Determination of the mitochondrial protonmotive force in isolated hepatocytes

The intracellular distribution of lipophilic ions and weak acids and bases was studied in intact hepatocytes as indicators of gradients of pH and membrane potential. Mitochondrial and plasma membrane potentials of 161 and 32.8 mV (negative inside), respectively, were calculated from the intracellular accumulation of triphenylmethylphosphonium (TPMP+) and thiocyanate in the intact hepatocyte, assuming a two-compartment model. The predominantly mitochondrial localization of TPMP+ was confirmed using disruption procedures that selectively break the plasma membrane but not the mitochondrial membrane. The intracellular distribution of 5,5-dimethyloxazolidine-2,4-dione and methylamine was studied with similar techniques. A cytosolic pH of 6.95 to 7.00 and a mitochondrial delta pH of 0.97 units was observed. From these results, a total mitochondrial protonmotive force in the intact cell in excess of -200 mV was obtained.