• Purity

  >80%, by SDS-PAGE under reducing conditions and visualized by Colloidal Coomassie? Blue stain at 5 μg per lane

• 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 >12 pmol/min/μg, as measured under the described conditions. See Activity Assay Protocol on .

• Source

  Chinese Hamster Ovary cell line, CHO-derived Thr25-Gly420, with C-terminal 6-His tag

• Accession #

• N-terminal Sequence    
  Analysis

  Thr25

• Predicted Molecular Mass

  44 kDa

• SDS-PAGE

  40-70 kDa

Assay Procedure

Materials

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

• Recombinant Human Napsin A (rhNAPSA) (Catalog # 8489-NA)

• 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: Gemini EM by Molecular Devices) or equivalent

1. Dilute rhNAPSA to 40 μg/mL in Assay Buffer.

2. Incubate 50 μL aliquot of 40 μg/mL rhNAPSA at room temperature for 20 minutes.

3. Dilute incubated rhNAPSA to 8 μg/mL in Assay Buffer.

4. Dilute Substrate to 20 μM in Assay Buffer.

5. Load 50 μL incubated rhNAPSA into a 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.

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

7. Calculate specific activity:

     
      *Adjusted for Substrate Blank

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

Per Well:

• rhNAPSA: 0.4 μg

• Substrate: 10 μM

Background: Napsin A

Human NAPSA (also called Napsin A) is a 38 kDa glycosylated aspartic protease (1-3). NAPSA/Napsin A is synthesized as a 420 amino acid (aa) proenzyme containing a signal peptide (up to 25 aa), an activation peptide (38 aa), and a 357 aa mature enzyme. Mature human NAPSA/Napsin A shares 74% and 73% aa sequence identity with mature mouse and rat NAPSA/Napsin A, respectively. The NAPSA/Napsin A proenzyme is expressed in lung epithelial cells and within the kidney (3). Kidney dysfunction is associated with a decrease in NAPSA/Napsin A excretion in urine (1). NAPSA/Napsin A expression is also used as a marker for clear cell ovarian carcinomas, pulmonary adenocarcinomas, and renal cell carcinomas (4-8). NAPSA/Napsin A is involved in processing surfactant protein B in type II pneumocytes (9).

• References:

1. Schauer-Vukasinovic, V. et al. (2001) Biochim. Biophys. Acta 1524:51.

2. Tatnell, P.J. et al. (1998) FEBS Lett. 441:43.

3. Schauer-Vukasinovic, V. et al. (1999) FEBS Lett. 462:135.

4. Yamashita, Y. et al. (2014) Mod. Pathol. PMID 24721826

5. Skirnisdottir, I. et al. (2013) BMC Cancer. 13:524.

6. Ordonez, N.G. (2012) Adv. Anat. Pathol. 19:66.

7. Jagirdar, J. (2008) Arch. Pathol. Lab. Med. 132:384.

8. Ao, M.H. et al. (2014) Hum. Pathol. 45:926.

9. Brasch, F. et al. (2003) J. Biol. Chem. 278:49006.

• Entrez Gene IDs:

  9476 (Human); 16541 (Mouse); 60575 (Rat)

• Alternate Names:

  Asp 4; ASP4; Aspartyl protease 4; EC 3.4.23; EC 3.4.23.-; EC 3.4.23.15; EC 3.4.23.3; EC 3.4.23.5; KAP; Kdap; NAP1napsin-A; NAPA; NAPApronapsin A; NAPSA; napsin A aspartic peptidase; Napsin A; Napsin-1; SNAPA; TA01/TA02