• 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 N-acetyl-L-Methione (Ac-Met). The specific activity is >4,000 pmol/min/µg, as measured under the described protocol. See Activity Assay Protocol on www.RnDSystems.com

• Source

  Mouse myeloma cell line, NS0-derived Met1-Ser408, with a C-terminal 10-His tag Accession # NP_079647

• Accession #

• N-terminal Sequence    
  Analysis

  No results obtained: Gln18 predicted    
   

• Predicted Molecular Mass

  45 kDa

• SDS-PAGE

  42 kDa, reducing conditions

Assay Procedure

Materials

• Assay Buffer: 0.1 M Potassium Phosphate, pH 7.0

• Recombinant Mouse Aminoacylase/ACY1 (rmACY1) (Catalog # 2700-ZN)

• Substrate: N-Acetyl-L-Methionine (N-Ac-Met) (Sigma, Catalog # 1310), 1 M stock in DMSO

• 96 well Clear UV Plate (Corning, Catalog # 3635)

• Plate reader (Model: SpectraMax Plus by Molecular Devices) or equivalent

1. Dilute Substrate to 40 mM in Assay Buffer.

2. In a plate load 90 µL of 40 mM Substrate to wells.

3. Incubate plate at 25 °C for 10 minutes.

4. Dilute rmACY1 to 200 µg/mL in Assay Buffer.

5. Load 10 µL of 200 µg/mL rmACY1 to each well containing 90 µL of Substrate. Include a Substrate Blank with 90 µL of 40 mM Substrate and 10 µL of Assay Buffer.

6. Incubate plate at 25 °C for 15 minutes.

7. Read at a wavelength of 238 nm (bottom read) in endpoint mode.

8. Calculate specific activity:

     *Adjusted for Substrate Blank
     **Derived using calibration standard N-Acetyl-L-Methionine (Sigma, Catalog # 1310).

Per Well:

• rmACY1: 2 µg

• Substrate: 36 mM

Background: Aminoacylase/ACY1

The mouse ACY1 gene encodes aminoacylase, a member of the M20 family of metalloproteases (1). ACY1 plays a general role in the cytosolic breakdown of acetylated amino acids generated during protein degradation. It also interacts with sphingosine kinase type 1, which is involved in promoting cell growth and inhibiting apoptosis of tumor cells (2). The full-length protein is expressed and the purified enzyme is active in the assay described in the Activity Assay Protocol.

• References:

1. Lindner, H.A. et al. (2003) J. Biol. Chem. 278:44496.

2. Maceyka, M. et al. (2004) FEBS Lett. 568:30.

• Entrez Gene IDs:

  95 (Human); 109652 (Mouse)

• Alternate Names:

  ACY1; ACY-1; ACY1D; acylase; aminoacylase 1; Aminoacylase; aminoacylase-1; EC 3.5.1; EC 3.5.1.14; N-acyl-L-amino-acid amidohydrolase