33] Enzymatic determination of long-chain fatty acyl-CoA

Abstract

Publisher Summary This chapter discusses the enzymatic determination of long-chain fatty acyl-CoA. The method consists of two main steps: enzymatic hydrolysis of longchain acyl-CoA (fatty acyl-CoA + H 2 O → CoA + fatty acid) and subsequent determination of the CoA produced. Several enzymes have been reported to release CoA from long-chain acyl-CoA—that is, pigeon and rat liver fatty acid synthase, pig brain pahnityl-CoA deacylase, Escherichia coli palmityl thioesterases, rat liver microsome acyl-CoA hydrolase, and pancreatic lipase. Rat liver fatty acid synthase can be prepared easily in a highly pure form and offers a convenient basis for the assay of long-chain acyl-CoA. Fatty acid synthase hydrolyzes palmityl- and stearyl- and oleyl-CoA. The concentration of long-chain acyl-CoA in the samples to be hydrolyzed should be below 5 μ M to avoid errors resulting from micelle formation. This low concentration of long-chain CoA necessitates the use of a cycling technique to measure the concentration conveniently in a spectrophotometer. The CoA released from long-chain acyl-CoA is measured with an enzymatic cycling procedure using the reactions catalyzed by carnitine acetyltransferase [acetyl-CoA:carnitine O-acetyltransferase] (CAT) and citrate synthase (CS).