Oxidative Amytal

Amytal (a barbiturate drug), rotenone (a plant product commonly used as an insecticide), and piericidin A (an antibiotic) inhibit electron flow from the Fe-S centers of Complex I to ubiquinone (Table 19-4) and therefore block the overall process of oxidative phosphorylation. 

The development by Chance of a dual wavelength spectrophotometer permitted easy observation of the state of oxidation or reduction of a given carrier within mitochondria.60 This technique, together with the study of specific inhibitors (some of which are indicated in Fig. 18-5 and Table 18-4), allowed some electron transport sequences to be assigned. For example, blockage with rotenone and amytal prevented reduction of the cytochrome system by NADH but allowed reduction by succinate and by other substrates having their own flavoprotein components in the chain. Artificial electron acceptors, some of which are shown in Table 18-5,... 

This scheme was supported and refined by examining the effects of specific inhibitors of individual steps in the electron-transport chain. If CO or CN was added in the presence of a reducing substrate and 02, all of the electron carriers became more reduced. This fits the idea that these inhibitors act at the end of the respiratory chain, preventing the transfer of electrons from cytochrome to 02. If amytal (a barbiturate) or rotenone (a plant toxin long used as a fish poison) was added instead, NAD+ and the flavin in NADH dehydrogenase were reduced, but the carriers downstream became oxidized. The antibiotic antimycin caused NAD+, flavins, and the b cytochromes to become more reduced, but cytochromes c, cx, a, and a3 all became more oxidized. The situation here is analogous to the construction of a dam across a stream When the gates are closed, the water level rises upstream from the dam, and falls downstream. The observation that antimycin did not inhibit reduction of UQ showed that the quinone fits into the chain upstream of cytochromes c, t i, a, and a3. 

It was shown by Strength et al. (261) that the oxidation of choline by a particulate preparation from rat liver was considerably enhanced upon addition of NAD. Others showed that choline oxidation by isolated rat liver mitochondria was completely inhibited by Amytal when oxygen, cytochrome c, ferricyanide, or methylene blue was the electron acceptor -264) Choline dehydrogenase activity of particles and soluble prepa-... 

D-10) Antimycin A, a fungal antibiotic, blocks oxidative phosphorylation at the step between cytochromes b and C. Rotenone, a toxic plant derivative used as a fish poison, and amytal, a baibituate sedative, both interrupt electron transport in the step between NADH and FMN. 

Oxidative phosphorylation is susceptible to inhibition at all stages of the process. Specific inhibitors of electron transport were invaluable in revealing the sequence of electron carriers in the respiratory chain. For example, rotenone and amytal block electron transfer in NADH-Q oxidoreductase and thereby prevent the utilization of NADH as a substrate (Figure 18.43). In contrast, electron flow resulting from the oxidation of succinate is unimpaired, because these electrons enter through QH2, beyond the block. AntimycinA interferes with electron flow from cytochrome h Q-cytochrome c... 

Numerous barbiturates and oral hypoglycemic sulfonyl-ureas also have aliphatic side chains that arc su.sceptible to oxidation. Note that the sedative hypnotic amobarbital (Amytal) undergoes extensive to - I oxidation to the corresponding 3 -hydroxylated metabolite.Other barbiturates, such as pentobarbital, thiamylal,and secobarbital," reportedly are metabolized by way of a and to - I oxidation. The ri-propyl side chain attached to the oral hypoglycemic agent chlorpropamide (Diabinc.se) undergoes extensive to -I hydroxylation to yield the secondary alcohol 2 -hydroxy-chlorpropamide as a major urinary metabolite in humans. " ... 

Rotenone is used experimentally as an inhibitor of mitochondrial respiration. It has little effect on the mitochondrial oxidation of succinate, but it powerfully inhibits all oxidations which operate via NADH dehydrogenase. The site of inhiUtion by rotenone has been located on the oxygen side of the nonheme iron of NADH dehydrogenase (see Respiratory chain). Piericidin and amytal appear to act at or very close to the same site. [W. W. Wainio The Mammalum Mitochondrial Respiratory Chain (Academic Press, 1970) T.P. Singer M. Gutman Adv. Enzmol. 34 (1971) 79-153 J.B.Harbome, T.J.Mabry H.Mabry eds. TTie Flavonoids (Chapman Hall, 1975)] rRNA ribosomal RNA (see Ribosomes). 

It is highly probable that the new compound plays a role in oxidative phosphorylation. It is not formed in the absence of succinate, and it can yield ATP from ADP in the presence of magnesium. Furthermore, the formation of the intermediate is inhibited by dinitro-phenol. Oligomycin has no effect on the formation of the intermediate but blocks the formation of ATP from the phosphorylated derivative. Amytal has an opposite effect it blocks the formation of the phosphorylated derivative but does not affect the transphosphorylation of ADP. 

Rotenone, the active ingredient of derris powder, an insecticide prepared from the roots of the leguminous plant Lonchocarpus nicou. It is an inhibitor of complex I (NADH — ubiquinone reduction). The same effect is seen in the presence of amytal (amobarbital), a barbiturate sedative drug, which again inhibits complex I. These two compounds inhibit oxidation of malate, which requires complex I, but not succinate, which reduces ubiquinone directly. The addition of the uncoupler... 

Oxidative phosphorylation may be inhibited at different stages by a variety of agents (Table 13.3) which have proved invaluable in its experimental investigation. Electron transport may be inhibited at a number of locations. Rotenone and amytal abrogate ATP synthesis driven by NADH-derived electrons but that initiated by FADHj continues. The sites of action of electron-transport inhibitors were identified by the crossover technique in which the carriers before the blockage become more reduced and those beyond more oxidized. 

Cyanide inhibits the action of one of the electron transfer chain proteins, but proton transport remains coupled to ATP production. Other inhibitors of oxidative phosphorylation are rotenone, amytal and carbon monoxide. 

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