Recombinant Mouse Aminoacylase/ACY1 Protein, CF

• Reactivity: Mu
• Applications: Enzyme Activity
• Format: Carrier-Free

Recombinant Mouse Aminoacylase/ACY1 Protein, CF Summary

• Details of Functionality: 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.
• Source: Mouse myeloma cell line, NS0-derived mouse Aminoacylase/ACY1 protein
  Met1-Ser408, with a C-terminal 10-His tag
  Accession # NP_079647
• Accession #: NP_079647
• N-terminal Sequence: No results obtained: Gln18 predicted
• Protein/Peptide Type: Recombinant Enzymes
• Gene: Acy1
• Purity: >95%, by SDS-PAGE under reducing conditions and visualized by silver stain
• Endotoxin Note: <1.0 EU per 1 μg of the protein by the LAL method.

Applications/Dilutions

• Dilutions:
  • Enzyme Activity
• Theoretical MW: 45 kDa.
  Disclaimer note: The observed molecular weight of the protein may vary from the listed predicted molecular weight due to post translational modifications, post translation cleavages, relative charges, and other experimental factors.
• SDS-PAGE: 42 kDa, reducing conditions

Packaging, Storage & Formulations

• Storage: Use a manual defrost freezer and avoid repeated freeze-thaw cycles.
  • 6 months from date of receipt, -20 to -70 °C as supplied.
  • 3 months, -20 to -70 °C under sterile conditions after opening.
• Buffer: Supplied as a 0.2 μm filtered solution in Tris and NaCl.
• Purity: >95%, by SDS-PAGE under reducing conditions and visualized by silver stain
• Assay Procedure:
  • 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:

  Specific Activity (pmol/min/µg) = (Adjusted Abs* (OD) x Conversion Factor** (pmol/OD)) / (Incubation time (min) x amount of enzyme (µg))

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

  Per Well:
  • rmACY1: 2 µg
  • Substrate: 36 mM

Notes

This product is produced by and ships from R&D Systems, Inc., a Bio-Techne brand.

Alternate Names for Recombinant Mouse Aminoacylase/ACY1 Protein, CF

• 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

Background

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.

1. Lindner, H.A. et al. (2003) J. Biol. Chem. 278:44496.
2. Maceyka, M. et al. (2004) FEBS Lett. 568:30.

Bioinformatics

• Gene Symbol: Acy1
• Uniprot:
  • NP_079647()