6. Dilute activated rhCathepsin A to 2.0 ng/µL in Assay Buffer.

7. Dilute Substrate to 20 µM in Assay Buffer.

8. 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.

9. Read at excitation and emission wavelengths of 320 nm and 405 nm (top read), respectively, in kinetic mode for 5 minutes.

10. Calculate specific activity:

     *Adjusted for Substrate Blank
     **Derived using calibration standard MCA-Pro-Leu-OH (Bachem, Catalog # M-1975)

Per Well:

• rhCathepsin A: 0.1 µg

• Substrate: 10 µM

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.

• References:

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.

• Entrez Gene IDs:

  5476 (Human); 19025 (Mouse)

• Alternate Names:

  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