详细说明
- Purity>95%, by SDS-PAGE visualized with Silver Staining and quantitative densitometry by Coomassie® Blue Staining.
- Endotoxin Level<1.0 EU per 1 μg of the protein by the LAL method.
- ActivityMeasured by its ability to reduce HMG-CoA with NADPH. The specific activity is >6,000 pmol/min/μg, as measured under the described conditions. See Activity Assay Protocol on .
- SourceE. coli-derived Ser426-Ala888, with an N-terminal Met and 6-His tag
- Accession #
- N-terminal Sequence
AnalysisMet - Predicted Molecular Mass51 kDa
- SDS-PAGE51-61 kDa, reducing conditions
| 9264-HM | | |
| Formulation Supplied as a 0.2 μm filtered solution in Tris, NaCl, DTT and Glycerol. | ||
| Shipping The product is shipped with dry ice or equivalent. Upon receipt, store it immediately at the temperature recommended below. | ||
| Stability & Storage: Use a manual defrost freezer and avoid repeated freeze-thaw cycles.
|
Assay Procedure
Materials
- 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 # )
- Fluorescent Plate Reader (Model: SpectraMax Plus by Molecular Devices) or equivalent
- Dilute rhHMGCR to 1 ng/µL in Assay Buffer.
- Prepare Reaction Mixture containing 1 mM of NADPH and 0.4 mM HMG-CoA in Assay Buffer.
- 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.
- Seal and incubate plate at 37 °C for 20 minutes.
- Read plate at 340 nm (absorbance) in endpoint mode.
- 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
- rhHMGCR: 0.05 µg
- NADPH: 0.5 mM
- HMG-CoA: 0.2 mM
Background: HMG-CoA Reductase/HMGCR
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).
- References:
- Goldstein, J. L. and Brown, M. S. (1990) Nature 343:425.
- Buhaescu, I. and Izzedine, H. (2007) Clin. Biochem. 40:575.
- Reynolds, G. A. et al. (1984) Cell 38:275.
- Brown, M. S. and Goldstein, J. L. (1980) J. Lipid Res. 21:505.
- Sweetman, S. C. (2009) Martindale: the complete drug reference (36th ed.) pp.1155–434.
- Istvan, E.S. et al. (2000) EMBO J. 19:819.
- Long Name:3-Hydroxy-3-Methylglutaryl-CoA Reductase
- Entrez Gene IDs:3156 (Human); 15357 (Mouse); 25675 (Rat)
- Alternate Names: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
400-6226-992