Abstract
For precise determination of the catalytic activity of 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase (EC 1.1.1.34), the HMG-CoA employed as substrate must be free of HMG, CoA, and other inhibitors of HMG-CoA reductase activity. The standard purification of HMG-CoA by paper chromatography gives poor resolution of HMG-CoA from CoA and may be accompanied by some decomposition of HMG-CoA. We describe a simplified procedure for synthesis and for isolation from the reaction mixture of homogeneous, high specific activity [3(-14)C]HMG-CoA free of HMG, CoA, or nonpolar contaminants. Isolation of HMG-CoA utilizes ion-exchange chromatography in a gradient of ammonium formate, which is subsequently removed by lyophilization. The methods are proposed for use in the preparation or isolation of HMG0CoA.
Highlights
T h e procedure given is for synthesis of [3-’4C]HMG-CoA of specific activity 14.3 dpm/pmol suitable for assay of HMG-CoA reductase activity
The chromatographic method rat liver HMG-CoA reductase
Summary
For all radioisotopic assays that measure the product (mevalonate), the nonpolarity of mevalonolactone relative to HMG o r HMG-CoA is exploited to effect resolution by T L C (2, 3) o r to partition mevalonolactone into a toluene-based scintillation fluor (4, .5). Sensitivity in either assay depends both on the specific radioactivity of the HMG-CoA used as substrate and on its purity (5). HMG-CoA generally is purified by paper chromatography in n-butanol-acetic acid-water (5, 7). While this removes nonpolar contaminants, resolution of HMG-CoA from CoA is poor, and application of sample to the paper may be accompanied by some decomposition of HMG-CoA.’ Ion-exchange chromatography on amininated supports (DEAE-cellulose or DEAE-Sephadex) has been employed to purify acetyl-CoA and malonyl-CoA (8).
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