Assay of 3-hydroxy-3-methylglutaryl CoA reductase activity using anionic-exchange column chromatography.
A rapid, easy, and sensitive method is described in this paper for the assay of 3-hydroxy-3-methylglutaryl CoA (HMG CoA) reductase, a key enzyme in cholesterol biosynthesis. [14C]HMG CoA was used as the substrate and the product formed, i.e., [14C]mevalonate, was allowed to be converted to its lactone form (mevalonolactone) in the presence of HCl. The reaction mixture was applied to a column containing an anionic exchanger. The column was made up of QAE-Sephadex (A25, formate form) packed to a height of 4 cm in Pasteur pipets. Under these conditions, mevalonolactone was not retained by the column and was eluted with ammonium formate solution while HMG CoA, being negatively charged, was retained by the gel and eluted by HCl above 0.05 M. Determination of the amount of radioactivity in mevalonolactone was then used to quantitate the activity of HMG CoA reductase. This assay has been successfully used for determining the activity of this enzyme in a microsomal fraction prepared from the liver of the rat.
Ong
K K
KK
Department of Biochemistry, Faculty of Medicine, University of Malaya, Kuala Lumpur.
Khor
H T
HT
Tan
D T
DT
D000838:Anions; D002852:Chromatography, Ion Exchange / Q000379:methods*; D002855:Chromatography, Thin Layer / Q000379:methods; D006903:Hydroxymethylglutaryl CoA Reductases / Q000302:isolation & purification / Q000378:metabolism*; D008798:Mevalonic Acid / Q000031:analogs & derivatives / Q000302:isolation & purification
D000838:Anions; C015367:mevalonolactone; D006903:Hydroxymethylglutaryl CoA Reductases; D008798:Mevalonic Acid