• Purity

  >95%, by SDS-PAGE visualized with Silver Staining and quantitative densitometry by Coomassie® Blue Staining.

• 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, Leu-AMC. The specific activity is >60 pmol/min/µg, as measured under the described conditions. See Activity Assay Protocol on .

• Source

  Mouse myeloma cell line, NS0-derived Ala37-Met941, with a C-terminal 10-His tag

• Accession #

• N-terminal Sequence    
  Analysis

  Ala37

• Predicted Molecular Mass

  105 kDa

• SDS-PAGE

  108 kDa, reducing conditions

Assay Procedure

Materials

• Assay Buffer: 25 mM Tris, pH 8.0

• Recombinant Human Aminopeptidase PILS/ARTS1 (rhARTS1) (Catalog # 2334-ZN)

• Substrate: Leu-AMC (Bachem, Catalog # I-1240)

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

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

1. Dilute rhARTS1 to 2 µg/mL in Assay Buffer.

2. Dilute Substrate to 200 µM in Assay Buffer.

3. Load 50 µL of 2 µg/mL rhARTS1 into the plate, and start the reaction by adding 50 µL of 200 µM Substrate. Include a Substrate Blank containing Assay Buffer and Substrate.

4. Read at excitation and emission wavelengths of 380 nm and 460 nm (respectively), in kinetic mode for 5 minutes.

5. Calculate specific activity:

     *Adjusted for Substrate Blank
     **Derived using calibration standard 7-amino, 4-Methyl Coumarin (Sigma, Catalog # A-9891).

Per Well:

• rhARTS1: 0.1 µg

• Substrate: 100 µM

Background: Aminopeptidase PILS/ARTS1

The name of Aminopetidase PILS (Puromycin-Insensitive Leucyl-Specific) describes the two basic properties of this zinc metalloprotease in vitro (1). Also known as ALAP (Adipocyte-derived Leucin AminoPeptidase), type 1 tumor necrosis factor receptor (TNFR) shedding aminopeptidase regulator and ER aminopeptidase ERAP1 or ERAAP, it is encoded by the ARTS1 gene (2-4). Aminopeptidase PILS has been identified to regulate antigen presentation, promote TNFR1 ectodomain shedding and associate with hypertension (2-5).

• References:

1. Schomburg, L. (2004) in Handbook of Proteolytic Enzymes (ed. Barrett, et al.) pp. 311, Academic Press, San Diego.

2. Cui, X. et al. (2002) J. Clin. Invest. 110:515.

3. York, I.A. et al. (2002) Nat. Immunol. 3:1177.

4. Serwold, T. et al. (2002) Nature 419:480.

5. Yamamoto, N. et al. (2002) Hum. Mutat. 19:251.

• Long Name:

  Aminopeptidase Puromycin-insensitive Leucyl-specific

• Entrez Gene IDs:

  51752 (Human); 80898 (Mouse)

• Alternate Names:

  Adipocyte-derived leucine aminopeptidase; A-LAP; A-LAPALAP; Aminopeptidase PILS; ARTS1; ARTS1aminopeptidase regulator of TNFR1 shedding; ARTS-1PILSAP; EC 3.4.11; EC 3.4.11.-; EC 3.4.11.1; endoplasmic reticulum aminopeptidase 1; endoplasmic reticulum aminopeptidase associated with antigen processing; ERAAP1; ERAP1; KIAA0525APPILS; PILS-AP; PILS-APERAAP; Puromycin-insensitive leucyl-specific aminopeptidase; Type 1 tumor necrosis factor receptor shedding aminopeptidase regulator