Recombinant Human Renalase Protein, CF

Recombinant Human Renalase (Catalog # 9887-RE) is measured by its ability to reduce Resazurin (Catalog # AR002).

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

Recombinant Human Renalase Protein, CF Summary

• Details of Functionality: Measured by its ability to reduce Resazurin.
  The specific activity is >3.5 pmol/min/μg, as measured under the described conditions.
• Source: E. coli-derived human Renalase protein
  Ala2-Ile342
  with substitution Glu37Asp, N-terminal Met and 6-His tag
• Accession #: Q5VYX0-1
• N-terminal Sequence: Met
• Protein/Peptide Type: Recombinant Enzymes
• 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: 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: 39 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, EDTA, 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 HEPES, 200 mM NaCl, 5 µM FAD (Flavin Adenine Dinucleotide), 0.05% Tween-20, pH 7.5
  • Recombinant Human Renalase (rhRenalase) (Catalog # 9887-RE)
  • beta -Nicotinamide adenine dinucleotide, reduced (NADH) (Sigma, Catalog # N8129), 20 mM stock in 0.1 M Sodium Borate, pH 9.0
  • Resazurin (Catalog # AR002) (MW = 251.17 Da)
  • F16 Black Maxisorp Plate (Nunc, Catalog # 475515)
  • Fluorescent Plate Reader (Model: SpectraMax Gemini EM by Molecular Devices) or equivalent
  1. Dilute rhRenalase to 40 µg/mL in Assay Buffer.
  2. Prepare a Substrate Mixture containing 400 µM of NADH and 20 µM Resazurin in Assay Buffer. Mix well and use immediately.
  3. Load 50 µL of 40 µg/mL rhRenalase into a plate, and start reaction by adding 50 µL Substrate Mixture. Include a Substrate Blank containing 50 µL Assay Buffer and 50 µL Substrate Mixture.
  4. Read plate at excitation and emission wavelengths of 540 and 585 nm, respectively, in kinetic mode for five minutes.
  5. Calculate specific activity:

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

  *Adjusted for Substrate Blank.
  **Derived using calibration standard Resorufin (Sigma, Catalog # R3257).

  Per Well:
  • rhRenalase: 2.0 µg
  • NADH: 200 µM
  • Resazurin: 10 µM

Notes

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

Alternate Names for Recombinant Human Renalase Protein, CF

• C10orf59
• C10orf59chromosome 10 open reading frame 59
• EC 1.4
• FLJ11218
• MAO-C
• Monoamine oxidase-C
• Renalase
• renalase, FAD-dependent amine oxidase
• RNLS

Background

Renalase has been reported to regulate cardiac function and blood pressure and initially proposed to metabolize ciculating catecholamines (1). Recent characterization has clarified the true enzymatic function of renalase is oxidation of NAD(P)H isomers to B-NAD(P)+ (2, 3). As these molecules are potent inhibitors of primary metabolism dehydrogenases, cellular renalase activity serves to prevent the inhibitory effects of these molecules on primary metabolism (2, 3). Renalase was initially found to have robust expression in the kidney (1) and later confirmed to have systemic expression in all tissues. Renalase is classified as a member of the flavoprotein superfamily but uniquely lacks an internal structural element that causes its active site to be solvent exposed (4). Renalase contains a putative secretory N-terminal signal sequence (aa 1-17) that also contains residues required for FAD cofactor binding, suggesting that the enzyme would require the N-terminus to remain functionally active (3). However, renalase is found both intracellularly and secreted extracellularly. Extracellular renalase is cleaved during secretion from the cell (5). Circulating renalase acts as a cytokine, resulting in extensive reported physiological effects (6). Increases in blood pressure was induced by renalase gene knockout (7) and gene deletion aggravates acute ischemic kidney (8). Polymorphisms in renalase are associated with hypertension and stroke in type 2 diabetes (9, 10). Full-length recombinant renalase as well as truncated, non-active, peptides were found to promote cell and organ survival in a process independent of enzymatic activity via signaling through the receptor PMCA4b, characterized as an ATPase involved in cell signaling and cardiac hypertrophy (11, 12). Renalase was significantly increased in pancreatic, bladder, breast and melanoma cancers where its signaling role favors cell survival and tumor growth (13). Consequently, renalase inhibition has been suggested to provide a novel therapeutic option for cancer treatment (13).

1. Xu, J. et al. (2005) J. Clin. Invest. 115:1275.
2. Beaupre, B. A. et al. (2015) Biochemistry 54:795.
3. Moran, G.R. et al. (2016) Biochim. Biophys. Acta 1864:177.
4. Milani, M. et al. (2011) J. Mol. Biol. 411:463.
5. Fedchenko, V. et al. (2016) Kidney Blood Press. Res. 41:593.
6. Guo, X. et al. (2014) Curr. Opin. Nephrol. Hypertens. 23:513.
7. Wu, Y. et al. (2011) Kidney International 79:853.
8. Lee, H.T. et al. (2013) J. Am. Soc. Nephrol. 24:445.
9. Buraczynska, M. et al. (2011) Neuromolecular Med. 13:321.
10. Orlowska-Baranowska, E. et al. (2017) PLoS One 125:e0186729.
11. Wang, L. et al. (2015) PLoS ONE 10:e0122932.
12. Wang, L. et al. (2014) J. Am. Soc. Nephrol. 25:1226.
13. Wang, Y. et al. (2017) J. Cell. Mol. Med. 21:1260.

Bioinformatics

• Uniprot:
  • Q5VYX0-1()