Effect of mitochondrial protein concentration on the efficiency of outer membrane removal by the cholesterol-selective detergent digitonin

The effects of different mitochondrial protein concentrations on the efficiency of digitonin titration of the outer mitochondrial membrane were investigated in isolated rat liver mitochondria. Isolated mitochondria were subjected to treatment with digitonin concentrations ranging from 0 to 0.40 mg digitonin per mg protein. This digitonin concentration range was used in incubations containing 5 to 50 mg mitochondrial protein per ml. Significant differences in the efficiency of outer membrane removal by digitonin titration were noted at protein concentrations of less than 20 mg per ml. Estimation of the effective concentration of digitonin required to remove 50% of the outer membrane indicated that in general, as the protein concentration decreases, the amount of digitonin required to remove the outer membrane increases. Significant differences were also noted in the amount of digitonin needed for removal of 95% of the outer membrane between 5, 10 and 20 mg/ml with the effect of protein concentration disappearing above 20 mg/ml. No effect of protein concentration was found on the disruption of the inner membrane by digitonin as judged by leakage of matrix marker enzyme activity and by release of inner membrane marker enzyme activity. The conclusions of these studies indicate that at relatively low mitochondrial protein concentrations (< 20 mg/ml), the efficiency of digitonin in removing the outer membrane is substantially reduced.