Determination of overt carnitine palmitoyltransferase by reversed-phase high-performance liquid chromatography

Abstract A method for the determination of carnitine palmitoyltransferase I (CPT I; EC 2.3.1.19) in isolated rat liver mitochondria by reversed-phase high-performance liquid chromatography is described. Enzyme activity is assayed by direct determination of coenzyme A (CoA) released from palmitoyl-CoA within 60 min by a linear gradient system. CPT I in rat liver mitochondria can be assayed from only 30 μg of mitochondrial protein per millilitre of assay mixture. The changes in the kinetic parameters of CPT I, including K i for malonyl-CoA, resulting from the fasting-feeding cycle are also discussed.