Evidence for a naturally occurring ATPase-inhibitor in Escherichia coli

Nieuwenhuis, F.J.R.M.; Drift, Jos A.M. v.d.; Voet, Andries B.; Dam, K. Van

doi:10.1016/0005-2728(74)90192-3

Cited by 28 publications

(7 citation statements)

References 11 publications

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“…Trypsin cleavage of F, has been used before to examine the topology as well as the importance of individual subunits for the functioning of this ATPase [e.g., Nelson et al (1974), Neiuwenhuis et al (1974), Bragg and Hou (1975), Laget (1978), andDunn et al (1980)]. Our results confirm much of the previous work and extend the protease digestion method to an analysis of the topology of the intact ATP synthase complex.…”

Section: Discussionsupporting

confidence: 83%

“…A third general approach used to study the role of subunits in F] is protease digestion of the complex. It has been shown in several laboratories that protease digestion of Fi leads to activation of ATPase activity but that the proteolytically modified enzyme no longer binds to F0 (Nelson et al, 1974;Neiuwenhuis et al, 1974;Bragg & Hou, 1975;Laget, 1978;Dunn et al, 1980). These effects have been related to cleavage of the a, 8, and t subunits.…”

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confidence: 99%

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Structure-function relationships of the Escherichia coli ATP synthase probed by trypsin digestion

Gavilanes‐Ruíz¹,

Tommasino²,

Capaldi³

1988

Biochemistry

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Trypsin cleavage has been used to probe structure-function relationships of the Escherichia coli ATP synthase (ECF1F0). Trypsin cleaved all five subunits, alpha, beta, gamma, delta, and epsilon, in isolated ECF1. Cleavage of the alpha subunit involved the removal of the N-terminal 15 residues, the beta subunit was cleaved near the C-terminus, the gamma subunit was cleaved near Ser202, and the delta and epsilon subunits appeared to be cleaved at several sites to yield small peptide fragments. Trypsin cleavage of ECF1 enhanced the ATPase activity between 6- and 8-fold in different preparations, in a time course that followed the cleavage of the epsilon subunit. This removal of the epsilon subunit increased multisite ATPase activity but not unisite ATPase activity, showing that the inhibitory role of the epsilon subunit is due to an effect on cooperativity. The detergent lauryldimethylamine oxide was found to increase multisite catalysis and also increase unisite catalysis more than 2-fold. Prolonged trypsin cleavage left a highly active ATPase containing only the alpha and beta subunits along with two fragments of the gamma subunit. All of the subunits of ECF1 were cleaved by trypsin in preparations of ECF1F0 at the same sites as in isolated ECF1. Two subunits, the beta and epsilon subunits, were cleaved at the same rate in ECF1F0 as in ECF1 alone. The alpha, gamma, and delta subunits were cleaved significantly more slowly in ECF1F0.(ABSTRACT TRUNCATED AT 250 WORDS)

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Section: Discussionsupporting

confidence: 83%

mentioning

confidence: 99%

Structure-function relationships of the Escherichia coli ATP synthase probed by trypsin digestion

Gavilanes‐Ruíz¹,

Tommasino²,

Capaldi³

1988

Biochemistry

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“…Under conditions that IFj caused ~75% inhibition of ATPase activity of complex V, no more than 10% of the ATP-Pj exchange activity was inhibited. (Nelson et al, 1972), and Escherichia coli (Nienvenhuis et al, 1974; Smith & Sternweis, 1977; Laget & Smith, 1979). The mitochondrial IFj has been extensively studied by various investigators (Horstman & Racker, 1970;Brooks & Senior, 1971; Ernster et al, 1973; Van de Stadt et al, 1974;Gomez-Puyou et al, 1977;Ferguson et al, 1977;Harris et al, 1979; for review, see Ernster et al, 1979).…”

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confidence: 99%

Mitochondrial adenosinetriphosphatase inhibitor protein: reversible interaction with complex V (ATP synthetase complex)

Galante¹,

Wong²,

Hatefi³

1981

Biochemistry

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Mitochondrial ATPase inhibitor protein (IF1) reacts reversibly with complex V and inhibits up to 90% of its ATPase activity. Both the rate and extent of inhibition are pH and temperature dependent and increase as the pH is lowered from pH 8 tp 6.7 (the lowest pH examined) or as the temperature is increased from 4 to 36 degrees C. Nucleotide triphosphates plus Mg2+ ions are required for inhibition of complex V ATPase activity by IF1. In the presence of Mg2+ ions, the effectiveness order of nucleotides is ATP greater than ITP greater than GTP greater than UTP. Highly purified complex V, which requires added phospholipids for expressing ATPase and ATP-Pi exchange activities, cannot be inhibited by IF1 plust ATP-Mg2+ unless phospholipids are also added. This indicates that the active state of the enzyme is necessary for the IF1 effect to be manifested, because F1-ATPase, which does not contain nor require phospholipids for catalyzing ATP hydrolysis, can be inhibited by IF1 plus ATP-Mg2+ in the absence of added phospholipids. The IF1-inhibited complex V, but not IF1-inhibited F1-ATPase, can be reactivated by incubation at pH greater than 7.0 in the absence of ATP-Mg2+. The reactivation rate is pH dependent and is influenced by temperature and enzyme concentration. Complex V preparations contain small and variable amounts of IF1. This endogenous IF1 behaves the same as added IF1 with respect to conditions described above for inhibition and reactivation and can result in 25-50% inhibition in different complex V preparations. However, complex V lacking endogenous IF1 can be reconstituted from F0, F1, oligomycin sensitivity conferring protein, and phospholipids. Inhibition of this reconstituted preparation in the presence of ATP-Mg2+ depends entirely on addition of IF1. In general, the ATP-Pi exchange activity of complex V is more sensitive to the chemical inhibitors of F1-AtPase tha its ATPase activity. This is not so, however, for IF1. Under conditions that IF1 caused approximately 75% inhibition of ATPase activity of complex V, no more than 10% of the ATP-Pi exchange activity was inhibited.

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“…When added, the concentrations of the following were as specified unless indicated otherwise: 3 mm ATP (disodium salt from Sigma), 0.1 mm 2,4-dinitrophenql, 2 mM MgCl2, 2 ,ug/ml oligomycin, 4.4 mg/ml Triton X-100, 200 ,ug/ml trypsin (type III from bovine pancreas, Sigma), 20 ,g/ml trypsin inhibitor (type 1-S from soybean, Sigma).…”

Section: Methodsmentioning

confidence: 99%

“…Trypsin-induced ATPase has also been described for particles from spinach chloroplasts (30) and several bacteria including Azotobacter vinelandii (3) and Escherichia coli (20).…”

mentioning

confidence: 99%