Attention:

Certain features of Sigma-Aldrich.com will be down for maintenance the evening of Friday August 18th starting at 8:00 pm CDT until Saturday August 19th at 12:01 pm CDT.   Please note that you still have telephone and email access to our local offices. We apologize for any inconvenience.

HMG-CoA Reductase (HMGR) Assay Kit

BioFiles 2007, 2.7, 2.

3-hydroxy-3-methylglutaryl-CoA reductase (HMGR) is a transmembrane glycoprotein, located on the endoplasmic reticulum.1 This enzyme catalyzes the four-electron reduction of HMG-CoA to coenzyme A and mevalonate, which is the ratelimiting step in sterol biosynthesis.2 The activity of HMGR is controlled through synthesis, degradation, and phosphorylation in order to maintain the concentration of mevalonate derived products. In addition to the physiological regulation of HMGR, the human enzyme has been targeted successfully by drugs in the clinical treatment of high serum cholesterol levels.3,4 Controlling serum cholesterol levels has an important therapeutic role as hypercholesterolemia often leads to the development of atherosclerosis and consequently to cardiovascular pathologies, which might result in myocardial infarction and stroke. Recent evidence suggests that a disturbance of cholesterol homeostasis contributes to the development of a chronic inflammatory state.5

back to top 

Kit Features and Benefits

  • Contains all the reagents for a simple and rapid measurement of HMGR enzyme activity
  • Includes an HMGR control enzyme, enabling screening of HMGR inhibitors and activators
  • Includes a control inhibitor
  • Sufficient for 30 assays of 1 mL or 100 assays of 200 μL

back to top 

Principle of the Assay

Reaction scheme:


The assay is based on a spectrophotometric measurement of the decrease in absorbance, which represents the oxidation of NADPH by the catalytic subunit of HMGR in the presence of the substrate HMG-CoA.

back to top 

Applications

  • Basic research of cholesterol and other related metabolic pathways
  • etection of HMGR activity as well as screening for different inhibitors/activators of the enzyme (which may play a crucial role in therapeutics)

Figure 1. Inhibition of HMG-CoA reductase (HMGR) activity by Pravastatin.

HMG-CoA reductase activity and inhibition assay was performed in a UV compatible 96 well plate, using the HMG-CoA Reductase Assay Kit. Approximately 6 µg of the enzyme were incubated at 37 °C with 400 µM NADPH, 0.3 mg/ml HMG-CoA and different concentrations of Pravastatin that is a specific inhibitor of HMG-CoA reductase.

back to top 

Materials

     

References

  1. Koning, A.J., et. al., Different subcellular localization of Saccharomyces cerevisiae HMG-CoA reductase isozymes at elevated levels corresponds to distinct endoplasmic reticulum membrane proliferations. Mol. Biol. Cell, 7, 769-789 (1996).
  2. Holdgate, G.A., et. al., Molecular mechanism for inhibition of 3-hydroxy-3-methylglutaryl CoA (HMG-CoA) reductase by rosuvastatin. Biochem. Soc. Trans., 31, 528-531 (2003).
  3. Istvan, E.S., et. al., Crystal structure of the catalytic portion of human HMG-CoA reductase: insights into regulation of activity and catalysis. EMBO J., 19, 819-830 (2000).
  4. Istvan, E.S., and Deisenhofer, J., The structure of the catalytic portion of human HMG-CoA reductase. Biochim. Biophys. Acta, 1529, 9-18 (2000).
  5. Kleemann, R., and Kooistra, T., HMG-CoA reductase inhibitors: effects on chronic subacute inflammation and onset of atherosclerosis induced by dietary cholesterol. Curr. Drug Targets Cardiovas. Haemat., Disord., 5, 441-453 (2005).

back to top 

Related Links