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.

Fig. 4. E1-E2 reaction cycle of the Na,K-pump with four major occluded conformations and ping-pong sequential cation translocation. The phosphoforms can occlude Na" and dephosphoforms can occlude or Rb. Na and K without brackets are cations bound to an open form such that they can exchange with medium cations [Na ] or [K ] within brackets are occluded and prevented from exchanging with medium cations. It is proposed that release of Na cxt accompanies transition from EiP[3Na] to E2P[2Na], since the capacity for occlusion of Na in the ouabain-stabilized E2P form is lower than in the EjP form prepared by incubation with CrATP [29] or oligomycin [89].

It is generally accepted that Na ions can be occluded in E]P forms. Occlusion of SNa ions per EP has been demonstrated in chymotrypsin cleaved enzyme and in the Cr-ADP-EiP[3Na] complex [29]. Three Na ions can also be occluded per EP in a complex stabilized by oligomycin in the absence of Mg or phosphate [97] while a maximum of two Na ions are occluded per a subunit in the ouabain complex. 

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. 

McCarron We had 3 mM Mg-ATP in the pipette, and in addition to using the uncoupler, oligomycin was present which should prevent ATPase activity. 

Dawley) (GO) decreased oligomycin rated startle response ... 

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). 

Curtis LR. 1988. Chlordecone is a potent in vitro inhibitor of oligomycin-insensitive magnesium-ATPase of rat bile canaliculi-enriched fraction. J Biochem Toxicol 3(Winter) 321-328. 

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. 

Between 1955 and 1960 various sub-mitochondrial preparations were developed to give vesicles comprising only sealed inner mitochondrial membranes. Cooper and Lehninger used digitonin extraction Lardy and Kielley Bronk prepared sub-mitochondrial particles by sonication. At this time, too, Racker and his colleagues isolated Fq/F1 particles from mitochondria and showed that a separated FI particle behaved as an ATPase. The F0 portion had no enzymic properties but conferred oligomycin sensitivity on the FI ATPase. The orientation of these sub-mitochondrial vesicles (inside-out or vice-versa) was shown by the position in electron micrographs of the dense (FI) particles which in normal intact mitochondria project into the matrix and so define the surface of the inner mitochondrial membrane. 

The ATP synthase (EC3.6.1.34, complex V) that transports H"" is a complex molecular machine. The enzyme consists of two parts—a proton channel (Fq, for oligomycin-sensitive ) that is integrated into the membrane and a catalytic unit (Fi) that protrudes into the matrix. The Fo part consists of 12 membrane-spanning c-peptides and one a-subunit. The head of the Fi part is composed of three a and three p subunits, between which there are three active centers. The stem between Fo and Fi consists of one y and one e subunit. Two more polypeptides, b and 8, form a kind of stator, fixing the a and p subunits relative to the Fo part. 

B. The ATP synthase inhibitor oligomycin binds directly to the enzyme complex and plugs up the H channel, which blocks ATP formation. 

A number of cases are known in which the properties of an enzyme are markedly altered by interaction with a membrane. Of course, in some cases the normal function of an enzyme is destroyed when it is removed from the membrane. For example, the mitochondrial coupling factor cannot synthesize ATP when removed from the membrane, since coupling to a proton gradient is required. The portion of the coupling factor that is easily solubilized (F,) is an ATPase. The steady-state kinetic properties of this solubilized ATPase are appreciably changed when it is reconstituted with mitochondrial membranes The turnover numbers and pH dependencies are different the solubilized enzyme is strongly inhibited by ADP, whereas the reconstituted enzyme is not and the reconstituted enzyme is inhibited by oligomycin, whereas the solubilized enzyme is not. 

The oligomycin inhibition requires interaction of F, with other polypeptide chains of the coupling factor that are associated with the membrane used for reconstitution. In a sense this is quite analogous to allosteric enzymes, where regulation is achieved by modulation of inter-subunit interactions. 

One striking characteristic of the coupling ATPase of energy-transducing membranes, apart from the extraordinarily large number of different polypeptide subunits, is the existence of two different polypeptides involved in the response of the enzyme to the inhibitor oligomycin. One binds the inhibitor, the other, separated in space from the former by possibly as much as 10 to 15 A, confers oligomycin sensitivity to the entire enzyme complex. How could the transfer of information between these two polypeptide subunits and their concerted interaction with the ATPase proper be visualized ... 

The activity of complex V (ATP synthase) can be conveniently measured in the reverse direction, ATP hydrolysis with a coupled assay thereafter described [72] (Fig. 3.8.6). The use of oligomycin, a specific inhibitor of the enzyme, allows discrimination of the mitochondrial enzyme from any nonmitochondrial ATPases. 

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. 

Chemiosmotic theory readily explains the dependence of electron transfer on ATP synthesis in mitochondria. When the flow of protons into the matrix through the proton channel of ATP synthase is blocked (with oligomycin, for example), no path exists for the return of protons to the matrix, and the continued extrusion of protons driven by the activity of the respiratory chain generates a large proton gradient. The proton-motive force builds up until the cost (free energy) of pumping... 

ATP is synthesized. Addition of cyanide (CN ), which blocks electron transfer between cytochrome oxidase and 02, inhibits both respiration and ATP synthesis, (b) Mitochondria provided with succinate respire and synthesize ATP only when ADP and P, are added. Subsequent addition of venturicidin or oligomycin, inhibitors of ATP synthase, blocks both ATP synthesis and respiration. Dinitrophenol (DNP) is an uncoupler, allowing respiration to continue without ATP synthesis. 

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