Reversal by uncouplers of oxidative phosphorylation and by Ca2+ of the inhibition of mitochondrial ATPase activity by the ATPase inhibitor protein of rat skeletal muscle.

Such treatment also released phospholipid.The specific activity of the purified inhibitor protein was determined to be 41,490.A monomer of about 6,500 daltons was present upon gel electrophoresis in sodium dodecyl sulfate and urea.Addition of 1 m~ eaZ+ to the electrophoresis system resulted in the formation of a dimeric form of the inhibitor protein.Much less of the dimer was present as Ca" was reduced to lo-' M, but dimer formation increased again as ea2+ was lowered to lo-' M in the presence of 1 m~ Mg2' but not in its absence.With 10 m~ MgZ' there was some dimer formed; none was formed at 1 m~ M 6 + .In conjunction with the biphasic pattern of inhibition of the ATPase complex by inhibitor protein, these data indicate that the dimer is the active form of the inhibitor protein.Similar results were obtained for the inhibitor protein of bovine heart purified by similar procedures.Dinitrophenol or carbonyl cyanide rn-chlorophenylhydrazone reversed inhibition of ATPase activity by the purified inhibitor protein.Reversal was observed at varying ea2+ concentrations.The ATPase content of submitochondrial particles of rat skeletal muscle was determined to be 0.78 nmol/mg of protein.It was then estimated that such particles contain between 3 and 5 nmol of active dimeric inhibitor protein/nmol of ATPase.These data indicate that stimulation of ATPase activity of the submitochondrial particles by classical uncouplers of oxidative phosphorylation or by Ca" is mediated by reversal of inhibitor protein activity.The separation of phosphorylation from oxidation in the energy-transducing membranes of mitochondria is known as uncoupling.Uncoupling is characterized by loss of respiratory control, loss of ATP synthesis, stimulation of ATPase activity, as well as other more subtle alterations (reviewed in Ref. 1).Uncoupling of mitochondria may result as a consequence of aging, injury, treatment with 2,4-dinitrophenol and similar agents (1, 2), or upon treatment with Ca2+ (reviewed in Ref.3).