Characterization of the inhibitory (e) subunit of the proton-translocating adenosine triphosphatase from escherichia colf

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Abstract

The inhibitory subunit (ε) of the F1 adenosine triphosphatase (ATPase) was purified to homogeneity from the ML 308-225 and K12(λ) strains of Escherichia coli. No tryptophan or cysteine was detected in the subunit from either strain. The highly active 6 from both strains was found to be a globular protein with a Stokes' radius of 18-19 A. Circular dichroism spectra suggested an α-helix content of ∼40%. The molecular weight of ε was ∼ 15 000-16 000 by sedimentation equilibrium centrifugation in the presence and absence of guanidinium hydrochloride, molecular sieve chromatography, and gel electrophoresis in the presence of sodium dodecyl sulfate and 8 M urea. The s20,w of ε was ∼ 1.6 s-1. Inhibition of the purified F1 ATPase by ε displayed noncompetitive kinetics with a Ki of ∼ 10 nM. The inhibition of the ATPase was rapidly reversed by diluting the enzyme-ε mixture. [125I]e which was incorporated into ECF1 was readily displaced by unlabeled ε. ε had no significant effect on the ATPase activity of "native" or reconstituted everted membrane vesicles under a variety of assay conditions. Combining the ε-inhibited F1 ATPase with its hydrophobic portion in everted membrane vesicles reconstituted the reversible proton-translocating ATPase and restored nearly full ATPase activity. These results suggest that ε inhibits the enzyme only when the F1 ATPase becomes detached from its hydrophobic subunits.

Original languageEnglish (US)
Pages (from-to)526-531
Number of pages6
JournalBiochemistry
Volume19
Issue number3
StatePublished - 1980

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Escherichia
Adenosine Triphosphatases
Protons
Membranes
Escherichia coli K12
Centrifugation
Guanidine
Molecular sieves
Enzymes
Circular Dichroism
Chromatography
Electrophoresis
Sedimentation
Tryptophan
Sodium Dodecyl Sulfate
Escherichia coli
Gel Chromatography
Cysteine
Urea
Assays

ASJC Scopus subject areas

  • Biochemistry

Cite this

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title = "Characterization of the inhibitory (e) subunit of the proton-translocating adenosine triphosphatase from escherichia colf",
abstract = "The inhibitory subunit (ε) of the F1 adenosine triphosphatase (ATPase) was purified to homogeneity from the ML 308-225 and K12(λ) strains of Escherichia coli. No tryptophan or cysteine was detected in the subunit from either strain. The highly active 6 from both strains was found to be a globular protein with a Stokes' radius of 18-19 A. Circular dichroism spectra suggested an α-helix content of ∼40{\%}. The molecular weight of ε was ∼ 15 000-16 000 by sedimentation equilibrium centrifugation in the presence and absence of guanidinium hydrochloride, molecular sieve chromatography, and gel electrophoresis in the presence of sodium dodecyl sulfate and 8 M urea. The s20,w of ε was ∼ 1.6 s-1. Inhibition of the purified F1 ATPase by ε displayed noncompetitive kinetics with a Ki of ∼ 10 nM. The inhibition of the ATPase was rapidly reversed by diluting the enzyme-ε mixture. [125I]e which was incorporated into ECF1 was readily displaced by unlabeled ε. ε had no significant effect on the ATPase activity of {"}native{"} or reconstituted everted membrane vesicles under a variety of assay conditions. Combining the ε-inhibited F1 ATPase with its hydrophobic portion in everted membrane vesicles reconstituted the reversible proton-translocating ATPase and restored nearly full ATPase activity. These results suggest that ε inhibits the enzyme only when the F1 ATPase becomes detached from its hydrophobic subunits.",
author = "Sternweis, {Paul C.}",
year = "1980",
language = "English (US)",
volume = "19",
pages = "526--531",
journal = "Biochemistry",
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T1 - Characterization of the inhibitory (e) subunit of the proton-translocating adenosine triphosphatase from escherichia colf

AU - Sternweis, Paul C.

PY - 1980

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N2 - The inhibitory subunit (ε) of the F1 adenosine triphosphatase (ATPase) was purified to homogeneity from the ML 308-225 and K12(λ) strains of Escherichia coli. No tryptophan or cysteine was detected in the subunit from either strain. The highly active 6 from both strains was found to be a globular protein with a Stokes' radius of 18-19 A. Circular dichroism spectra suggested an α-helix content of ∼40%. The molecular weight of ε was ∼ 15 000-16 000 by sedimentation equilibrium centrifugation in the presence and absence of guanidinium hydrochloride, molecular sieve chromatography, and gel electrophoresis in the presence of sodium dodecyl sulfate and 8 M urea. The s20,w of ε was ∼ 1.6 s-1. Inhibition of the purified F1 ATPase by ε displayed noncompetitive kinetics with a Ki of ∼ 10 nM. The inhibition of the ATPase was rapidly reversed by diluting the enzyme-ε mixture. [125I]e which was incorporated into ECF1 was readily displaced by unlabeled ε. ε had no significant effect on the ATPase activity of "native" or reconstituted everted membrane vesicles under a variety of assay conditions. Combining the ε-inhibited F1 ATPase with its hydrophobic portion in everted membrane vesicles reconstituted the reversible proton-translocating ATPase and restored nearly full ATPase activity. These results suggest that ε inhibits the enzyme only when the F1 ATPase becomes detached from its hydrophobic subunits.

AB - The inhibitory subunit (ε) of the F1 adenosine triphosphatase (ATPase) was purified to homogeneity from the ML 308-225 and K12(λ) strains of Escherichia coli. No tryptophan or cysteine was detected in the subunit from either strain. The highly active 6 from both strains was found to be a globular protein with a Stokes' radius of 18-19 A. Circular dichroism spectra suggested an α-helix content of ∼40%. The molecular weight of ε was ∼ 15 000-16 000 by sedimentation equilibrium centrifugation in the presence and absence of guanidinium hydrochloride, molecular sieve chromatography, and gel electrophoresis in the presence of sodium dodecyl sulfate and 8 M urea. The s20,w of ε was ∼ 1.6 s-1. Inhibition of the purified F1 ATPase by ε displayed noncompetitive kinetics with a Ki of ∼ 10 nM. The inhibition of the ATPase was rapidly reversed by diluting the enzyme-ε mixture. [125I]e which was incorporated into ECF1 was readily displaced by unlabeled ε. ε had no significant effect on the ATPase activity of "native" or reconstituted everted membrane vesicles under a variety of assay conditions. Combining the ε-inhibited F1 ATPase with its hydrophobic portion in everted membrane vesicles reconstituted the reversible proton-translocating ATPase and restored nearly full ATPase activity. These results suggest that ε inhibits the enzyme only when the F1 ATPase becomes detached from its hydrophobic subunits.

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