Subunit specific antisera to the Escherichia coli ATP synthase: Effects on ATPase activity, energy transduction, and enzyme assembly

Jeffrey B. Smith, Paul C. Sternweis

Research output: Contribution to journalArticle

21 Scopus citations

Abstract

We obtained antisera to each of the five subunits (α, β, γ, δ, and ε{lunate}) of the F1 portion of the proton-translocating ATPase from Escherichia coli (ECF1). No cross-reaction between the antiserum to a given subunit and any of the other four subunits was observed by Ouchterlony immunodiffusion. The α antiserum reacted only with the denatured α chain. Antibodies to either subunit β or subunit γ inhibited the ATPase activity of the enzyme. The ATPase activity of the holoenzyme in the everted membrane vesicles was just as sensitive as purified ECF1 to inhibition by the anti-β or anti-γ serum. A prolonged digestion of ECF1 with trypsin removed intact γ from ECF1, but did not alter the sensitivity of the ATPase to inhibition by the anti-γ serum. Proteolytic fragments were isolated from the trypsinized enzyme. They gave an immunoprecipitation band with the anti-γ serum, but none of the other subunit antisera. The antiδ serum detached ECF1 from everted membrane vesicles and completely blocked both the ATP- and respiration-dependent pyridine nucleotide transhydrogenase, an energylinked membrane function. The δ antiserum had no effect on the ATPase activity of the ECF1. The e antiserum stimulated the ATPase activity of purified ECF1 as shown previously (P. P. Laget and J. B. Smith, Arch. Biochem. Biophys. 197, 83, 1979), but strongly inhibited the holoenzyme in membrane vesicles. The α antiserum completely blocked the ATP-driven transhydrogenase. The same antiserum maximally inhibited the respiratory chain-driven reaction by only 35%. These observations indicate that the antiserum selectively affected energy transduction mediated by the ATPase. The protonmotive force generated by substrate oxidation was probably not dissipated by the ε{lunate} antiserum. Adsorbing the δ or ε{lunate} antiserum with everted membrane vesicles selectively removed those antibodies that reacted with membrane-bound ATPase. The adsorbed sera still reacted strongly with purified ECF1, and prevented it from restoring ATP-dependent proton translocation in ECF1-depleted vesicles. Therefore, it appears that more of the δ and the ε{lunate} subunit is exposed in the purified ECF1 molecule than in the membrane-bound enzyme.

Original languageEnglish (US)
Pages (from-to)376-387
Number of pages12
JournalArchives of Biochemistry and Biophysics
Volume217
Issue number1
DOIs
StatePublished - Aug 1982

ASJC Scopus subject areas

  • Biophysics
  • Biochemistry
  • Molecular Biology

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