HMG-CoA Reductase Enzyme Activity Assay

HMG CoA reductase enzymatic activity was assessed by a colorimetric assay. HMG CoA reductase reactions were set up in 96 well plates, with each well containing a total volume of 100 µL Buffer E, supplemented with 400 µM NADPH (sigma) and 2 µL of either WT or mutant-purified HMG CoA reductase protein (0.6 mg/mL), WT enzyme with pravastatin, or a no enzyme control. Reactions were initiated by the addition of HMG-CoA substrate to a final concentration of 0 to 400 µM. Immediately after the addition of HMG-CoA, plates were analyzed for 340 nm absorbance using an Infinite M200 plate reader (Tecan). Absorbance was monitored every 30 s for a total of 90 min. For repeat experiments, HMG CoA was added to either WT or mutant reactions in an alternating fashion in order to minimize technical variations of V₀.

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Yogev Y., Shorer Z., Koifman A., Wormser O., Drabkin M., Halperin D., Dolgin V., Proskorovski-Ohayon R., Hadar N., Davidov G., Nudelman H., Zarivach R., Shelef I., Perez Y, & Birk O.S. (2023). Limb girdle muscular disease caused by HMGCR mutation and statin myopathy treatable with mevalonolactone. Proceedings of the National Academy of Sciences of the United States of America, 120(7), e2217831120.

Publication 2023

NADPH Infinite M200

Assay Buffer Colorimetric Enzymatic activity Enzyme Hmg coa Hmg coa reductase M200 Mutant protein Nadph Pravastatin

Corresponding Organization :

Other organizations : Ben-Gurion University of the Negev

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Variable analysis

independent variables

HMG-CoA substrate concentration (0 to 400 µM)

dependent variables

Absorbance at 340 nm (measured every 30 s for 90 min)

control variables

Buffer E (total volume of 100 µL)
NADPH (400 µM)
WT or mutant-purified HMG CoA reductase protein (0.6 mg/mL)
WT enzyme with pravastatin

controls

Positive control: WT HMG CoA reductase protein
Negative control: No enzyme

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