Partial purification of active delta and epsilon subunits of the membrane ATPase from Escherichia coli

J. B. Smith, P. C. Sternweis, L. A. Heppel

Research output: Chapter in Book/Report/Conference proceedingChapter

46 Scopus citations

Abstract

The authors purified active delta and epsilon subunits of the E. coli membrane bound Mg2+ ATPase (ECF1). Treating purified ECF1 with 50% pyridine precipitates the major subunits (α, β, and γ) of the enzyme, but the two minor subunits (δ and ε), which are present in relatively small amounts, remain in solution. The delta and epsilon subunits were then resolved from one another by anion exchange chromatography. The partially purified epsilon strongly inhibits the hydrolytic activity of ECF1. The epsilon fraction inhibits both the highly purified five subunit ATPase and the enzyme deficient in the δ subunit. The latter result indicates that the delta subunit is not required for inhibition by epsilon. By contrast, two subunit enzyme, consisting chiefly of the α and β subunits, was insensitive to the ATPase inhibitor, suggesting that the γ subunit may be required for inhibition by epsilon. The partially purified delta subunit restored the capacity of ATPase deficient in delta to recombine with ATPase depleted membranes and to reconstitute ATP dependent transhydrogenase. Previously the authors reported that a fraction containing both the delta and epsilon subunits of ECF1 restored the capacity of ATPase missing delta to recombine with depleted membranes and to function as a coupling factor in oxidative phosphorylation and for the energized transhydrogenase. These reconstitution experiments using isolated subunits provide rather substantial evidence that the delta subunit is essential for attaching the ATPase to the membrane and that the epsilon subunit has a regulatory function as an inhibitor of the ATPase activity of ECF1.

Original languageEnglish (US)
Title of host publicationJournal of Supramolecular and Cellular Biochemistry
Pages248-255
Number of pages8
Volume3
Edition3
StatePublished - 1975

ASJC Scopus subject areas

  • Biochemistry

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    Smith, J. B., Sternweis, P. C., & Heppel, L. A. (1975). Partial purification of active delta and epsilon subunits of the membrane ATPase from Escherichia coli. In Journal of Supramolecular and Cellular Biochemistry (3 ed., Vol. 3, pp. 248-255)