Isolation and characterization of an inhibitory subunit of the Mg2+-Ca2+-ATPase of Escherichia coli

Nieuwenhuis, F.J.R.M.; Bakkenist, A.R.J.

doi:10.1016/0005-2728(77)90057-3

Cited by 26 publications

(2 citation statements)

References 32 publications

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“…On the other hand, the amino acid composition of e differs markedly from that of the mitochondrial ATPase inhibitor which is composed of over 25% basic amino acids and lacks threonine, proline, and methionine (Brooks & Senior, 1971). Recently, Nieuwenhuis & Bakkenist (1977) reported the amino acid composition of the e subunit of the ATPase from the A428 strain of E. coli. The subunit was purified by sodium dodecyl sulfate gel electrophoresis, appeared to lack tyrosine, and had different ratios of other amino acids compared to e from the two strains we studied.…”

Section: Resultsmentioning

confidence: 99%

Characterization of the inhibitory (.epsilon.) subunit of the proton-translocating adenosine triphosphatase from Escherichia coli

Sternweis¹,

Smith²

1980

Biochemistry

130

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The inhibitory subunit (epsilon) of the F1 adenosine triphosphatase (ATPase) was purified to homogeneity from the ML 308-225 and K12 (lambda) strains of Escherichia coli. No tryptophan or cysteine was detected in the subunit from either strain. The highly active epsilon from both strains was found to be a globular protein with a Stokes' radius of 18--19 A. Circular dichroism spectra suggested an alpha-helix content of approximately 40%. The molecular weight of epsilon was approximately 15000--16000 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 epsilon was approximately 1.6 s-1. Inhibition of the purified F1 ATPase by epsilon displayed noncompetitive kinetics with a Ki of approximately 10 nM. The inhibition of the ATPase was rapidly reversed by diluting the enzyme--epsilon mixture. [125I]epsilon which was incorporated into ECF1 was readily displaced by unlabeled epsilon. epsilon had no significant effect on the ATPase activity of "native" or reconstituted everted membrane vesicles under a variety of assay conditions. Combining the epsilon-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 epsilon inhibits the enzyme only when the F1 ATPase becomes detached from its hydrophobic subunits.

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Section: Resultsmentioning

confidence: 99%

Characterization of the inhibitory (.epsilon.) subunit of the proton-translocating adenosine triphosphatase from Escherichia coli

Sternweis¹,

Smith²

1980

Biochemistry

130

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“…The pattern cross-linked products formed by reaction of EDAC or EEDQ of loss of ATPase activity of F[ with increasing amounts of with subunits of Fj, one of them, the 0y dimer, did not ac-(14C)PITC-IFi indicated that the ATPase activity is fully cumulate when IF] was added to F, prior to cross-linking, inhibited when 1 mol of IFt is bound to 1 mol of F,. As Ft e natural ATPase inhibitor (IFi),1 *discovered by Pullman & Monroy (1963) in preparations of beef heart mitochondrial ATPase, was later found in a number of H+-linked ATPases, namely, yeast mitochondrial ATPase (Satre et al, 1975; Landry & Goffeau, 1975;Ebner & Maier, 1977), liver mitochondrial ATPase (Chan & Barbour, 1976; Cintron & Pedersen, 1979), chloroplast ATPase (Nelson et al, 1972), and bacterial ATPase (Nieuwenhuis & Bakkenist, 1977; Smith & Sternweis, 1977). It has been shown that IF, plays a regulatory function in oxidative phosphorylation by controlling both the reverse flow of energy from ATP to ATP-driven reactions (Asami et al, 1970;Ernster et al, 1973;Van de Stadt et al, 1973;Van de Stadt & Van Dam, 1974) and the rate of ATP synthesis (Harris et al, 1979).…”

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confidence: 99%

Radiolabeling of natural adenosine triphosphatase inhibitor with phenyl[14C]isothiocyanate and study of its interaction with mitochondrial adenosine triphosphatase. Localization of inhibitor binding sites and stoichiometry of binding

Klein¹,

Satre²,

Dianoux³

et al. 1980

Biochemistry

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The natural ATPase inhibitor (IF1) from beef heart mitochondria was labeled with phenyl (14C)isothiocyanate [(14C)PITC]. Chemical labeling by (14C)PITC does not modify the inhibitory properties of IF1, provided the number of residues of (14C)PITC bound per molecule of IF1 is lower than five to six, which corresponds to the average labeling of roughly half of the available lysine residues in IF1. This partially labeled, fully active, IF1 was used to determine the binding stoichiometry of IF1 with respect to F1 and to localize the inhibitor binding sites in F1-ATPase. The pattern of loss of ATPase activity of F1 with increasing amounts ot (14C)PITC-IF1 indicated that the ATPase activity is fully inhibited when 1 mol of IF1 is bound to 1 mol of F1. As F1 contains at least 2 beta subunits, this points to a half-site reactivity of F1 with respect to IF1. Sites of interaction between (14C)PITC-IF1 and F1 subunits were investigated by the use of two cross-linking reagents which act as "zero length" cross-linkers, 1-ethyl-3-[(dimethylamino)propyl]carbodiimide (EDAC) and N-(ethoxycarbonyl)-2-ethoxydihydroquinoline (EEDQ); the products of cross-linking were analyzed by NaDodSO4-polyacrylamide gel electrophoresis. IF1 was found to bind preferentially to the beta subunit of F1. Among the cross-linked products formed by reaction of EDAC or EEDQ with subunits of F1, one of them, the beta gamma dimer, did not accumulate when IF1 was added to F1 prior to cross-linking.

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Structure-Function Relationships of Micrococcus lysodeikticus Membranes: A Bacterial Membrane Model System

Saltón

1980

Subcellular Biochemistry

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Isolation and characterization of an inhibitory subunit of the Mg2+-Ca2+-ATPase of Escherichia coli

Cited by 26 publications

References 32 publications

Characterization of the inhibitory (.epsilon.) subunit of the proton-translocating adenosine triphosphatase from Escherichia coli

Characterization of the inhibitory (.epsilon.) subunit of the proton-translocating adenosine triphosphatase from Escherichia coli

Radiolabeling of natural adenosine triphosphatase inhibitor with phenyl[14C]isothiocyanate and study of its interaction with mitochondrial adenosine triphosphatase. Localization of inhibitor binding sites and stoichiometry of binding

Structure-Function Relationships of Micrococcus lysodeikticus Membranes: A Bacterial Membrane Model System

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