Characterization of the purified membrane attachment (δ) subunit of the proton translocating adenosine triphosphatase from Escherichia coli

Paul C. Sternweis, Jeffrey B. Smith

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Abstract

Some of the physical and functional characteristics of the purified δ subunit obtained from the proton-translocating 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 f/f0 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. δ 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 δ 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
StatePublished - 1977

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Escherichia coli
Adenosine Triphosphatases
Protons
Membranes
Molecular sieves
Chromatography
Gel Chromatography
Molecular Weight
Molecular weight
Molecules
Oxidative Phosphorylation
Guanidine
Circular Dichroism
Electrophoresis
Sedimentation
Sodium Dodecyl Sulfate
Monomers
Amino Acids
Weights and Measures
Enzymes

ASJC Scopus subject areas

  • Biochemistry

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Characterization of the purified membrane attachment (δ) subunit of the proton translocating adenosine triphosphatase from Escherichia coli. / Sternweis, Paul C.; Smith, Jeffrey B.

In: Biochemistry, Vol. 16, No. 18, 1977, p. 4020-4025.

Research output: Contribution to journalArticle

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abstract = "Some of the physical and functional characteristics of the purified δ subunit obtained from the proton-translocating 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 f/f0 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. δ 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 δ 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.",
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N2 - Some of the physical and functional characteristics of the purified δ subunit obtained from the proton-translocating 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 f/f0 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. δ 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 δ 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.

AB - Some of the physical and functional characteristics of the purified δ subunit obtained from the proton-translocating 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 f/f0 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. δ 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 δ 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.

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