The proton adenosine triphosphatase complex of rat liver mitochondria. Temperature-dependent dissociation-reassociation of the F1-ATPase subunits

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Abstract
The soluble F1 moiety of the rat liver mitochondrial proton ATPase dissociates into 2 easily separable fractions when cold treated and then warmed. One fraction is soluble in potassium phosphate buffer, pH 7.4, whereas the other is insoluble. Neither of these 2 fractions alone can catalyze ATP hydrolysis under assay conditions optimal for the native F1-ATPase. The insoluble fraction when resolved via sodium dodecyl sulfate-polyacrylamide gel electrophoresis is composed of only .alpha. and .gamma. subunits. When this fraction is chromatographed on Sephadex G-75, it is resolved into an .alpha..gamma. complex and into the .alpha. subunit alone. The soluble fraction when resolved in the same electrophoretic system contains the remaining subunits, .beta., .delta., .epsilon. and some .gamma.. This fraction is resolved into 2 major components by chromatography on Sepharose CL-6B, a .beta..gamma. complex and .beta. subunit alone. The cold-dissociated enzyme can be readily associated when the temperature is raised to 20.degree. C. In the presence of either ATP or MgATP the enzyme completely regains its original ATPase specific activity. In contrast, Mg2+ is only .apprx. 15% effective in restoring ATPase activity. The results presented here define conditions for the dissociation and reassociation of the major subunits comprising the F1-ATPase of rat liver and thus provide a unique system among mammalian enzymes for testing the function of individual subunits. In addition, they strongly indicate that neither the .alpha. nor .beta. subunits, nor complexes of these subunits with the .gamma. subunit, are capable of catalyzing ATP hydrolysis. Finally, they provide evidence that the .gamma. subunit interacts with both the .alpha. and .beta. subunits. As the most likely subunit stoichiometry of the rat liver F1 is .alpha.3.beta.3.gamma..delta..epsilon., the .gamma. subunit may tag one of 3 .alpha..beta. pairs in the molecule. The possible significance of this unusual type of subunit interaction to the function of the mitochondrial proton ATPase is discussed.