Uncoupler nigericin

Using the protocol of titrations of qE against the ionophoric uncoupler nigericin in p>ea and spinach chloroplasts, and using 9-aminoacridine fluorescence as a monitor for the proton gradient, we have investigated some relationships between ApH, "redox state", and zeaxanthin content, in their effects up>on the magnitude of qE. 

The fact that the anions of the uncouplers are large, often aromatic, and therefore soluble in the lipid bilayer supports this interpretation the protonated uncouplers can diffuse into the mitochondria and the anion can diffuse back out. Mitochondria can also be uncoupled by a combination of ionophores, e.g., a mixture of valinomycin (Fig. 8-22), which carries K+ into the mitochondria, plus nigericin, which catalyzes an exchange of K+ (out) for H+ (in).172... 

Fig. 10.6. The effect of respiration and membrane potential (Ai )) on Cl permeation in brown adipose tissue mitochondria. When brown fat mitochondria were incubated in KCl in the presence of the ionophore, nigericin, they swelled (A, B). If a respiratory substrate (here G-3-P glycerol-3-phosphate) was added to the expanded mitochondria, they contracted, and this contraction ceased immediately and swelling was reintroduced if azide (NaNj) and an uncoupler (FCCP) were added (Fig. A). The passive halide ion permeability can be inhibited by GDP (cf.. Fig. 10.5), but respiration-driven contraction in KCl-expanded mitochondria was only partially inhibited by the presence of GDP (Fig. B) if again azide and uncoupler were added during the contraction, the mitochondria did not swell, indicating that the thermogenin channel was closed by GDP. This behaviour can partly be explained by the fact that the Cl permeation is driven by the membrane potential. Indeed, when, under similar conditions, the rate of contraction was plotted as a function of the membrane potential, it was seen that the rate was membrane potential dependent. It should, however, he noted that at low membrane potentials GDP nearly totally abolished the Cl permeation but when the membrane potential was increased above 30 mV, the inhibitory effect of GDP was apparently partially lost. The basis for this phenomenon is not understood it is not even known if there is a lower affinity of thermogenin for GDP in the energized membrane, as measurements of GDP affinities always refer to the non-energized situation. (Adapted from Nicholls et al. [27] (A, B) and Nicholls [94] (C).)...

A group of antibiotics (e.g., valinomycin, nigericin, and gramicidin A) transport cations across the cell membrane. Such agents, known as ionophores, are widely used to probe membrane structure and function. Ionophores uncouple oxidative phosphorylation. Valinomycin, a cyclic peptide (Figure 14-17), forms a lipid-soluble complex with K+ that readily passes through the inner membrane, whereas K+ by itself does not. In the valinomycin-K complex, hydrophobic groups, present on the outside, facilitate transport of the complex in the lipid environment ... 

NO3 > Br > Cl > I acetate isethionate. The same is true for the valinomycin-stimulated ATPase reaction. The vesicular gradient for was abohshed by a combination of a K -ionophore and a protonophore or a K - H exchange ionophore (nigericin). Under these circumstances the ATPase is uncoupled and does not generate proton transport. 

Many different protonophores, each with a different effectiveness, are available like 2,4-dinitrophenol (DNP) or 5-chloro-3-tert-butyl-2 -chloro-4 -nitro-salicyl-anilide (CCCP). Manipulation of the two components of the A/Ih can be effected with two potassium ionophores. Valinomycin increases the electrogenic permeability of a membrane for potassium and leads to the dissipation of the if a high concentration of potassium is present (more than 10 mM). Nigericin catalyzes an electroneutral potassium proton exchange and thus dissipates the A pH under the same conditions. In combination these two ionophores function as an uncoupler. 

It is a very interesting to know the role of electrical potential under conditions in which ApH across the thylakoid membrane is removed and electrical potential is not damaged, so as to eliminate the enhancement of iipH induced by a decline of electrical potential. Such a condition was obtained by the addition of Nig. or NHi Cl. The result was shown in Fig.5. Nigericin, at such concentration that electron transport is uncoupled from phosphorylation, enhanced the light-induced swelling of thylakoid membrane significantly. This effect was inhibited by Val. It could be deduced that in addition to the H accumulated inside the thylakoid membranes enhanced by decline of electrical potential, electrostatic repulsion between membranes... 

Effects of nigericin and NH Cl on PSP Md electron transport. At low concentrations, nigericin (<3xlO"°M) and NH Cl (<10 M) stimulated PSP in steady state obviously but had no such effects under jus flashes (Fig.la,b). At these concentrations (not uncoupling concentrations) NH Cl also enhanced coupling or basal electron transport (Fig.lc), nigericin had similar effect (data not shown). 

Since isolated thyleikoids may be partly uncoupled in dependence of the leaf material and the isolation procedure, an optimal activation of hydrolysis by uncouplers demands for the titration shown in Fig. 2. The nigericin concentration was varied from zero to 5 pM, the maximum activity was observed at 0.6 pM. When 125 pM dibucaine was added in addition to nigericin, the rates of hydrolysis increased up to about 0.6 pM nigericin, then declined. In the presence of dibucaine alone, hydrolysis increased. Chlorpromazine showed a similar effect. 

Table 1. Stimulation by uncoupler, ionophore and protonophore on the membrane bound chromatophore, chloroplast and mitochondrial PPase activities. 1.5 yM FCCP, 10 yg/ml valinomycin and 10 yg/ml nigericin were used for chromatophore activity. 5 yM valinomycin and 5 yM nigericin were used for chloroplast and mitochondrial activities. 1 yM FCCP - for mitochondrial PPase and 5 yM FCCP - for chloroplast PPase activity. The concentration of NaF, when added, was 10 mM. In the case of valinomycin and nigericin 15 mM KCl was added to the assay medium.

H/e ratio in dependence on the e-flow increase induced by addition of the uncoupling agents nigericin, FCCP, and gramicidin for actinic light of 100 Wm-2 intensity. 

Nigericin in the presence of Is a potent uncoupler of the steady-state photophosphorylation in chloroplasts. It is generally thought that in flashing light or in the initial period of illumination photophosphorylation is insensitive to nigericin. We observed that even very low concentrations of nigericin (10 nM) inhibited flash-induced ATP formation but did not suppress the dark hydrolysis of ATP and the concomitant increase of the ApH (Table 2). That confirms the necessity of a critical value of this ApH to observe the ATP synthesis. 

A number of other uncouplers like FCCP, DNP, gramicidin or valinomycin was tested but failed to show comparable effects. Also combinations of FCCP and valinomycin (that should mimic nigericin-induced exchange) did not induce... 

The steady state membrane potential is increased by low concentrations of methylamine (Giersch, 1981) and practically not affected by 10 nM nigericin (Ch. Giersch, unpublished). From the relative magnitude of the alterations of ApH and Ai it is evident that the pmf is decreased by low concentrations of amines. The decrease of the pmf is small with both types of uncouplers and does not exceed about 3-5 mV in the presence of lO nM nigericin or 0.5 mM methylamine. It is concluded that the stimulation of the rate of phosphorylation occurs at a diminished pmf. This conclusion is confirmed by the observation that lowering of the steady-state membrane potential by valinomycin/K does not affect the methylamine induced stimulation of phosphorylation (Giersch, 1981). 

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