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 e fficiency of digitonin titration of the outer mitochondrial membrane w ere investigated in isolated rat liver mitochondria. Isolated mitochon dria were subjected to treatment with digitonin concentrations ranging from 0 to 0.40 mg digitonin per mg protein. This digitonin concentrat ion range was used in incubations containing 5 to 50 mg mitochondrial protein per ml. Significant differences in the efficiency of outer mem brane removal by digitonin titration were noted at protein concentrati ons of less than 20 mg per ml. Estimation of the effective concentrati on of digitonin required to remove 50% of the outer membrane indicated that in general, as the protein concentration decreases, the amount o f digitonin required to remove the outer membrane increases. Significa nt differences were also noted in the amount of digitonin needed for r emoval of 95% of the outer membrane between 5, 10 and 20 mg/ml with th e effect of protein concentration disappearing above 20 mg/ml. No effe ct of protein concentration was found on the disruption of the inner m embrane by digitonin as judged by leakage of matrix marker enzyme acti vity and by release of inner membrane marker enzyme activity. The conc lusions 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.