Recombinant Human ASAHL Protein, CF

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

Recombinant Human ASAHL Protein, CF Summary

• Details of Functionality: Measured by its ability to hydrolyze the substrate palmitoylethanolamide into palmitate and ethanolamine. The specific activity is >300 pmol/min/µg, as measured under the described conditions.
• Source: Chinese Hamster Ovary cell line, CHO-derived human ASAHL/N-acylethanolamine-hydrolyzing Acid Amidase protein
  Met1-Lys359 (Val151Ile), with a C-terminal 10-His tag
• Accession #: Q02083
• N-terminal Sequence: Ser29
• Protein/Peptide Type: Recombinant Enzymes
• Gene: NAAA
• 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: 39 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 20 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, NaCl and Glycerol.
• Purity: >95%, by SDS-PAGE under reducing conditions and visualized by silver stain
• Assay Procedure:
  • Assay Buffer: 0.1 M NaOAc, 0.1% (v/v) NP-40 substitute (Fluka, Catalog # 74385), pH 4.0
  • Recombinant Human ASAHL/N‑acylethanolamine-hydrolyzing Acid A (rhASAHL) (Catalog # 4494-AH)
  • Palmitoyl Ethanolamide (PEA) (Tocris, (Tocris, Catalog # 0879), 25 mM stock in dimethyl formamide
  • Dithiothreitol (DTT) (Sigma, Catalog # D0632), 1 M stock in deionized water
  • NaOH (Sigma, Catalog # 221465), 2 M stock in deionized water
  • beta -mercaptoethanol (Sigma, Catalog # M-7154)
  • 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 by dissolving 10 µL of 25 mM stock in 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 rhASAHL to 1.25 µg/mL in Assay Buffer.
  3. Combine 200 µL of 50 µM PEA, 50 µL of 1.25 µg/mL rhASAHL, and 2.5 µL of 1 M DTT.
  4. Incubate reaction tubes at 37 °C for 1 hour.
  5. Dilute NaOH to 0.2 M in deionized water.
  6. Combine 3.84 mL of 0.2 M NaOH with 4 µL beta -mercaptoethanol and 160 µL of 50 mg/mL o-PA.
  7. Stop the reactions by adding 250 µL of the o-PA mixture (step 6) to all the vials and mix well.
  8. Incubate at room temperature for 10 minutes.
  9. Combine 250 µL of o-PA mixture, 50 µL of 1.25 µg/mL rhASAHL, and 200 µL of 50 µM PEA in this order for a control.
  10. Load 200 µL of reaction mixtures and control in a plate.
  11. Read at excitation and emission wavelengths of 330 nm and 450 nm (top read), respectively, in endpoint mode.
  12. 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:
  • rhASAHL: 0.025 µg
  • Palmitoyl Ethanolamide: 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 Human 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 human NAAA gene encodes N-acylethanolamine-hydrolyzing acid amidase, also known as ASAH-like protein (ASAHL), 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 cholylglycine hydrolase family of enzymes, and is structurally similar to acid ceramidase (3). NAAA is both a lysosomal and a secreted enzyme, and like acid ceramidase, has been observed to be proteolytically processed during maturation (3). NAAA can be distinguished from anandamide amidohydrolase by its lack of inhibition by methyl arachidonyl fluorophosphonate (2).

1. Hong, S.B. et al. (1999) Genomics 62:232.
2. Ueda, N. et al. (2001) J. Biol. Chem. 276:35552.
3. Tsuboi, K. et al. (2005) J. Biol. Chem. 280:11082.

Bioinformatics

• Gene Symbol: NAAA
• Uniprot:
  • Q02083()