Recombinant Human HMG-CoA Reductase/HMGCR Protein, CF

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Product Details

Summary
Reactivity HuSpecies Glossary
Applications Enzyme Activity
Format
Carrier-Free

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Recombinant Human HMG-CoA Reductase/HMGCR Protein, CF Summary

Details of Functionality
Measured by its ability to reduce HMG-CoA with NADPH. The specific activity is >6,000 pmol/min/μg, as measured under the described conditions.
Source
E. coli-derived human HMG-CoA Reductase/HMGCR protein
Ser426-Ala888, with an N-terminal Met and 6-His tag
Accession #
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
51 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
51-61 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 KCl, 1 mg/mL BSA, pH 7.0
  • Recombinant Human HMG-CoA Reductase/HMGCR (rhHMGCR) (Catalog # 9264-HM)
  • Acceptor Substrate: NADPH (Sigma, Catalog # N7505), 10 mM stock in deionized water
  • Donor Substrate: DL-3-Hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) (Sigma, Catalog # H6132), 10 mM stock in deionized water
  • 96-well Clear Plate (Catalog # DY990)
  • Fluorescent Plate Reader (Model: SpectraMax Plus by Molecular Devices) or equivalent
  1. Dilute rhHMGCR to 1 ng/µL in Assay Buffer.
  2. Prepare Reaction Mixture containing 1 mM of NADPH and 0.4 mM HMG-CoA in Assay Buffer.
  3. Load 50 µL of 1 ng/µL rhHMGCR into wells of a plate, and start the reaction by adding 50 μL of Reaction Mixture. Include a Substrate Blank containing 50 μL of Reaction Mixture and 50 µL of Assay Buffer.
  4. Seal and incubate plate at 37 °C for 20 minutes.
  5. Read plate at 340 nm (absorbance) in endpoint mode. 
  6. Calculate specific activity:

     Specific Activity (pmol/min/µg) =

Adjusted Abs* (OD) x well volume (L) x 1012 pmol/mol x (-1)
Inc. time (min) x epsilon ** (M-1cm-1) x path corr.*** (cm) x amount of enzyme (µg)

      * Adjusted for Substrate Blank
      **Using extinction coefficient 6220 M-1cm-1
      ***Using the path correction 0.32 cm

Per Well:

  • rhHMGCR:  0.05 µg
  • NADPH:  0.5 mM
  • HMG-CoA:  0.2 mM

Notes

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

Alternate Names for Recombinant Human HMG-CoA Reductase/HMGCR Protein, CF

  • 3-hydroxy-3-methylglutaryl CoA reductase (NADPH)
  • 3-hydroxy-3-methylglutaryl-CoA reductase
  • 3-hydroxy-3-methylglutaryl-Coenzyme A reductase
  • EC 1.1.1
  • EC 1.1.1.34
  • HMG-CoA Reductase
  • HMGCR
  • Hydroxymethylglutaryl-CoA Reductase
  • LDLCQ3

Background

HMG-CoA reductase (HMGCR) is a transmembrane glycoprotein of the endoplasmic reticulum. It is the rate-limiting enzyme in cholesterol biosynthesis and converts HMG-CoA to mevalonate (1). Mevalonate can be converted to cholesterol through a series of enzymatic reactions, and can also serve as the precursor for several nonsterol isoprenoid compounds such as ubiquinone, dolichol, and the isopentenyl group of tRNA (2). The activity of HMGCR is finely regulated by a negative feedback mechanism in which cholesterol and the other end products of the metabolic pathway suppress the enzyme in a multivalent fashion. Cholesterol suppresses the reductase activity primarily by inhibiting the rate of transcription of the reductase gene (3). Cytosolic cholesterol is derived from the internalization and degradation of low density lipoprotein (LDL) via the LDL receptor. Competitive inhibitors of the reductase induce the expression of LDL receptors in the liver, which in turn increases the catabolism of plasma LDL and lowers the plasma concentration of cholesterol, an important determinant of atherosclerosis (4). The well-known inhibitors for HMGCR are statins, a class of hypolipidemic agents used as pharmaceuticals to lower cholesterol levels in individuals at risk from cardiovascular disease due to hypercholesterolemia (5). The recombinant human HMGCR contains only the catalytic domain of the enzyme (6).
  1. Goldstein, J. L. and Brown, M. S. (1990) Nature 343:425.
  2. Buhaescu, I. and Izzedine, H. (2007) Clin. Biochem. 40:575.
  3. Reynolds, G. A. et al. (1984) Cell 38:275.
  4. Brown, M. S. and Goldstein, J. L. (1980) J. Lipid Res. 21:505.
  5. Sweetman, S. C. (2009) Martindale: the complete drug reference (36th ed.) pp.1155–434.
  6. Istvan, E.S. et al. (2000) EMBO J. 19:819.

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