Recombinant Human Aldo-keto Reductase 1C4/AKR1C4 Protein, CF

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

Recombinant Human Aldo-keto Reductase 1C4/AKR1C4 Protein, CF Summary

• Details of Functionality: Measured by its NADP⁺-dependent oxidation of cholic acid. The specific activity is >300 pmol/min/μg, as measured under the described conditions.
• Source: E. coli-derived human Aldo-keto Reductase 1C4/AKR1C4 protein
  Met1-Tyr323
• Accession #: NP_001809
• N-terminal Sequence: Met1
• Protein/Peptide Type: Recombinant Enzymes
• Gene: AKR1C4
• Purity: >90%, by SDS-PAGE under reducing conditions and visualized by Colloidal Coomassie® Blue stain at 5 μg per lane
• Endotoxin Note: <1.0 EU per 1 μg of the protein by the LAL method.

Applications/Dilutions

• Dilutions:
  • Enzyme Activity
• Theoretical MW: 37 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: 36-38 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 Tris, NaCl, Glycerol and DTT.
• Purity: >90%, by SDS-PAGE under reducing conditions and visualized by Colloidal Coomassie® Blue stain at 5 μg per lane
• Assay Procedure:
  • Assay Buffer: 50 mM Tris, 500 mM NaCl, pH 8.5
  • Recombinant Human Aldo-keto Reductase 1C4/AKR1C4 (rhAKR1C4) (Catalog # 6957-DH)
  • Sodium Cholate (Sigma, Catalog # C1254), 464.5 mM (20% w/v) stock in deionized water
  • beta -Nicotinamide adenine dinucleotide phosphate (NADP+) (Sigma, Catalog # N5755), 50 mM stock in deionized water
  • UV Plate (Costar, Catalog # 3635)
  • Plate Reader (Model: SpectraMax Plus by Molecular Devices) or equivalent
  1. Dilute rhAKR1C4 to 6 ng/μL in Assay Buffer.
  2. Prepare a Reaction Mixture containing 2 mM sodium cholate and 1 mMNADP+ in Assay Buffer.
  3. In a plate, load 50 μL of 6 ng/μL rhAKR1C4, and start the reaction by adding 50 μL of Reaction Mixture.
  4. Include a Substrate Blank containing 50 μL of Assay Buffer and 50 μL of Reaction Mixture.
  5. Read at an absorbance of 340 nm in kinetic mode for 5 minutes.
  6. Calculate specific activity:

  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 6270 M^-1cm^-1
  ***Using the path correction 0.32 cm
  Note: the output of many spectrophotometers is in mOD Per Well:
  • rhAKR1C4: 0.3 μg
  • Sodium cholate: 1 mM
  • NADP+: 0.5 mM

Notes

Coomassie is a registered trademark of Imperial Chemical Industries Ltd.

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

Alternate Names for Recombinant Human Aldo-keto Reductase 1C4/AKR1C4 Protein, CF

• 3-alpha-HSD
• AKR1C4
• Aldo-keto Reductase 1C4
• aldo-keto reductase family 1, member C4
• AldoketoReductase 1C4
• C11
• CDR
• CHDR
• DD4
• DD-4
• HAKRA

Background

Aldo-keto Reductase 1C4 (AKR1C4), also known as dihydrodiol dehydrogenase 4, is a member of the aldo-keto reductase (AKR) superfamily. It catalyzes the reversible oxidoreduction of various 3 alpha ‑hydroxysteroids and prostaglandins as well as the oxidation of trans‑dihydrodiols of aromatic hydrocarbons and alicyclic alcohols (1‑4). AKR1C4 is important for steroid hormone metabolism primarily through synthesis and clearance of testosterone. Its high expression in liver and the ability to reduce xenobiotic carbonyl compounds suggests an important role in xenobiotic metabolism, as is manifested by the detoxification of the pesticide chlordecone in humans (5). AKR1C4 also plays a role in the pharmacokinetics of various drugs and prodrug activation (6, 7).

1. Penning, P. M. et al. (1990) Steroids 47:221.
2. Takikawa, H. et al. (1992) Hepatology 16:365.
3. Binstock, J. M. et al. (1992) Biochem. Biophys. Res. Commun. 187:760.
4. Ohara, H. et al. (1994) Biochim. Biophys. Acta. 1215:59.
5. Winters, C. J. et al. (1990) Biochemistry 29:1080.
6. Jin, Y. et al. (2009) J. Biol. Chem. 284:10013.
7. Breyer-Pfaff, U. et al. (2004) J. Pharm. Pharmacol. 56:1601.
   
   

Bioinformatics

• Gene Symbol: AKR1C4
• Uniprot:
  • NP_001809()