Recombinant Mouse PSMA/FOLH1 Protein, CF

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

Recombinant Mouse PSMA/FOLH1 Protein, CF Summary

• Details of Functionality: Measured by its ability to hydrolyze the substrate N-acetyl-L-Asp-L-Glu into N-acetyl-L-Asp and L-Glu. The L-Glu product is measured by fluorescence after its derivatization by ortho-phthaldialdehyde. The specific activity is >350 pmol/min/µg, as measured under the described conditions.
• Source: Chinese Hamster Ovary cell line, CHO-derived mouse PSMA/FOLH1/NAALADase I protein
  Ile44-Ala752, with an N-terminal 6-His tag
• Accession #: Q0VDM5
• N-terminal Sequence: His
• Protein/Peptide Type: Recombinant Enzymes
• Gene: Folh1
• 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: 80 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: 100 kDa, reducing conditions

Packaging, Storage & Formulations

• Storage: Use a manual defrost freezer and avoid repeated freeze-thaw cycles.
  • 6 months from date of receipt, -70 °C as supplied.
  • 3 months, -70 °C under sterile conditions after opening.
• Buffer: Supplied as a 0.2 μm filtered solution in MES and NaCl.
• Purity: >95%, by SDS-PAGE under reducing conditions and visualized by silver stain
• Assay Procedure:
  • Assay Buffer: 50 mM HEPES, 0.1 M NaCl, pH 7.5
  • o-PA Buffer: 0.2 M Sodium Hydroxide containing 0.1% mercaptoethanol (v/v)
  • Recombinant Mouse PSMA/FOLH1/NAALADase I (rmNAALADase I) (Catalog # 4946-ZN)
  • Substrate: Ac-Asp-Glu (Sigma, Catalog # A5930), 10 mM stock in 40 mM Sodium Hydroxide
  • o-phthaldialdehyde (o-PA) (Sigma, Catalog # P0657) - 50 mg/mL (0.373 M) stock in DMSO
  • F16 Black Maxisorp Plate (Nunc, Catalog # 475515)
  • Fluorescent Plate Reader (Model: SpectraMax Gemini EM by Molecular Devices) or equivalent
  1. Dilute rmNAALADase I to 0.2 µg/mL in Assay Buffer.
  2. Dilute Substrate to 40 µM with Assay Buffer.
  3. In reaction tube, mix 125 µL of rmNAALADase-1 and 125 µL of Substrate. For a Substrate Blank (no enzyme activity control), deactivate 125 µL of rmNAALADase-1 by heating it at 95 °C for 5 minutes, then add 125 µL of Substrate.
  4. Incubate the reaction tubes and blank control at 37 °C for 60 minutes.
  5. Stop the reaction tubes by heating at 95 °C for 5 minutes, then cool to room temperature.
  6. Prepare a 15 mM o-PA solution in o-PA Buffer.
  7. Add 250 µL of o-PA solution to each reaction and blank. Vortex and incubate at room temperature for 10 minutes.
  8. Remove two 200 µL aliquots from each reaction and control and transfer to the wells of a black microplate.
  9. Read at excitation and emission wavelengths of 330 nm and 450 nm (top read), respectively, in endpoint mode.
  10. Calculate specific activity:

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

  *Adjusted for Substrate Blank
  **Derived using calibration standard L-Glutamic Acid (Sigma, Catalog # G8415).

  Per Well:
  • rmNAALADase I: 0.010 µg
  • Substrate: 10 µM
  • o-PA: 7.5 mM

Notes

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

Alternate Names for Recombinant Mouse PSMA/FOLH1 Protein, CF

• Cell growth-inhibiting gene 27 protein
• cell growth-inhibiting protein 27
• EC 3.4.17.21
• FGCP
• folate hydrolase (prostate-specific membrane antigen) 1
• Folate hydrolase 1
• FOLH1
• FOLHNAALADase I
• Folylpoly-gamma-glutamate carboxypeptidase
• GCP2
• GCPII
• GCPIImGCP
• glutamate carboxylase II
• glutamate carboxypeptidase 2
• Glutamate carboxypeptidase II
• Membrane glutamate carboxypeptidase
• mopsm
• NAALAD1
• NAALAD1N-acetylated-alpha-linked acidic dipeptidase I
• NAALADase I
• NAALAdase
• N-acetylated alpha-linked acidic dipeptidase 1
• prostate specific membrane antigen variant F
• Prostate-specific membrane antigen
• PSMA
• PSMAFGCP
• PSMPteroylpoly-gamma-glutamate carboxypeptidase

Background

Prostate-specific membrane antigen (PSMA), a tumor marker in prostate cancer encoded by the FOLH1 gene, is a type II transmembrane zinc metallopeptidase that is most highly expressed in the nervous system, prostate, kidney, and small intestine (1, 2). The enzyme is also known as glutamate carboxypeptidase II (GCPII), folate hydrolase 1, folylpoly-(-glutamate carboxypeptidase (FGCP), and N-acetylated-alpha-linked acidic dipeptidase-1 (NAALADase1). In the brain, PSMA hydrolyzes the neurotransmitter N-acetyl-Asp-Glu to produce glutamate, another neurotransmitter. Inhibition of brain PSMA activity is considered to be a promising approach for the treatment of neurological disorders associated with glutamate excitotoxicity, such as stroke, chronic pain, and amyotrophic lateral sclerosis (3). Intestinal PSMA hydrolyzes folylpoly-gamma -glutamates, facilitating the uptake of folate (4).

1. Silver, D.A. et al. (1997) Clin. Cancer Res. 3:81.
2. Carter, R.E. et al. (1996) Pro. Natl. Acad. Sci. USA. 93:749.
3. Jackson, P.F. and Slusher, B.S. (2001) Curr. Med. Chem. 8:949.
4. Heston, W.D. (1997) Urology 49:104.

Bioinformatics

• Gene Symbol: Folh1
• Uniprot:
  • Q0VDM5()