Mitochondrial transport, inhibitors

In mitochondria, the allelochemlcals acted primarily as electron transport inhibitors. Malate oxidation was more sensitive than either succinate or NADH oxidation. No evidence for interaction with a specific membrane complex was obtained. Instead, Inhibition of substrate oxidation seems to result from alterations and perturbations produced in the inner membrane as reflected in interference with the behavior of transport processes. The compounds did not act as uncouplers or directly inhibit ATP synthesis. However, naringenin, some of the flavones, and the cinnamic acids dj inhibit the hydrolysis of ATP catalyzed by mitochondrial Mg -ATPase. 

The development of toxic symptoms on plants treated with pure electron transport inhibitors, such as simazine, diuron, and the uracils, can be prevented if the plants are supplied exogenously with a respirable carbohydrate (2). This observation suggests that the glycolytic or the mitochondrial system can provide sufficient energy to prevent the appearance of phyto-... 

TOXICANT-INDUCED MITOCHONDRIAL APOPTOSIS/NECROSIS 17.4.1 Electron Transport Inhibitors... 

Simple stilbenes (Phe-CH=CH-Phe) include the Vitis vinifera (grape) (Vitaceae) cytotoxic resveratrol (4,3, 5 -trihydroxystilbene), the mitochondrial electron transport inhibitor oxyresveratrol (3,5,2, 4 -tetrahydroxystilbene) and the protein kinase inhibitor piceatannol (3,4,3, 5 -tetrahydroxystilbene), all these compounds having antifungal activity. The isoprenylated stilbene chlorophorin (4-geranyl-3,5,2, 4 -tetrahydroxystilbene) is an antioxidant free radical scavenger (AO/FRS). 

Figure 18.38 Structure of mitochondrial transporters. The structure of the ATP-ADP translocase is shown. Notice that this structure comprises three similar units (shown in red, biue, and yellow) that come together to form a binding site, here occupied by an inhibitor of this transporter. Other members of the mitochondrial transporter family adopt similar tripartite structures. [Drawn from lOKC.pdb.]...

The electron transport inhibitors do not directly affect photophosphorylation or interfere with mitochondrial electron transport and phosphorylation ( 1), However, the inhibitory uncouplers, in addition to interfering with electron transport in thylakoids, uncouple photophosphorylation and oxidative phosphorylation, and inhibit mitochondrial electron transport. 

For most mitochondrial transport systems inhibitors are available and these have been very useful in studies with isolated mitochondria, not only in the study of their kinetic properties but also for the isolation of some of the translocator proteins. In addition, some of the inhibitors can be used in studying the role of mitochondrial transport systems in metabolic processes as they occur in the intact cell. Fig. 1 gives a list of inhibitors commonly used. For a more complete list the reader is referred to [2,3,5-7]. 

Secondly, the transport inhibitor must be able to pass the cell membrane. The inability of benzene-1,2,3-tricarboxylate to inhibit gluconeogenesis from lactate in perfused pigeon liver, a tissue in which mitochondrial efflux of phosphoenolpyruvate is obligatory for glucose synthesis, is presumably due to lack of penetration through the plasma membrane [16], This observation is interesting since it shows that the ability of an inhibitor to penetrate the cell membrane may vary from tissue to tissue benzene-1,2,3-tricarboxylate does inhibit lipogenesis in hepatocytes of neonatal chicks, as discussed above. Another example is the apparent relative impermeability of the plasma membrane of isolated foetal rat hepatocytes, as compared with that from adult rats, for atractyloside, the inhibitor of the adenine nucleotide translocator [17]. 

An important question is whether mitochondrial pyruvate transport can regulate pyruvate metabolism. One way to approach this problem is to carry out careful titrations of pyruvate-dependent processes with the transport inhibitors. So far, such experiments have not been done. Inspection of the kinetic properties of the pyruvate translocator, however, shows that limitation of pyruvate metabolism by its transport into the mitochondria is possible. For liver the average reported is 70 nmol/min/mg mitochondrial protein, which is 900 jumol/g dry weight of liver tissue/h (Table 1). The maximum rate of glucose synthesis from lactate in hepato-cytes is about 430 jumol/g dry weight/h [86], so that flux through the pyruvate translocator under these conditions is 2 X 430 = 860 jumol/g dry weight/h, which is close to its In the presence of lactate plus ethanol mitochondrial pyruvate... 

Mitochondrial complex III electron transport inhibitors (Coupling site II)... 

Inhibition of chitin synthase (CS, EC 2.4.1.16) is widely recognized as a largely untapped, intrinsically manunalian-safe, arthropodicidal mechanism of action. Certain classes of cyanine dyes were observed as hits in a high-throughput Lepidopteran (tobacco budworm, Heliothis virescem) chitin synthase assay. Optimization produced in v/vo-active analogs however, ultimately these were shown to be off-target, active instead as mitochondrial electron transport inhibitors (METl) at Complex 1. An overview of the chitin synthase project and chemistry is presented. 

Synthetic analogs were evaluated for potency as mitochondrial electron transport inhibitors in a 96-well plate assay, based on oxidation of NADH by mitochondrial fragments and the associated reduction in absorbance at 340 nm. Table I shows that, based on I50 values, V is a potent mitochondrial inhibitor across a broad spectrum of fungal pathogens, including the important cereal... 

Abstract The transport mechanism of the bovine heart mitochondrial ADP/ATP carrier was studied using submitochondrial particles. The modifications of the cysteine residues of the carrier by the SH-reagents eosin-5-maleimide (EMA) and Af-ethyhnaleimide (NEM), and disulfide bond formation between these cysteine residues catalyzed by copper-o-phenanthroline (Cu(OP)2) under various conditions were studied. In particular, the effects of the transport inhibitors carboxyatractyloside (CATR) and bongkrekic acid (BKA), and fluorescein derivatives were... 

Considerable evidence now supports the hypothesis that the inner membrane of a rat liver mitochondrion contains a specific permease or carrier for Ca, which makes possible the inward transport of this cation from regions of low concentration in response to electrochemical gradients generated by electron transport. The carrier has a very hig affinity for Ca + and it also has the ability to transport Sr + and Mn + but not Mg +. Lehninger has discussed mitochondrial transport of calcium he has also shown " that La + is a specific inhibitor of the Ca + carrier and is therefore a potential probe for calcium transport. The dye Ruthenium Red also inhibits calcium transport and, since this dye reacts specifically with mucopolysaccharides, it has been concluded that the latter (in the form of mucoproteins or muco- or glyco-lipids) are at the active centre of the sites of mediation of mitochondrial Ca + transport. Fluorescence enhancement of 8-anilino-l-naphthalene has also been used as a probe for calcium transport. 

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. 

Mechanistic studies have shown that TBT and certain other forms of trialkyltin have two distinct modes of toxic action in vertebrates. On the one hand they act as inhibitors of oxidative phosphorylation in mitochondria (Aldridge and Street 1964). Inhibition is associated with repression of ATP synthesis, disturbance of ion transport across the mitochondrial membrane, and swelling of the membrane. Oxidative phosphorylation is a vital process in animals and plants, and so trialkyltin compounds act as wide-ranging biocides. Another mode of action involves the inhibition of forms of cytochrome P450, which was referred to earlier in connection with metabolism. This has been demonstrated in mammals, aquatic invertebrates and fish (Morcillo et al. 2004, Oberdorster 2002). TBTO has been shown to inhibit P450 activity in cells from various tissues of mammals, including liver, kidney, and small intestine mucosa, both in vivo and in vitro (Rosenberg and Drummond 1983, Environmental Health Criteria 116). 

The reaction of choline with mitochondrial bound acetylcoenzyme A is catalysed by the cytoplasmic enzyme choline acetyltransferase (ChAT) (see Fig. 6.1). ChAT itelf is synthesised in the rough endoplasmic reticulum of the cell body and transported to the axon terminal. Although the precise location of the synthesis of ACh is uncertain most of that formed is stored in vesicles. It appears that while ChAT is not saturated with either acetyl-CoA or choline its synthesising activity is limited by the actual availability of choline, i.e. its uptake into the nerve terminal. No inhibitors of ChAT itself have been developed but the rate of synthesis of ACh can, however, be inhibited by drugs like hemicholinium or triethylcholine, which compete for choline uptake into the nerve. 

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