Resolution and Reconstitution of H⁺-ATPase Complex from Beef Heart Mitochondria

Abstract

Mitochondrial H⁺-ATPase complex, purified by the lysolecithin extraction procedure, has been resolved into a “membrane” (NaBr-F₀) and a “soluble” fraction by treatment with 3.5 M sodium bromide. The NaBr-F₀ fraction is completely devoid of p, 8, and e subunits of the F, ATPase and largely devoid of α and γ subunits of F₁, where F₀ is used to denote the membrane fraction and F₁, coupling factor 1. This is confirmed by complete loss of ATPase and P₁-ATP exchange activities. The addition of F₁ (400 μg · mg⁻¹ F₀) results in complete restoration of oligomycin sensitivity without any reduction in the F₁-ATPase activity. Presumably, this is due to release of ATPase inhibitor protein from the F₁-F₀ complex consequent to sodium bromide extraction. Restoration of Pf-ATP exchange and H⁺-pumping activities require coupling factor B in addition to FpATPase. The oligomycin-sensitive ATPase and 32P₁ATP exchange activities in reconstituted Fr F₀ have the same sensitivity to uncouplers and energy transfer inhibitors as in starting submitochondrial particles from the heavy layer of mitochondria and F₁-F₀ complex. The data suggest that the altered properties of NaBr-F₀ observed in other laboratories are probably inherent to their F₁F₀ preparations rather than to sodium bromide treatment itself.

The H⁺-ATPase (F₁-F₀) complex of all known prokaryotic (3, 8, 9, 10, 21, 32, 34) and eukaryotic (11, 26, 30, 33, 35–37) phosphorylating membranes contain two functionally and structurally distinct entities. The hydrophilic component F₁, composed of five unlike subunits, shows ATPase activity that is cold labile as well as uncoupler-and oligomycin-insensitive. The membrane-bound hydrophobic component F₀, having no energy-linked catalytic activity of its own, is indirectly assayed by its ability to regain oligomycin sensitive ATPase and P₁-ATP exchange activities on binding to F¹-ATPase (33). The purest preparations of bovine heart mitochondrial F₀ show seven or eight major components in polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate or SDS-PAGE (1, 2, 12, 14), ranging from 6 to 54 ku in molecular weight (12). The precise structure and polypeptide composition of mitochondrial Fo is not known.

The F₀ preparations from bovine heart reported so far have been derived from H⁺-ATPase preparations isolated in the presence of cholate and deoxycholate (11, 33, 36, 37). The ATPase and P₁-ATP exchange activity of the preparations so obtained are low, dependent upon additional phospholipids and coupling factors; they show altered sensitivity to energy transfer inhibitors as compared to submitochondrial particles from the heavy layer of the mitochondria or ETPh (1. 2, 12, 14, 29, 33). Recently, lysolecithin has been successfully employed to extract highly active H⁺-ATPase from beef (17, 19, 28) and pig (24) heart mitochondria. The beef heart H⁺-ATPase preparation has the same ratio of ATPase to PrATP exchange activity and apparently the same sensitivity to energy transfer inhibitors as submitochondrial particles (17). The present communication describes resolution of this F₁-F₀ preparation using sodium bromide (NaBr) and reconstitution of ATPase and Pr ATP exchange activities. The NaBr-F₀ prepared from this preparation shows no dependence on lipids, and the same or increased sensitivity to energy transfer inhibitors when reconstituted with F₁-ATPase. Furthermore, F₁ ATPase activity does not decrease on binding of F₁ to NaBr-F₀, even though the reconstituted ATPase activity is 99% sensitive to oligomy-cin and dicyclohexylcarbodiimide. These properties are in contrast to the properties of F₀ reported by other workers (12, 14).