• 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-PLGL-Dpa-AR-NH    ₂ (Catalog # ). The specific activity is >450 pmol/min/µg, as measured under the described conditions. See Activity Assay Protocol on www.RnDSystems.com

• Source

  Mouse myeloma cell line, NS0-derived Leu21-Leu412, with a C-terminal 10-His tag

• Accession #

• N-terminal Sequence    
  Analysis

  Leu21

• Structure / Form

  Pro form

• Predicted Molecular Mass

  44 kDa

• SDS-PAGE

  50 kDa, reducing conditions

Assay Procedure

Materials

• Assay Buffer: 0.1 M NaOAc, 0.2 M NaCl, pH 3.5

• Recombinant Human Cathepsin D (rhCathepsin D) (Catalog # 1014-AS)

• Substrate: MCA-Pro-Leu-Gly-Leu-DPA-Ala-Arg-NH₂ (Catalog # ), 2 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 rhCathepsin D to 20 µg/mL in Assay Buffer.

2. Aliquot 50 µL of 20 µg/mL rhCathepsin D.

3. Incubate at 37 °C for 30 minutes.

4. Dilute incubated rhCathepsin D to 1 ng/µL in Assay Buffer.

5. Dilute Substrate to 20 µM in Assay Buffer.

6. Load 50 µL of 1 ng/µL rhCathepsin D in 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.

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

8. Calculate specific activity:

     *Adjusted for Substrate Blank

     **Derived using calibration standard MCA-Pro-Leu-OH (Bachem, Catalog # M-1975).

Per Well:

• rhCathepsin D: 0.050 µg

• Substrate: 10 µM

Background: Cathepsin D

Cathepsin D is a lysosomal aspartic protease of the pepsin family (1). Human cathepsin D is synthesized as a precursor protein, consisting of a signal peptide (residues 1‑18), a propeptide (residues 19‑64), and a mature chain (residues 65‑412) (2‑4). The mature chain can be processed further to the light (residues 65‑161) and heavy (residues 169‑412) chains. It is expressed in most cells and overexpressed in breast cancer cells (5). It is a major enzyme in protein degradation in lysosomes, and also involved in the presentation of antigenic peptides. Mice deficient in this enzyme showed a progressive atrophy of the intestinal mucosa, a massive destruction of lymphoid organs, and a profound neuronal ceroid lipofucinosis, indicating that cathepsin D is essential for proteolysis of proteins regulating cell growth and tissue homeostasis (6). Cathepsin D secreted from human prostate carcinoma cells are responsible for the generation of angiostatin, a potent endogeneous inhibitor of angiogenesis (6).

• References:

1. Conner et al. in Handbook of Proteolytic Enzymes Barrett (1998) Academic Press, San Diego, p. 828.

2. Faust, et al. (1985) Proc. Natl. Acad. Sci. USA 82:4910.

3. Westley and May (1987) Nucl. Acid Res. 15:3773.

4. Redecker, et al. (1991) DNA Cell Biol. 10:423.

5. Rochefort, et al. (2000) Clin. Chim. Acta. 291:157.

6. Tsukuba, et al. (2000) Mol. Cells 10:601.

• Entrez Gene IDs:

  1509 (Human); 13033 (Mouse)

• Alternate Names:

  cathepsin D (lysosomal aspartyl protease); Cathepsin D; CPSD; CTSD; EC 3.4.23; EC 3.4.23.5; lysosomal aspartyl peptidase; lysosomal aspartyl protease; MGC2311; neuronal 10