Characterization of the Purified Membrane Attachment (δ) Subunit of the Proton Translocating Adenosine Triphosphatase from Escherichia coli

Paul C. Sternweis, Jeffrey B. Smith

Research output: Contribution to journalArticle

69 Scopus citations

Abstract

Some of the physical and functional characteristics of the purified δ subunit obtained from the protontranslocating ATPase of Escherichia coli (ECF1) have been examined. The subunit has a molecular weight of about 18 500 as measured by sodium dodecyl sulfate electrophoresis and by sedimentation equilibrium either with or without 6 M guanidine hydrochloride, δ therefore exists as a monomer and the apparent high molecular weight of about 33 000 obtained from molecular sieve chromatography suggests that the protein is a rather elongated molecule with a calculated ƒ/ƒo of 1.4. Circular dichroism spectra indicate that δ has a high degree of secondary structure with an α-helix content of about 55- 70%. The amino acid composition was determined, attaches δ-deficient ECF1 to inverted membrane vesicles depleted of ECF1 and restores oxidative phosphorylation. About 1 part δ by weight in the presence of excess ECF1 lacking reconstituted depleted membranes to the same extent as 20 parts of completely reconstitutive ECF1. This indicates that only one δ of mol wt 18 500 is needed per functional ECF1 molecule of mol wt 370 000. δ associates rapidly with δ-deficient enzyme to yield a stable five-subunit complex which was separated from excess δ by molecular sieve chromatography and was fully active in reconstituting depleted membranes, δ has no effect on activities in depleted or partially reconstituted membrane vesicles and binds only poorly, if at all, to these membranes. It appears then that reconstitution with δ is an ordered process in which δ first combines with δ-deficient ECF1 to yield a complex which then binds to membranes.

Original languageEnglish (US)
Pages (from-to)4020-4025
Number of pages6
JournalBiochemistry
Volume16
Issue number18
DOIs
StatePublished - Sep 1 1977

ASJC Scopus subject areas

  • Biochemistry

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