[22] 3-Hydroxy-3-methylglutaryl-CoA synthase from bakers' yeast

This chapter discusses the determination of 3-hydroxy-3-methylglutaryl-CoA synthase from bakers' yeast. The spectrophotometric assay is based on the absorption of the enol form of acetoacetyl-CoA at 303 nm. The synthase activity is measured by observing the acetyl-CoA stimulated decrease in this absorption, Mg2+ is routinely included in the assay to increase the apparent extinction coefficient of the acetoacetyl-CoA. Mg2+ has no direct effect on the enzyme activity. A unit of enzymatic activity is defined as the amount of enzyme necessary to transform 1 μmole of acetoacetyl-CoA into product per minute. Specific activity is expressed in units per milligram of protein. In the course of purification procedure, Fresh bakers' yeast is crumbled into a stainless steel beaker and toluene added to 110 ml/kg of yeast. The mixture is heated in a water bath at 55° until the yeast has warmed to 38°. The yeast is then allowed to autolyze at 38° for 8–12 hours. Within these time limits the yield and specific activity of the HMG-CoA synthase remain approximately constant; further incubation causes losses. At the end of this period, an equal volume of cold deionized water (900 ml/kg of yeast) is added with stirring and the cell debris removed by centrifugation at 15,000 g for 20 minutes.