2,4-Dinitrophenol, uncoupler

The most consistent and most powerful inhibitor of amino acid incorporation and of peptide and protein synthesis is 2,4-dinitrophenol. This is the strongest evidence in favor of the participation of high-energy phosphate bonds, because 2,4-dinitrophenol uncouples many (but not all) oxidation and phosphorylation reactions. " " The coupling of phos-... 

A. Pentachlorophenol and dinitrophenols uncouple oxidative phosphorylation in the mitochondria. Substrates are metabolized but the energy produced is dissipated as heat instead of producing adenosine triphosphate (ATP). The basal metabolic rate increases, placing increased demands on the cardiorespiratory system. Excess lactic add results from anaerobic glycolysis. 

Dinitrophenol uncouples oxidative phosphorylation by leaking protons Into the matrix... 

Whereas sodium participates in metabolism mainly by its cationic properties, potassium is more directly involved in metabolism. Potassium stimulates the activity of a specific enzyme— pyruvic kinase—and is required for the phosphorylation of fructose-1-phosphate to fructose-1,6-diphosphate. Similarly, potassium stimulates acetyl kinase activity. Many alterations in the bioenergetic pathways of the cell are accompanied by changes in the intracellular concentration of potassium. After insulin administration, some of the potassium of the extracellular fluid is transferred inside the cells. During oxidative phosphorylation, potassium accumulates inside the mitochondria, and dinitrophenol uncouples the ion penetration and the oxidation. 

Many inhibitors of substrate oxidations, substrate transport, electron transport, and ATP synthesis are known including many well-known toxins (see Sherratt, 1981 Harold, 1986 Nicholls and Ferguson, 1992). These are not discussed here except to mention specific uncouplers of oxidative phosphorylation. Classic uncouplers such as 2,4-dinitrophenol have protonated and unprotonated forms, both of which are lipid soluble and cross the inner mitochondrial membrane discharging the proton gradient. This prevents ATP synthesis and stimulates respiration. 

The action of uncouplers is to dissociate oxidation in the respiratory chain from phosphorylation. These compounds are toxic in vivo, causing respiration to become uncontrolled, since the rate is no longer limited by the concentration of ADP or Pj. The uncoupler that has been used most frequently is 2,4-dinitrophenol, but other compounds act in a similar manner. The antibiotic oligomycin completely blocks oxidation and phosphorylation by acting on a step in phosphorylation (Figures 12-7 and 12-8). 

Figure 12-8. Principles of the chemiosmotic theory of oxidative phosphorylation. The main proton circuit is created by the coupling of oxidation in the respiratory chain to proton translocation from the inside to the outside of the membrane, driven by the respiratory chain complexes I, III, and IV, each of which acts as a protonpump. Q, ubiquinone C, cytochrome c F Fq, protein subunits which utilize energy from the proton gradient to promote phosphorylation. Uncoupling agents such as dinitrophenol allow leakage of H" across the membrane, thus collapsing the electrochemical proton gradient. Oligomycin specifically blocks conduction of H" through Fq.

Uncouplers (eg, dinitrophenol) are amphipathic (Chapter 14) and increase the petmeabihty of the lipoid inner mitochondrial membrane to protons (Figure 12—8), thus teducing the electtochemical potential and shott-citcuiting the ATP synthase. In this way, oxidation can proceed without phosphotylation. 

Dinitrophenol is a member of the aromatic family of pesticides, many of which exhibit insecticide and fungicide activity. DNP is considered to be highly toxic to humans, with a lethal oral dose of 14 to 43mg/kg. Environmental exposure to DNP occurs primarily from pesticide runoff to water. DNP is used as a pesticide, wood preservative, and in the manufacture of dyes. DNP is an uncoupler, or has the ability to separate the flow of electrons and the pumping of ions for ATP synthesis. This means that the energy from electron transfer cannot be used for ATP synthesis [75,77]. The mechanism of action of DNP is believed to inhibit the formation of ATP by uncoupling oxidative phosphorylation. 

In this chapter, the voltammetric study of local anesthetics (procaine and related compounds) [14—16], antihistamines (doxylamine and related compounds) [17,22], and uncouplers (2,4-dinitrophenol and related compounds) [18] at nitrobenzene (NB]Uwater (W) and 1,2-dichloroethane (DCE)-water (W) interfaces is discussed. Potential step voltammetry (chronoamperometry) or normal pulse voltammetry (NPV) and potential sweep voltammetry or cyclic voltammetry (CV) have been employed. Theoretical equations of the half-wave potential vs. pH diagram are derived and applied to interpret the midpoint potential or half-wave potential vs. pH plots to evaluate physicochemical properties, including the partition coefficients and dissociation constants of the drugs. Voltammetric study of the kinetics of protonation of base (procaine) in aqueous solution is also discussed. Finally, application to structure-activity relationship and mode of action study will be discussed briefly. 

Mitochondria do three things oxidize substrates, consume oxygen, and make ATP. Uncouplers prevent the synthesis of ATP but do not inhibit oxygen consumption or substrate oxidation. Uncouplers work by destroying the pH gradient. The classic uncoupler is dinitrophenol (DNP). This phenol is a relatively strong acid and exists as the phenol and the phenolate anion. 

Sophisticated isotope experiments were also performed using H2180 (Mildred Cohn) and 32P, and various exchange reactions identified between ATP, ADP, and Pr Analysis of the mode of action of two inhibitors was also relevant. Dinitrophenol (DNP) uncoupled the association between oxidation and ATP generation (Lardy and Elvejhem, 1945 Loomis and Lipmann, 1948). Oligomycin inhibited reaction (ii) above, blocking the terminal phosphorylation to give ATP, but not apparently the formation of A C. 

Dinitrophenol (1 mM) and potassium cyanide (KCN) (5 mM) are used as uncouplers of oxidative phosphorylation from electron transport (105). 

Because the rate of the ETC increases, with no ATP synthesis, energy is released as heat. Important uncouplers include 2,4-dinitrophenol (2,4-DNP) and aspirin (and other salicylates). Brown adipose tissue contains a natural uncoupling protein (UCP, formerly called thernio-genin), which allows energy loss as heat to maintain a basal temperature around the kidneys, neck, breastplate, and scapulae in newborns. 

Answer C. The toxic agent (example, 2,4-dinitrophenol) would uncouple oxidative phosphorylation, leading to a fall in ATP levels, increased respiration, and increased substrate utilization. 

During the First World War, trinitrotoluene (abbreviated to TNT) was the explosive used in shells and bombs. It was noticed that many of the women who packed the explosive into shells suffered from fever and loss of body weight. In some cases the fever was fatal. The cause was not known. Some time later, it was discovered that a very similar chemical, namely dinitrophenol, was an uncoupling agent. The fever and loss of weight were due to uncoupling caused by TNT (i.e. causing a leak of protons back across the mitochondrial membrane). 

Toxicology. 2,4-Dinitrophenol (2,4-DNP) uncouples oxidative phosphorylation from electron transport, resulting in diminished production of ATP, with the energy dissipated as heat, which can lead to fatal hyperthermia. ... 

Moreover, uncoupHng experiments using Escherichia coli cells were described. An addition of glucose to stationary E. coli cells leads to an increase of fluorescence intensity in the region of NAD(P)H, because it is formed in the glycolysis. This increase was stopped abruptly by addition of 2,4 dinitrophenol (DNP), which effects a decrease in the fluorescence signal according to the theory of uncoupled oxidative phosphorylation. The dynamics of this process. 

Dinitrophenols had been used as insecticides since 1892 but it was not until the 1930s that their value as herbicides was discovered and 4,6-dinitro-o-cresol (DNOC) was introduced. The trouble with dinitrophenols was their toxicity to all living organisms that respire. Their mode of action is through the uncoupling of oxidative phosphorylation, an effect that leads to a rapid death of any organism that comes into contact with the chemical, including the operator. 

Depletion of ATP is caused by many toxic compounds, and this will result in a variety of biochemical changes. Although there are many ways for toxic compounds to cause a depletion of ATP in the cell, interference with mitochondrial oxidative phosphorylation is perhaps the most common. Thus, compounds, such as 2,4-dinitrophenol, which uncouple the production of ATP from the electron transport chain, will cause such an effect, but will also cause inhibition of electron transport or depletion of NADH. Excessive use of ATP or sequestration are other mechanisms, the latter being more fully described in relation to ethionine toxicity in chapter 7. Also, DNA damage, which causes the activation of poly(ADP-ribose) polymerase (PARP), may lead to ATP depletion (see below). A lack of ATP in the cell means that active transport into, out of, and within the cell is compromised or halted, with the result that the concentration of ions such as Na+, K+, and Ca2+ in particular compartments will change. Also, various synthetic biochemical processes such as protein synthesis, gluconeogenesis, and lipid synthesis will tend to be decreased. At the tissue level, this may mean that hepatocytes do not produce bile efficiently and proximal tubules do not actively reabsorb essential amino acids and glucose. 

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