Changes in Endogenous Substrates of Isolated Rabbit Heart Mitochondria during Storage

Rabbit heart mitochondria were isolated in the presence of low concentrations (150 to 250 µm) of pyruvate-2- or -3-14C to label the endogenous substrates. Chromatographic analyses of protein-free filtrates of the mitochondria indicated that the principal labeled endogenous substrates were citrate, malate, glutamate, and alanine. During storage of the mitochondria under anoxic conditions at 0°, radioactive citrate disappeared while aspartate and succinate increased. Continual changes in the composition of the endogenous substrates occurred during storage. When air was supplied to the mitochondrial suspension, the changes were similar to, but more rapid than, those occurring under anoxic conditions. Under aerobic conditions, however, radioactive succinate did not appear. Changes in total citrate, alanine, glutamate, and aspartate paralleled the changes in their radioactivity. Although partially metabolized, the endogenous substrates were largely retained by the mitochondria during repeated washing. During storage, in addition to metabolism, partial loss of some substrates into the medium was evident. Possible relationships of the metabolism of the endogenous substrates to the metabolic properties of isolated mitochondria are discussed.