Recombinant Human 15-PGDH/HPGD Protein, CF

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

Recombinant Human 15-PGDH/HPGD Protein, CF Summary

• Details of Functionality: Measure by the production of NADH during the oxidation of PGF2 alpha . The specific activity is >1,500 pmol/min/μg, as measured under the described conditions.
• Source: E. coli-derived human 15-PGDH/HPGD protein
  Met1-Gln266, with a C-terminal 6-His tag
• Accession #: P15428
• N-terminal Sequence: Met1
• Structure / Form: Noncovalently-linked homodimer
• Protein/Peptide Type: Recombinant Enzymes
• Gene: HPGD
• Purity: >95%, by SDS-PAGE visualized with Silver Staining and quantitative densitometry by Coomassie® Blue Staining.
• Endotoxin Note: <1.0 EU per 1 μg of the protein by the LAL method.

Applications/Dilutions

• Dilutions:
  • Enzyme Activity
• Theoretical MW: 30 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: 28 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 HEPES, NaCl, DTT and Glycerol.
• Purity: >95%, by SDS-PAGE visualized with Silver Staining and quantitative densitometry by Coomassie® Blue Staining.
• Assay Procedure:
  • Assay Buffer: 50 mM Tris, 100 mM NaCl, 2 mM Dithiothreitol (DTT), pH 9.0
  • Recombinant Human 15‑PGDH/HPGD (rhHPGD) (Catalog # 5660-DH)
  • beta -Nicotinamide adenine dinucleotide ( beta -NAD) (Sigma, Catalog # N6522), 100 mM stock in deionized water
  • Prostaglandin F2 alpha (PGF2 alpha ) (Sigma, Catalog # P0424), 10 mM stock in absolute ethanol
  • 96-well Clear Plate (Costar, Catalog # 92592)
  • Plate Reader (Model: SpectraMax Plus by Molecular Devices) or equivalent
  1. Prepare the Substrate Mixture.
  1. Dilute beta -NAD to 1 mM in Assay Buffer.
  2. Dilute PGF2 alpha to 0.4 mM in Assay Buffer.
  3. Mix equal volumes of each for a final concentration of 0.5 mM beta -NAD and 0.2 mM PGF2 alpha .
  2. Dilute rhHPGD to 1.0 ng/μL in Assay Buffer.
  3. Load in a plate 50 μL of 1.0 ng/μL rhHPGD, and start the reaction by adding 50 μL of Substrate Mixture.
  4. Include a Substrate Blank containing 50 μL of Assay Buffer and 50 μL of Substrate Mixture.
  5. Read at 339 nm in kinetic mode for 5 minutes.

  Specific Activity (pmol/min/µg) = (Adjusted Vmax* (OD/min) x well volume (L) x 10^12 pmol/mol) / (ext. coeff** (M^-1cm^-1) x path corr.*** (cm) x amount of enzyme (µg))

  
  *Adjusted for Substrate Blank
  **Using the extinction coefficient 6220 M^-1cm^-1
  ***Using the path correction 0.32 cm
  Note: the output of many spectrophotometers is in mOD Per Well:
  • rhHPGD: 0.050 µg
  • beta -NAD: 0.25 mM
  • PGF2 alpha : 0.1 mM

Notes

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

Alternate Names for Recombinant Human 15-PGDH/HPGD Protein, CF

• 15-hydroxyprostaglandin dehydrogenase [NAD+]
• 15PGDH
• 15-PGDH
• EC 1.1.1
• EC 1.1.1.141
• HPGD
• hydroxyprostaglandin dehydrogenase 15-(NAD)
• NAD+-dependent 15-hydroxyprostaglandin dehydrogenase
• PGDH1
• PGDH1PGDH
• Prostaglandin dehydrogenase 1
• SDR36C1
• short chain dehydrogenase/reductase family 36C, member 1

Background

HPGD, or 15-hydroxyprostaglandin dehydrogenase, is a NAD⁺-linked dehydrogenase that oxidizes the hydroxyl group at position 15 of prostaglandins to a ketone, resulting in a loss of biological activity (1). HPGD is a major enzyme for the catabolism of prostaglandins. The enzyme is a member of the short-chain alcohol dehydrogenase family of enzymes (2). HPGD is a cytosolic enzyme expressed in most tissues, with highest expression levels in placenta, lung, and kidney (3). It is inhibited by aspirin and nonsteroidal anti-inflammatory drugs (4). Defects in HPGD are a cause of hypertrophic osteoarthropathy (5).

1. Anggard, E. and B. Samuelsson (1964) J. Biol. Chem. 239:4097.
2. Krook, M. et al. (1990) Biochemistry 29:738.
3. Tai, H.H. (1976) Biochemistry 15:4586.
4. Mak, O.T. et al. (1982) Biosci. Rep. 2:503.
5. Uppal, S. et al. (2008) Nat. Genet. 40:789.

Bioinformatics

• Gene Symbol: HPGD
• Uniprot:
  • P15428(Human)