A radiochemical assay for acetoacetyl-CoA synthetase

✍ Scribed by James D. Bergstrom; John Edmond

• Book ID: 102984920
• Publisher: Elsevier Science
• Year: 1985
• Tongue: English
• Weight: 643 KB
• Volume: 149
• Category: Article
• ISSN: 0003-2697
• DOI: 10.1016/0003-2697(85)90583-4

✦ Synopsis

A sensitive radiochemical assay is described for the assay ofacetoacetyl-CoA synthetase activity in cytosolic extracts. Enzyme activity is measured by the incorporation of '% from acetoacetate into acetyl camitine as mediated by acetoacetyl-CoA synthetase, endogenous acetoacetyl-CoA thiolase, and exogenous camitine acetyl transferase. Separation of "C-labeled reactants from "C-labeled acetyl carnitine is achieved by cation-exchange chromatography. The assay is sensitive with less than 10 pmol of product readily detected. Acetoacetyl-CoA synthetase activity was measured in human fibroblasts, 0.12 nmol min-' mg cytosolic protein-', and was found to be more than two orders of magnitude below the activity level of acetoacetyl-CoA synthetase in rat liver cytosol, 18.4 nmol mitt-' mg cytosolic protein -I. An HPLC method is also described for the purification of [3-'Qacetoacetate.