Transfer of C2-Units Across the Mitochondrial Membrane Direct Recording of Citrate, Acetate and Acetoacetate Production Rates

Abstract

Methods are described for the direct optical assay of citrate, acetate, and acetoacetate production by isolated, incubated rat liver mitochondria. Each metabolite is converted into acetyl-CoA, using ATP:citrate lyase [EC 4.1.3.8] or acetyl-CoA synthetase [EC 6.2.1.1] or acetoacetyl-CoA synthetase and acetyl-CoA acetyltransferase [EC 2.3.1.9], respectively. Arylamine acetyltransferase [EC 2.3.1.5] acts as an auxiliary enzyme. Isolated rat liver mitochondria produce citrate, acetate and acetoacetate and production rates are stimulated by pyruvate and hexanoate. These 3 products might contribute to the transport of acetyl units across the mitochondrial membrane and thus serve as precursors in fatty acid synthesis. The rate of acetyl transfer does not seem to be rate-limiting with regard to the overall process of fatty acid synthesis from carbohydrates.