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
AnalysisNo results obtained: Gln18 predicted
Predicted Molecular Mass
45 kDa
SDS-PAGE
42 kDa, reducing conditions
2700-ZN |
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Formulation Supplied as a 0.2 μm filtered solution in Tris and NaCl. | ||
Shipping The product is shipped with polar packs. Upon receipt, store it immediately at the temperature recommended below. | ||
Stability & Storage: Use a manual defrost freezer and avoid repeated freeze-thaw cycles.
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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
Dilute Substrate to 40 mM in Assay Buffer.
In a plate load 90 µL of 40 mM Substrate to wells.
Incubate plate at 25 °C for 10 minutes.
Dilute rmACY1 to 200 µg/mL in Assay Buffer.
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.
Incubate plate at 25 °C for 15 minutes.
Read at a wavelength of 238 nm (bottom read) in endpoint mode.
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
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:
Lindner, H.A. et al. (2003) J. Biol. Chem. 278:44496.
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
