A combination of methods for the preparation of highly intact mitoplasts from beef heart mitochondria

Beef heart mitochondria are noted for difficulty of fractionation compared to mitochondria from other tissues. A method that would give the desired efficiency while leaving the inner membrane intact did not seem to be available. Four variables, alone and in all possible combinations, were evaluated for their effectiveness in fractionation. These variables were treatment with digitonin, hypotonic swelling, phospholipase A2 predigestion, and addition of high concentrations of EDTA. The efficiency of fractionation was evaluated by electron microscopy and marker enzyme assays. Highly intact mitoplasts were obtained when the mitochondria were treated with digitonin together with intense hypotonic swelling, or moderate hypotonic swelling and phospholipase A2 predigestion, or EDTA addition and phospholipase predigestion. The enzyme GTP:AMP phosphotransferase was found to be located exclusively in the matrix compartment.