Selective disaggregation of the H+-translocating ATPase. Isolation of two discrete complexes of the rutamycin-insensitive ATPase differing in mitochondrial membrane-binding properties.

The H+-translocating ATPase from rat liver mitochondria can be disaggregated selectively to yield two distinct, stable complexes of the rutamycin-insensitive ATPase. The two ATPase complexes can be purified to homogeneity by zone sedimentation in a glycerol gradient. Based on their electrophoretic mobility in 5% polyacrylamide gels, the aggregates have been designated as type I (Rf = 0.49) ATPase and type II (Rf = 0.56) ATPase. These two complexes of the ATPase differ in ATP hydrolytic activity, in stability, in mobility on 5% polyacrylamide gel electrophoresis, in subunit composition, and in ability to reassociate with submitochondrial particles which are highly depleted in ATPase activity. The type II ATPase is similar to the F1-ATPase, but the type I ATPase contains a 26.5-kilodalton subunit not present in the type II enzyme. This 26.5-kilodalton subunit is equimolar with the gamma subunit of the ATPase (based on Coomassie blue dye binding); its presence seems to be correlated to the altered properties of the type I ATPase. Type I ATPase reconstitutes rutamycin-sensitive ATPase activity in submitochondrial particles treated with trypsin, urea, ammonia, and 1.5% silicotungstic acid. The type II ATPase does not reconstitute rutamycin-sensitive ATPase activity in these ATPase-depleted submitochondrial particles unless it is supplemented with the 26.5-kilodalton subunit isolated from the type I ATPase. The 26.5-kilodalton protein has thus been functionally identified as important for the binding of the ATPase to the membrane by providing a direct link to the membrane or by binding to the ATPase putting it in an appropriate conformation for binding.