Oxidation State of Respiratory Carriers and the Mechanism of Oxidative Phosphorylation

Wadkins, Charles L.; Lehninger, Albert L.

doi:10.1021/ja01561a075

Cited by 25 publications

(19 citation statements)

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“…Discussion. The "recombination" of the DNP-insensitive soluble ATP-ADP exchange enzyme with phosphorylating digitonin particles to reconfer DNPsensitivity is fully consistent with and provides further support for a general mechanism of respiratory energy coupling postulated earlier: [2][3][4][5] (1) Carrier --X + P1 i P X + Carrier (2) P-X+± E ±P--E+X (3) P -E + ADP T ATP + E where Carrier '-X is the chemical species of the electron carrier in which is conserved oxido-reduction energy, and X and E are group-transferring enzymes. E is the enzyme catalyzing the ATP-ADP exchange reaction, which has another active site reactive with P -X.…”

supporting

confidence: 82%

“…6 The soluble form of the enzyme is completely insensitive to DNP. 2,6 These findings thus show that DNP-sensitivity is not intrinsic but is conferred on the ATP-ADP exchange in fresh mitochondria or membrane fragments because it is in functional equilibrium with an earlier enzymatic reaction of the respiratory energy coupling mechanism which is sensitive to DNP, from which it can be dissociated by aging, azide, or physical separation. This paper describes the restoration of DNP-sensitivity to the soluble, DNPinsensitive ATP-ADP exchange enzyme separated from rat liver mitochondria, by recombining it with mitochondrial membrane fragments prepared by the action of digitonin.7 Such "recombination," which is specific, indicates that the soluble ATP-ADP exchange enzyme we have isolated is a participant in the mechanism of respiratory energy-coupling and differentiates it from other mitochondrial enzymes catalyzing ATP-ADP exchanges2 which are not relevant to oxidative phosphorylation.…”

mentioning

confidence: 90%

“…'-5 The ATP-ADP exchange reaction is inhibited by dinitrophenol only in freshly prepared particles capable of phosphorylation; aging of the particles causes no loss of activity of the exchange but results in loss of its DNP-sensitivity. 2 Similarly, azide has no inhibitory effect on the ATP-ADP exchange, but abolishes its sensitivity to DNP. 2 The stability of the ATP-ADP exchange enzyme made possible its extraction in soluble form2 and its purification to a high degree.…”

mentioning

confidence: 99%

“…2 Similarly, azide has no inhibitory effect on the ATP-ADP exchange, but abolishes its sensitivity to DNP. 2 The stability of the ATP-ADP exchange enzyme made possible its extraction in soluble form2 and its purification to a high degree. 6 The soluble form of the enzyme is completely insensitive to DNP.…”

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confidence: 99%

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Reconstitution of the Dinitrophenol-Sensitive Atp-Adp Exchange Reaction of Oxidative Phosphorylation

Wadkins¹,

Lehninger²

1960

Proc. Natl. Acad. Sci. U.S.A.

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Intact rat liver mitochondrial as well as fragments of mitochondrial membranes capable of oxidative phosphorylation2 catalyze an exchange reaction between ATP and ADP which is inhibited by 2,4-dinitrophenol (DNP). The exchange enzyme has been postulated to catalyze the terminal reaction of oxidative phosphorylation by which ATP is formed.'-5 The ATP-ADP exchange reaction is inhibited by dinitrophenol only in freshly prepared particles capable of phosphorylation; aging of the particles causes no loss of activity of the exchange but results in loss of its DNP-sensitivity.2 Similarly, azide has no inhibitory effect on the ATP-ADP exchange, but abolishes its sensitivity to DNP.2 The stability of the ATP-ADP exchange enzyme made possible its extraction in soluble form2 and its purification to a high degree.6 The soluble form of the enzyme is completely insensitive to DNP.2,6These findings thus show that DNP-sensitivity is not intrinsic but is conferred on the ATP-ADP exchange in fresh mitochondria or membrane fragments because it is in functional equilibrium with an earlier enzymatic reaction of the respiratory energy coupling mechanism which is sensitive to DNP, from which it can be dissociated by aging, azide, or physical separation. This paper describes the restoration of DNP-sensitivity to the soluble, DNPinsensitive ATP-ADP exchange enzyme separated from rat liver mitochondria, by recombining it with mitochondrial membrane fragments prepared by the action of digitonin.7 Such "recombination," which is specific, indicates that the soluble ATP-ADP exchange enzyme we have isolated is a participant in the mechanism of respiratory energy-coupling and differentiates it from other mitochondrial enzymes catalyzing ATP-ADP exchanges2 which are not relevant to oxidative phosphorylation.Methods.-The ATP-ADP exchange rate was measured with p32-or C'4-labeled ADP using the paper chromatographic separation described before.2 Initial incorporation rates under conditions of substrate saturation were measured. Phosphorylating digitonin particles from rat liver mitochondria were prepared according to Devlin and Lehninger.7 Soluble ATP-ADP exchange enzyme was prepared according to Wadkins and Lehninger.2 The purification of the ATP-ADP ex-

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supporting

confidence: 82%

mentioning

confidence: 90%

mentioning

confidence: 99%

mentioning

confidence: 99%

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Reconstitution of the Dinitrophenol-Sensitive Atp-Adp Exchange Reaction of Oxidative Phosphorylation

Wadkins¹,

Lehninger²

1960

Proc. Natl. Acad. Sci. U.S.A.

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“…The ADP-ATP exchange reaction was investigated in the mast cell fraction by the procedure described by Wadkins and Lehninger (11). Each sample of reaction mixture contained 2 mm ATP, 2 mm ADP (0.5 11C), 154 mm NaCI, 1 mm CaC12i 10 mm Tris-HC1 (pH 7.3), 0.05 `,'o BSA and mast cells (0.4-0.6 mg protein 2-3 x 10' mast cells); the final volume was 0.5 ml.…”

Section: Methodsmentioning

confidence: 99%

Significance of Atp-Splitting Activity of Rat Peritoneal Mast Cells in the Histamine Release Induced by Exogenous Atp

Sugiyama

1971

Jpn.J.Pharmacol

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It has previously been reported that exogenous adenosine-5'-triphosphate (ATP) evokes a marked histamine release from isolated rat mast cells with accompanying morphologi cal changes including degranulation when Cat²⁺ is present in the medium (1, 2). Since it has been suggested that the energy requiring process is involved in the histamine release (3, 4), it seems to be reasonable to assume that ATP can be utilized as a source of energy supply in the process of histamine release induced by this compound, and that Ca²⁺ plays a role as a cofactor of ATPase of mast cell membrane. Diamant and Krüger (5, 6) and Diamant (7) MATERIALS AND METHODSPreparation of cell fractions. The peritoneal fluid of Wistar rats (250-300 g, males), obtained as described before (2), was placed gently over the two layers of gum arabic solu tions of different specific gravities, 1.060 and 1.070, which were dissolved in 0.9' NaC1 in a conical centrifugation tube. Centrifugation was made at 650 for 10 minutes.Mast cells were precipitated beneath the lower layer of gum arabic. They were collected with a pipette and used as the mast cell fraction. The other cells were concentrated in the interphase between the upper and lower layers. They were mononuclear cells, lym phocytes, granulocytes and a few mast cells, of which more than 80",0 were mononuclear cells and lymphocytes.Hereafter the suspension of these mixed cells is referred to as the macrophage fraction.The mast cells and macrophages (cells of macrophage frac tion) obtained from 10-15 rats were washed 3 times with 6 nil of the incubation medium described below and resuspended in the same medium and used for each experiment. The mast cells in samples were counted in a hemocytometer after staining with toluidine blue.The protein contents of the cell fraction were determined by the method of Lowry et al. (8).

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Mechanisms of Synthesis of Adenosine Triphosphate

Racker¹

1961

Advances in Enzymology - And Related Areas of Molecular Biology

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Oxidation State of Respiratory Carriers and the Mechanism of Oxidative Phosphorylation

Cited by 25 publications

References 2 publications

Reconstitution of the Dinitrophenol-Sensitive Atp-Adp Exchange Reaction of Oxidative Phosphorylation

Reconstitution of the Dinitrophenol-Sensitive Atp-Adp Exchange Reaction of Oxidative Phosphorylation

Significance of Atp-Splitting Activity of Rat Peritoneal Mast Cells in the Histamine Release Induced by Exogenous Atp

Mechanisms of Synthesis of Adenosine Triphosphate

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