Purification and reconstitution of the F1F0-ATP synthase from alkaliphilic bacillus firmus OF4: Evidence that the enzyme translocates H+ but not Na+

David B. Hicks, Terry A. Krulwich

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Abstract

The F1F0-ATP synthase from the alkaliphilic Bacillus firmus OF4 was purified in a reconstitutively active form, in good yield and with a high specific ATPase activity when appropriately activated. The purification procedure involved octyl glucoside extraction of washed membrane vesicles in the presence of 20% glycerol and asolectin followed by ammonium sulfate fractionation and sucrose density gradient centrifugation. The purified preparation was resolved into seven bands by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, corresponding to the five F1 subunits, α, β, γ, δ, and ε, and to the b and c subunits of the F0. Two-dimensional sodium dodecyl sulfate-polyacrylamide gel analysis revealed a candidate for the a subunit of F0. The MgATPase activity of B. firmus OF4 F1F0 was barely detectable but could be stimulated, optimally more than 100-fold, by sulfite, methanol, and octyl thioglucoside. The enzyme was inhibited by N,N′-dicyclohexylcarbodiimide and sodium azide, but not by aurovertin, an inhibitor of the F1 from Escherichia coli. The F1F0 reconstituted into proteoliposomes catalyzed ATPase activity, ATP-P1 exchange, and ATP-dependent ΔpH and Δψ formation. ATP hydrolysis was stimulated by protonophores while the other activities were abolished by protonophores. These activities were neither dependent on added sodium ions nor significantly affected by them. F1F0 proteoliposomes made from crude octyl glucoside extracts that also contained the Na+/H+ antiporter were shown to catalyze ATP-dependent Na+ uptake that was completely sensitive to carbonyl cyanide m-chlorophenylhydrazone; Na+ uptake activity was absent in proteoliposomes containing more purified F1F0 but lacking the Na+/H+ antiporter. These data show that the F1F0 translocates protons and does not substitute Na+ for H+ in energy coupling.

Original languageEnglish
Pages (from-to)20547-20554
Number of pages8
JournalJournal of Biological Chemistry
Volume265
Issue number33
StatePublished - 25 Nov 1990
Externally publishedYes

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