Recombinant Mouse ASAHL Protein, CF

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

Recombinant Mouse ASAHL Protein, CF Summary

• Details of Functionality: Measured by its ability to hydrolyze the substrate palmitoylethanolamide into palmitate and ethanolamine. The specific activity is >100 pmol/min/µg, as measured under the described conditions.
• Source: Mouse myeloma cell line, NS0-derived mouse ASAHL/N-acylethanolamine-hydrolyzing Acid Amidase protein
  Val33-Ser362, with a C-terminal 10-His tag
• Accession #: AAH04572
• N-terminal Sequence: Val33
• Protein/Peptide Type: Recombinant Enzymes
• Gene: Naaa
• Purity: >90%, 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: 38 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: 50 kDa, 33 kDa and 19 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 MES and NaCl.
• Purity: >90%, by SDS-PAGE under reducing conditions and visualized by silver stain
• Assay Procedure:
  • Assay Buffer: 0.1 M Sodium Acetate, 0.1% (v/v) NP-40 substitute (Fluka, Catalog # 74385), pH 4.0
  • Recombinant Mouse ASAHL/N‑acylethanolamine-hydrolyzing Acid A (rmASAHL) (Catalog # 4886-AH)
  • Palmitoyl Ethanolamide (PEA) (Tocris, Catalog # 0879), 25 mM stock in dimethyl formamide
  • Dithiothreitol (DTT) (Sigma, Catalog # D0632), 1 M stock in deionized water
  • Sodium Hydroxide
  • beta -mercaptoethanol (Sigma, Catalog # M7154)
  • o-phthaldialdehyde (o-PA) (Sigma, Catalog # P0657), 50 mg/mL stock in DMSO
  • F16 Black Maxisorp Plate (Nunc, Catalog # 475515)
  • Fluorescent Plate Reader (Model: SpectraMax Gemini EM by Molecular Devices) or equivalent
  1. Dilute PEA to 50 µM in Assay Buffer. Dissolve 10 µL of 25 mM stock into 4.99 mL of Assay Buffer (Note: Preheat assay buffer to
     37 °C and vortex for 30 seconds to completely solubilize the PEA).
  2. Dilute rmASAHL to 1.25 µg/mL in Assay Buffer.
  3. Set up reactions in 1.5 mL microtubes. Mix 200 µL of 50 µM PEA, 50 µL of 1.25 µg/mL rmASAHL, and 2.5 µL of 1 M DTT.
  4. Incubate reaction tubes at 37 °C for 1 hour.
  5. Prepare an o-PA solution. Mix 3.84 mL of 0.2 M Sodium Hydroxide, 4 µL beta -mercaptoethanol, and 160 µL 50 mg/mL o-PA.
  6. Add 250 µL of the o-PA mixture (step 5) to all reaction vials. Mix well and incubate at room temperature for 10 minutes.
  7. Create a control vial by combining, in this order, 250 µL o-PA mixture (step 5), 50 µL of 1.25 µg/mL rmASAHL, and 200 µL of 50 µM PEA.
  8. Load in a plate 200 µL in duplicate of reaction mixtures and control.
  9. Read at excitation and emission wavelengths of 330 nm and 450 nm (top read), respectively, in endpoint mode.
  10. Calculate specific activity (Average duplicates):

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

  *Adjusted for control
  **Derived using calibration standard ethanolamine (Sigma, Catalog # E9508).

  Per Well:
  • rmASAHL: 0.025 µg
  • PEA: 20 µM
  • o-PA: 1 mg/mL

Notes

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

Alternate Names for Recombinant Mouse ASAHL Protein, CF

• Acid ceramidase-like protein
• ASAHL
• ASAH-like protein
• EC 3.5.1.-
• NAAA
• N-acylethanolamine acid amidase
• Nacylethanolaminehydrolyzing Acid Amidase
• N-acylethanolamine-hydrolyzing acid amidase
• N-acylsphingosine amidohydrolase (acid ceramidase)-like
• N-acylsphingosine amidohydrolase-like
• PLT

Background

The mouse ASAHL gene encodes N-acylethanolamine-hydrolyzing Acid Amidase (NAAA), a fatty acid amidase with maximal activity at acidic pH (1). NAAA hydrolyzes a number of N-acyl ethanolamines, including N-myristoyl-, N-stearoyl-, N-oleoyl-, and N-arachidonoyl, but is most active against N-palmitoylethanolamine (2). NAAA is a member of the choloylglycine hydrolase family of enzymes, and is structurally similar to acid ceramidase (1). NAAA is both a lysosomal and a secreted enzyme, and like acid ceramidase, has been observed to be proteolytically processed during maturation (1). Through its amidase activity, ASAHL may play a role in the termination of the actions of a variety of N-acylethanolamides (3). NAAA can be distinguished from anandamide amidohydrolase by its lack of inhibition by methyl arachidonoyl fluorophosphonate (2).

1. Tsuboi, K. et al. (2005) J. Biol. Chem. 280:11082.
2. Ueda, N. et al. (2001) J. Biol. Chem. 276:35552.
3. Sun, Y. X. et al. (2005) Biochim. Biophys. Acta 1736:211.

Bioinformatics

• Gene Symbol: Naaa
• Uniprot:
  • AAH04572()