Solubilization of 3-hydroxy-3-methylglutaryl coenzyme A reductase from rat and chicken liver microsomes

✍ Scribed by Zafarul H. Beg; John A. Stonik; H.Bryan Brewer Jr.

• Publisher: Elsevier Science
• Year: 1978
• Tongue: English
• Weight: 312 KB
• Volume: 86
• Category: Article
• ISSN: 0003-2697
• DOI: 10.1016/0003-2697(78)90779-0

✦ Synopsis

A simple, efficient, freeze-thaw procedure for the solubilization of liver 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase has been developed. Microsomes of chicken or rat liver were prepared by homogenization in buffer containing 100 mM sucrose, 50 mM KCI, 40 mM KH,PO,, 30 mM EDTA. and 2 mM DTT, pH 7.2 (buffer A). The homogenate was centrifuged at 12,OOOg (15 min), and the microsomes were separated from the supernatant by centrifugation at 100,OOOg (60 min). The isolated microsomes were frozen, either by dry ice-acetone or by storage in a freezer at -20°C. The frozen microsomes were permitted to thaw at room temperature, homogenized in buffer A, and centrifuged at lOO,OOOg (60 min). The extraction was repeated and the combined supernatants contained 70 to 90% of the microsomal HMG-CoA reductase activity. The yield of enzyme activity by the freeze-thaw technique is equal to or greater than previously reported methodologies and is significantly easier to perform. This procedure is particularly suited to the preparation of large quantities of solubilized enzyme for isolation and characterization of HMG-CoA reductase. In addition, this method does not require the use of detergents. sonification, or other procedures which might partially inactivate or alter the molecular properties of the enzyme.