Oxidative Oligomycin

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.

Polarographic studies of a mitochondrial fraction from Hymenolepis diminuta showed that of four substrates tested, DL-glycerol-3-phosphate was the most rapidly oxidized, but the highest respiratory control ratio (1.7) was obtained with dl-isocitric acid. With isocitrate as substrate oxyclozanide at 1.61 nM stimulated O uptake and relieved oligomycin inhibition of adinosine diphosphate-stimulated respiration, but at concentrations above 2 pM progressively inhibited O uptake. Rafoxanide, niclosamide, 3,4,5-tribromo-salicylanilide, nitroxynil, resorantel, di-chlorophen, and 2,4-dinitrophenol exhibited effects similar to those of oxyclozanide on the respiration in cestode mitochondria. The relative potencies were compared and the possible mode of action discussed [38]. 

Oligomycin ADP phosphorylation Blocks phosphorylation of ADP. Does not inhibit uncoupled oxidations. 

Mitochondrial oligomycin-sensitive mg2+ATPase is thought to play a major role in oxidative phosphorylation (Boyer et al. 1977). It has been suggested that impairment of mitochondrial energy metabolism by chlordecone may contribute to the decreases in body weight observed following exposure to this chemical (Desaiah 1981). 

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. 

The ADP produced by the hydrolysis of ATP is continuously used up by added purified pyruvate kinase, which in the presence of phosphoenol pyruvate produces pyruvate and ATP (Fig. 3.8.6). Pyruvate is then utilized by added lactate dehydrogenase, which in the presence of NADH produces lactate and NAD+. Complex V activity is estimated from the rate of NADH oxidation at 340 nm (e 4870-M 1-cm 1 isosbestic point 380 nm), after subtracting the oligomycin-resistant activity. It should be kept in mind that oligomicyn sensitivity requires the preserved attachment of the Fr component of the enzyme to the membranous F0 component. The attachment is readily lost upon freeze-thaw cycles. Consequently, it is reasonable to measure the activity on fresh material only. 

Figure I. Effect of oligomycin (O), benzylamine-Jluorescamine compound ( ), and 2,4-dinitrophenoI (A)on oxidative phosphorylation. C = concentration of inhibitor in nmoles I mg ofprotein, R = initial rate of inhibited A TP synthesis expressed as percent of the uninhibited rate (110.4 nmoles of ATP min 1 mg-1 of protein).

ADP phosphorylation is tightly coupled to electron transport. Shutting down one shuts down the other. It is well known that if ADP phosphorylation is inhibited by such compounds as oligomycin, electron transport also ceases. If the proton gradient is broken by a proton ionophore, however, such as 2,4-dinitrophenol, electron transport resumes at a rapid pace and no phosphorylation takes place. Such proton ionophores are also termed "uncouplers" of electron transport and ADP phosphorylation. Under normal conditions, the factors limiting ATP production are the pH gradient across the inner mitochondrial membrane and the cellular ADP/ATP ratio. An increase in the proton gradient shuts down phosphorylation and electron transport, whereas an increase in the ADP/ATP ratio stimulates both. Stimulation of oxidative phosphorylation by increases in cellular ADP concentration is termed respiratory control. 

Fig. 10.2. The respiratory pattern of isolated brown fat mitochondria. A. When substrate (succinate) is added to brown fat mitochondria (here isolated from cold-acclimated or control guinea-pigs), they respire rapidly. Upon ADP addition the rate is initially increased (normal State 2-3 transition), but the ensuing State 4 rate is lower than State 2. Successive ADP additions result in a successively decreased State 4 rate. Numbers indicate respiratory rates in nmol oxygen-min -mg protein. (Adapted from Pedersen and Flatmark [93] for details see this paper.) B. The specific coupling effect of purine nucleotides (here ADP) can be demonstrated after addition of oligomycin so that the respiratory stimulation due to ATP synthesis is eliminated. Addition of the uncoupler FCCP results in a respiratory rate identical to that prior to ADP, indicating that the ADP effect is on coupling, and not due to inhibition of substrate oxidation. (Adapted from Cannon et al. [23] for details see this paper.)...

The spheres removed from SMPs do not support ATP synthesis but do hydrolyze ATP to ADP and phosphate. Thus, ATP synthesis is carried out by Fq/Fi—ATPase (ATP synthase). The subscript o in Fq indicates that it contains the site at which a potent antibiotic inhibitor, oligomycin, binds and inhibits oxidative phosphorylation. Oligomycin does not bind Fi-ATPase and does not inhibit ATP hydrolysis to ADP and phosphate. 

Oxidative Phosphorylation Antagonists, Oligomycin, and Antimycin Abolished the Hypoxic... 

Inhibition of the synthesis of ATP during oxidative phosphorylation by oligomycin is thought to be due to... 

The answer is d. (Murray, pp 123-148. Scriver, pp 2367-2424. Sack, pp 159-175. Wilson, pp 287-317.) Oligomycin inhibits mitochondrial ATPase and thus prevents phosphorylation of ADP to ATP It prevents utilization of energy derived from electron transport for the synthesis of ATP Oligomycin has no effect on coupling but blocks mitochondrial phosphorylation so that both oxidation and phosphorylation cease in its presence. 

Since sodium azide or oligomycin, inhibitors of mitochondrial oxidative phosphorylation, did not affect the Ca " release, it became generally accepted that the intracellular pool corresponds to the SR. 

The antibiotic oligomycin binds to a specific protein of the FO complex, blocks the flow of protons through the FO channel, and inhibits oxidative phosphorylation directly. 

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