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

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

Principle of the Assay

Reaction scheme:

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.

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)
Inhibition of HMG-CoA reductase

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.

Materials
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References

1.
Koning AJ, Roberts CJ, Wright RL. 1996. Different subcellular localization of Saccharomyces cerevisiae HMG-CoA reductase isozymes at elevated levels corresponds to distinct endoplasmic reticulum membrane proliferations.. MBoC. 7(5):769-789. http://dx.doi.org/10.1091/mbc.7.5.769
2.
Holdgate G, Ward W, McTaggart F. 2003. Molecular mechanism for inhibition of 3-hydroxy-3-methylglutaryl CoA (HMG-CoA) reductase by rosuvastatin. 31(3):528-531. http://dx.doi.org/10.1042/bst0310528
3.
Istvan ES, Palnitkar M, Buchanan SK, Deisenhofer J. 2000. Crystal structure of the catalytic portion of human HMG-CoA reductase: insights into regulation of activity and catalysis. EMBO J. 19(5):819-830. http://dx.doi.org/10.1093/emboj/19.5.819
4.
Istvan E. 2000. The structure of the catalytic portion of human HMG-CoA reductase. 1529(1-3):9-18. http://dx.doi.org/10.1016/s1388-1981(00)00134-7
5.
Kleemann R, Kooistra T. 2005. HMG-CoA Reductase Inhibitors: Effects on Chronic Subacute Inflammation and Onset of Atherosclerosis Induced by Dietary Cholesterol. CDTCHD. 5(6):441-453. http://dx.doi.org/10.2174/156800605774962077

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