Reconstitution of the malate/aspartate shuttle from mitochondria

The isolated aspartate/glutamate carrier and oxoglutarate carrier from mitochondria were coreconstituted into phospholipid vesicles. Reconstitution of the functionally active carrier proteins with high protein/lipid ratios was achieved by detergent removal on hydrophobic ion-exchange columns. A simplified version of the mitochondrial malate/aspartate shuttle was constructed by inclusion of glutamate-oxaloacetate transaminase and the substrates aspartate and oxaloacetate within the interior of the liposomes. Addition of external glutamate led to internal production of oxoglutarate which could be exchanged against externally added labeled malate. The reconstitution procedure was characterized with respect to the optimum ratio of reconstituted carrier proteins, the lipid concentration, and the concentration of internal substrates.