Radiometric assay of citrate condensing enzyme (citrate synthetase) in liver tissue and mitochondria.

Citrate synthetase activity was determined in samples of commercially available citrate condensing enzyme. We measured the amount of radioactive citrate formed from the condensation of carbon-14 labeled acetyl CoA and oxaloacetate. The labeled citrate was oxidized by oxidative bromination; 14C02 evolved was collected and represented the amount of citrate synthesized. Enzyme activity as low as 1O tmol/min was measured reproducibly with this procedure. Results obtained with this assay compared closely with those of a widely used spectrophotometric assay in which ElIman’s SH-coupling reagent [5,5-dithio-bis(2-nitrobenzoate)] is coupled with C0A released during the reaction. Accuracy of labeled citrate determination in the presence of labeled acetate or pyruvate was excellent. The method can be used to determine citrate synthetase activity in skeletal muscle, liver, and isolated liver mitochondria, or to study the fate of labeled citrate injected into animals.