Radiometric Assay of Citrate Condensing Enzyme (Citrate Synthetase) in Liver Tissue and Mitochondria

摘要

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; 14CO2 evolved was collected and represented the amount of citrate synthesized. Enzyme activity as low as 10-4 μmol/min was measured reproducibly with this procedure. Results obtained with this assay compared closely with those of a widely used spectrophotometric assay in which Ellman’s SH-coupling reagent [5,5-dithio-bis-(2-nitrobenzoate)] is coupled with CoA 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.