Recombinant Human Aldo-keto Reductase 1C1/AKR1C1 Protein, CF

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

Recombinant Human Aldo-keto Reductase 1C1/AKR1C1 Protein, CF Summary

• Details of Functionality: Measured by its ability to oxidize S-1-indanol in the presence of NADP⁺. The specific activity is >150 pmol/min/μg, as measured under the described conditions.
• Source: E. coli-derived human Aldo-keto Reductase 1C1/AKR1C1 protein
  Met1-Tyr323
• Accession #: Q04828
• N-terminal Sequence: Met1
• Protein/Peptide Type: Recombinant Enzymes
• Gene: AKR1C1
• 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: 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, 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, 500 mM NaCl, pH 8.5
  • Substrate Buffer: 50 mM Tris, 500 mM NaCl, 20% (v/v) ethanol, pH 8.5
  • Recombinant Human Aldo-keto Reductase 1C1/AKR1C1 (rhAKR1C1) (Catalog # 6529-DH)
  • S-1-Indanol (Sigma, Catalog # 323128), 200 mM in DMSO
  • beta -Nicotinamide adenine dinucleotide phosphate (NADP⁺) (Sigma, Catalog # N5755), 50 mM in deionized water
  • 96-well Clear Plate (Costar, Catalog # 92592)
  • Plate Reader (Model: SpectraMax Plus by Molecular Devices) or equivalent
  1. Dilute rhAKR1C1 to 40 ng/μL in Assay Buffer.
  2. Dilute Indanol to 4 mM in Substrate Buffer.
  3. Dilute NADP⁺ to 2 mM in Substrate Buffer.
  4. Combine equal volumes of 4 mM Indanol and 2 mM NADP⁺ to form the Substrate Mixture.
  5. Load 50 μL of 40 ng/μL rhAKR1C1 into the microplate and start the reaction by adding 50 μL of Substrate Mixture. Include a Substrate Blank containing 50 μL of Assay Buffer and 50 μL of Substrate Mixture.
  6. Read at an absorbance of 340 nm in kinetic mode for 5 minutes.
  7. 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:
  • rhAKR1C1: 2 μg
  • Indanol: 1 mM
  • NADP⁺: 0.5 mM

Notes

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

Alternate Names for Recombinant Human Aldo-keto Reductase 1C1/AKR1C1 Protein, CF

• 20 alpha-hydroxysteroid dehydrogenase
• 20-ALPHA-HSD
• 20-alpha-hydroxysteroid dehydrogenase
• 2-ALPHA-HSD
• AKR1C1
• Aldo-keto Reductase 1C1
• aldo-keto reductase family 1 member C1
• aldo-keto reductase family 1, member C1 (dihydrodiol dehydrogenase 1; 20-alpha(3-alpha)-hydroxysteroid dehydrogenase)
• AldoketoReductase 1C1
• C9
• Chlordecone reductase homolog HAKRC
• DD1/DD2
• DD1MGC8954
• DDH1
• DDHH-37
• dihydrodiol dehydrogenase 1
• Dihydrodiol dehydrogenase 1/2
• dihydrodiol dehydrogenase isoform DD1
• EC 1.1.1
• EC 1.1.1.-
• EC 1.1.1.112
• EC 1.1.1.149,2-ALPHA-HSD
• EC 1.3.1.20
• HAKRC
• HAKRCDDH1aldo-keto reductase C
• HBAB
• hepatic dihydrodiol dehydrogenase
• High-affinity hepatic bile acid-binding protein
• Indanol dehydrogenase
• MBAB
• Trans-1,2-dihydrobenzene-1,2-diol dehydrogenase
• type II 3-alpha-hydroxysteroid dehydrogenase

Background

AKR1C1 (20-alpha -hydroxysteroid dehydrogenase, 20‑ alpha ‑HSD) is a member of aldo-keto reductase (AKR) superfamily. AKRs perform the NAD(P)H-dependent reduction of carbonyl groups (1). Four AKR1C isoforms (AKR1C1-C4) are known to exist in humans. They are all highly expressed in the liver. Three isoforms, excluding AKR1C4, have a wider expression pattern including prostate, testes, uterus, mammary gland, and haemopoietic progenitors (2). These enzymes are able to accept various natural steroids as substrates, including 3-, 7-, and 20‑ketosteroids (3). They can also activate prodrugs such as synthetic steroid hormone tibolone by converting it into active 3 alpha / beta -hydroxy form (4). They are recognized as phase I drug-metabolizing enzymes involved in the maintenance of steroid homeostasis, prostaglandin metabolism, and metabolic activation of polycyclic aromatic hydrocarbons (5). Their reactions introduce a hydroxyl group into the product making it available for sulfonation and glucuronidation by phase II enzyme. Elevated expression of these enzymes is related to cancer with hormone-dependent malignancies (6, 7). Increased levels of expression of AKR1C1 parallels increased cell proliferation activity in human colon cancer cells. It has been shown to be associated with oncogenic potential and proproliferative effects. It is also involved in cancer cell chemoresistance.

1. Jez, J.M. et al. (1997) Biochem J. 326:499.
2. Penning, T.M. et al. (2000) Biochem. J. 351:67.
3. Rizner, T.L. et al. (2003) Endocrinology. 144:2922.
4. Steckelbroeck, S. et al. (2006) J. Pharmacol. Exp. Ther. 316:1300.
5. Penning, T.M. et al. (2004) Mol. Cell. Endocrinol. 1784:1342.
6. Penning, T.M. and M.C. Byrns (2009) Ann. N. Y. Acad. Sci. 1155:33.
7. Baumann, D.R. et al. (2004) Drug News Perspect. 17:563.

Bioinformatics

• Gene Symbol: AKR1C1
• Uniprot:
  • Q04828()