Proton transport inhibition

The electrochemical potential difFetence across the membrane, once established as a tesult of proton translocation, inhibits further transport of teducing equivalents through the respiratory chain unless discharged by back-translocation of protons across the membtane through the vectorial ATP synthase. This in turn depends on availability of ADP and Pj. 

The properties are as follows, (i) The activity of the protein (i.e. the inward transport of protons) is inhibited by ATP. (ii) The activity of the protein is increased by the presence of long-chain fatty acids, since they relieve the ATP inhibition, (iii) When mitochondria, isolated from brown adipose tissue, are incubated in the presence of fatty acids, there is a sharp increase in the rates of electron transfer, substrate utilisation and oxygen consumption, whereas the rate of ATP generation remains low. These studies indicate that the rate of proton transport, by the uncoupling protein, depends on the balance between the concentrations of ATP and fatty acids, (iv) In adipocytes isolated from brown adipose tissue, the rate of oxygen consumption (i.e. electron transfer) is increased in the presence of catecholamines. 

Note A problem in the interpretation of the amiloride inhibition of proton release is the amiloride inhibition of di-ferric transferrin and ferricyanide reduction (Sun et al., 1987). The question arises does inhibition of the exchanger inhibit electron transport or does inhibition of electron transport inhibit a H release not dependent on the exchanger Exchanger inhibition is most likely because of the Na+ dependence for part of the H release (Sun et al., 1988). As an alternative, proton transport may be necessary for electron transport (Stahl and Anst, 1993). 

Activation of the Na+/H+ exchanger is revealed by the characteristic inhibition by amiloride and N-substituted amilorides, lack of effect by benzamil, and specific requirement for external Na+ or Li+ with low activity with Cs+. With pineal and HeLa cells, the inhibitions indicate that a major part of the proton transport is based on activation of the exchanger (Table 2). 

A number of substances inhibit oxidative phosphorylation at specific locations. These may be divided into agents that affect electron transport, those that affect complex V, and those that collapse proton gradients (proton ionophores). Such substances have been used as research tools to unravel the complexities of these pathways, as poisons, and as antibiotics. Inhibition of electron transport inhibits phosphorylation, the extent of which depends on the location of the inhibition site. Thus, if complex I is inactivated, electron transport can still take place using FADH2 as an electron donor. The donor P/O ratio is then 2. 

The activated membrane-bound ATPase is functionally coupled to proton movements. Thus, a transmembrane pH gradient (acid inside) of a magnitude similar to that observed during light-induced coupled electron flow is developed during ATP hydrolysis. ATP hydrolysis is stimulated, while the coupled proton transport is inhibited, by the addition of uncouplers, indicating that the rate of ATP hydrolysis is also partially limited by the electrochemical gradient which it creates. Nevertheless, attempts to measure H /ATP ratios in this system yielded numbers much below the expected ratio of 3. 

The difference between omeprazole and SCH 28080 in their ability to inhibit gastric H /K -ATPase is dependent on their inhibition kinetics. In contrast to omeprazole, SCH 28080 competes with the high affinity K -site on the gastric H /K -ATPase. Its effect on Na /K -ATPase activity is much less pronounced in comparison with its effect on gastric H /K -ATPase activity [159, 160]. SCH 28080 is a protonatable weak base (pK = 5.6) which accumulates in acidic compartments in the same way as omeprazole on the lumenal, acidic side of the parietal cell membrane in a protonated form [161]. However, SCH 28080 is chemically stable and active by itself after protonation [162] and does not need an acid-induced transformation such as required by omeprazole-like irreversible inhibitors. Therefore, in proton transport studies, SCH 28080 inhibits the initial rate of HVK" -ATPase mediated H accumulation and the steady state proton concentration. This is in contrast to omeprazole, which first needs accumulation of acid within gastric vesicles to generate an interior of low pH to facilitate the acid-induced transformation prior to being able to inhibit the HVK -ATPase [163]. SCH 28080 binds to the lumenal side of H /K" -ATPase [161,... 

The authors further found that y-subunit rotation was blocked when the Fq complex was modified by dicyclohexylcarbodiimide (DCCD), the lipid-soluble carboxyl reagent that is known to inhibit proton transport by reacting with a carboxylate residue on one of the c-subunits in Fq (Glu in MFo Asp in EcFq). These results demonstrate that the reconstituted EcFi can rebind to Fq to form a functional, membrane-bound EcFo F complex and that y-subunit rotation in F, is functionally coupled to Fq. This is also consistent with the long-held notion of a long-range conformational interaction between Fq within the membrane and the catalytic nucleotide-binding site on the extrinsic F complex. 

The anion-sensitive ATPase can generally be inhibited by thiocyanate [4,5]. Since this anion inhibits gastric acid secretion, this finding has been used as an argument in favour of a role of the enzyme in proton transport. The concentration of thiocyanate required for maximal inhibition usually amounts to 5- 10 mM. The residual activity, which sometimes occurs, can be attributed to an anion-insensitive ATPase. The enzyme from brush border shows again a deviating behaviour in that it is relatively insensitive towards thiocyanate, the inhibition being less than 30% [17,37]. 

Other arguments against such a role are the fact that the enzyme is not activated, but is rather non-specifically inhibited by the binding of anions to a single anionbinding site [9] (see Section 2c). Moreover, the molar ratio between proton transport and ATP hydrolysis in lizard gastric mucosa lies between 0.06 and 0.17 [9], which is far below that of the 3 1 Na" /ATP ratio for (Na" +K )-ATPase and the 2 1 Ca /ATP ratio for (Ca " +Mg )-ATPase. 

Strong evidence for this assumption has been provided by Sachs and coworkers [58,66,78,87]. They found that the rate of proton uptake depends on the nature of the cation present and that the sequence of the stimulating effect of these cations is the same as for the ATPase reaction. The only exception is T1, which strongly stimulates the ATPase but inhibits proton transport [71]. The substrate specificity for the proton transport is also the same as for the (K +H" )-ATPase activity. Most inhibitors of the enzyme reaction, described in Section 3g, also inhibit the proton transport process. 

As illustrated in Figure 2.1b, ideal locations of Pt particles are at the true triple-phase boundary, highlighted by the big star. Catalyst particles with nonoptimal double-phase contacts are indicated by the smaller stars. Pt gas interfaces are inactive due to the inhibited access to protons. Bulky chunks of ionomer on the agglomerate surface build the percolating network for proton conduction in secondary pores. Only individual or loosely connected ionomer molecules seem to be able to penetrate the small primary pores. It is unlikely that they could sustain notable proton conductivity. They merely act as a binder. Proton transport inside agglomerates, thus, predominantly occurs via water-filled primary pores, toward Pt water interfaces. 

The hght-induced proton translocation by bacteriorhodopsin at the planar interface of octane/water [10,12,19,20] and in octane-water emulsions [64] has been studied. A retinotoxin thought to form a stable shift base with retinol in rhodopsin, inhibited the light-actived proton transport [64]. 

Lorentzon P, Jackson R, Wallmark B, et al. Inhibition of (H + K )-ATPase by omeprazole in isolated gastric vesicles requires proton transport. Biochim Biophys Acta 1987 897 41-51. 

Another important class of proteins that contain water channels are the aquaporins, which regulate the flow of water in and out of cells. They will let water through but not salts or other dissolved substances, and as such, they act as molecular water filters. Water transport occurs via a chain of nine hydrogen-bonded molecules (Fig. 6.13). But if this chain were to permit rapid transmembrane proton motion, that would disturb the delicate charge balance across the membrane. So aquaporin must somehow disrupt the potential proton wire that threads through it. The mechanism has been much debated, but it now seems that the inhibition of proton transport is dominated by electrostatic repulsion by positively charged groups in a narrow constriction in the middle of the pore [72]. 

The sulphenamides bind covalently to the H, K -ATPase in the secretory membranes, inhibiting the proton transport. 

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. 

Attempts to measure ATP-induced ApH formation in these reconstituted liposomes were unsuccessful (Dewey, Hammes, 1981 Shahak, Pick, 1983). We considered the possibility that residues of ammonium, carried over with the enzyme from the (NH4)2S04 percipitation step, were responsible for inhibiting the formation of ApH, by buffering the protons transported into the liposomes. This will inhibit the formation of ApH while facilitating Aifj formation. Indeed, removing the ammonium by passing the proteoliposomes through a Sephadex column preequilibrated with valinomycin and K, enabled us to measure ATP-dependent ApH formation in CFq-CFj proteoliposomes. 

Like the spinach enzyme, the pea ATPase is activated equally by Mg2+ and Mn2+ and hydrolyzes a broad range of nucleoside triphosphates, but not ADP, AMP, or monophosphorylated substrates. Although pea chloroplast envelope membranes have ADPase and pyrophosphatase activity, we conclude that the activities are distinct from the ATPase activity. The envelope ATPase differs from putative transport ATPases characterized in other plant membranes in that it is not inhibited by vanadate or DCCD, nor is it stimulated by potassium. However, a role for this activity in proton efflux and ion transport cannot be ruled out, because the envelope vesicles may be sufficiently leaky that protons and ions can diffuse freely across the membrane. This might limit any stimulatory effect of K+ and uncouplers. Evidence supporting a role for the ATPase in proton transport will depend on further characterization of the envelope vesicles, and/or purification and reconstitution of the ATPase into artificial lipid vesicles. 

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