Validity of the digitonin method for metabolite compartmentation in isolated hepatocytes

Abstract

1. A modification of the digitonin method of Zuurendonk & Tager (1974) (Biochim. Biophys. Acta 333, 393-399) (i.e. the ‘convaentional’ method) was developed that allows the fractionation of isolated hepatocytes at −5 degrees C (i.e. ‘low-temperature’ method). 2. With respect to compartmentation of adenine nucleotides, glutamate and citrate, the two methods yielded very similar results. 3. In contrast, the mitochondrial amounts of aspartate and malate, as revealed by the low-temperature method, were about twice as high as those found by the conventional procedure. No change in the total cellular content occurred. 4. With n-butylmalonate and glisoxepid present in the conventional digitonin medium, significantly higher amounts of malate and aspartate respectively were found in the mitochondrial pellets. The results obtained by the low-temperature method, however, were not influenced by the these inhibitors. 5. It is concluded that under the conventional conditions of cell fractionation no appreciable redistribution of adenine nucleotides, glutamate and citrate occurs.