Combination Of Valinomycin Research Articles

Binding of an intrinsic ATPase inhibitor to the interface between alpha- and beta-subunits of F1FoATPase upon de-energization of mitochondria.

Yeast mitochondrial F1FoATPase has three regulatory subunit proteins: ATPase inhibitor, 9K protein, and 15K protein. Mutant yeasts lacking one or more of these protein factors were constructed by gene disruption [Ichikawa, N. et al. (1990) J. Biol. Chem. 265, 6274-6278; Yoshida, Y. et al. (1990) Eur. J. Biochem. 193, 49-53]. Dissipation of the electrochemical potential of protons of the mitochondrial inner membrane by an uncoupler or by a combination of valinomycin and potassium ions induced ATP-hydrolyzing activity of F1FoATPase in mitochondria of all the mutants, as in those of wild-type cells. However, the ATPase activity was inactivated within a minute in normal mitochondria, but was not suppressed in inhibitor-deficient mitochondria, and in mitochondria lacking either 9K or 15K protein, the inactivation of ATPase was slow and incomplete. Covalent binding of inhibitor protein to the enzyme was achieved with a zero length cross-linker, EEDQ, in uncoupled normal mitochondria, in which the inhibitor linked directly to both the alpha- and beta-subunits. This result strongly suggests that the binding site of the inhibitor protein is located at the interface between the two subunits.

Organic cation transport by rat hepatocyte basolateral membrane vesicles.

Hepatocyte basolateral membrane possesses transport systems for mediated uptake of organic cations, the first step in the subsequent biliary excretion and/or metabolism of these compounds. The purpose of these studies was to evaluate potential mechanisms for transport of this class of solutes across this membrane by measuring 3H-labeled tetraethylammonium ([3H]TEA) transport into rat hepatocyte basolateral membrane vesicles. [3H]TEA uptake was stimulated by an outwardly directed proton gradient consistent with TEA-proton exchange. Proton gradient-stimulated [3H]TEA uptake was inhibited by quinidine and by the combination of valinomycin and carbonyl cyanide m-chlorophenylhydrazone (CCCP) but not by CCCP alone or by N1-methylnicotinamide (NMN). An outwardly directed TEA gradient also stimulated uptake of [3H]TEA with values at early time points exceeding those at equilibrium. This trans-stimulation or countertransport was saturable with an apparent Michaelis constant of 106 microM and maximal velocity of 434 pmol.mg-1.15 s-1. TEA countertransport was cis-inhibited by quinidine, cimetidine, and thiamine and by low temperature, but not by NMN. Thiamine was also capable of trans-stimulating [3H]TEA uptake. An outwardly directed potassium gradient enhanced and an inwardly directed potassium gradient reduced TEA countertransport but had no effect on [3H]TEA uptake occurring in the absence of other electrochemical driving forces. These studies indicate that there are at least two potential mechanisms in the hepatocyte basolateral membrane for transport of organic cations; organic cation-organic cation exchange (countertransport) and organic cation-proton exchange. Furthermore, the results are consistent with the existence of more than one transporter with different substrate affinities in each of these categories.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of monensins in on the receptor-mediated endocytosis of 125I-labelled IgG by guinea-pig yolk sac in vitro

The effects of the carboxylic ionophore, monensin, on the receptor-mediated binding and uptake of 125I-labelled IgG by the guinea-pig yolk sac have been studied in vitro. Exposure of tissue to 10 μm monensin resulted in a rapid inhibition of uptake which correlated with a time- and time-dependent loss of cell-surface receptor activity. Monensin appeared to bring about a change in receptor distribution since the lost activity could be detected after permeabilizing the tissue with saponin. Electron microscopic examination of monensin-treated tissue revealed that the apical plasma membrane of endoderms cell was depleted of coated and uncoated pits and that the apical cytoplasm contained numerous large vacuoles. Dilatation of the Golgi apparatus was also observed. Normal surface receptor activity and ultrastructural features could be largely recovered by removal of monensin. Recovery of receptor activity was unaffected by the presence of cycloheximide. These results are consistent with a model in which IgG receptors are recycled and in which monensin blocks this process by causing receptors to be trapped intracellularly. Ammonium chloride or a combination of valinomycin and carbonyl cyanide p-(trifluoromethoxy)-phenyllhydrazone also brought about a loss of surface IgGreceptors, lending support to the idea that inhibition of recycling was the result of pertubation of an intracellular acidification event and implying that passage through an acidic compartment may be improtant for correct receptor processing.

Energy coupling to ATP synthesis and pyridine nucleotide transhydrogenase in chromatophores from photosynthetic bacteria A ‘dual-consumer’ test for localised interactions with electron transport components

The rate of ATP synthesis and the rate of pyridine nucleotide transhydrogenase were recorded in parallel experiments in steady-state conditions following the onset of illumination in chromatophores of Rhodobacter capsulatus (formerly Rhodopseudomonas capsulata). The transhydrogenase rate was more resistant than the ATP synthesis rate to inhibition by antimycin A and myxothiazol, the uncoupler, carbonyl cyanide p-trifluoromethoxyphenylhydrazone and the combination of valinomycin with nigericin in the presence of K +. The relationship between the transhydrogenase rate and the ATP synthesis rate was not influenced by the nature of the inhibitor. These data suggest that the ATP synthase and the transhydrogenase are differentially regulated by Δp but not by local interactions with components of the electron transport chain.

DCCD-sensitive Na+-transport in the membrane vesicles of Halobacterium halobium.

The effects of N,N'-dicyclohexylcarbodiimide (DCCD) and various ionophores on light-induced 22Na+-transport were studied in right-side-out membrane vesicles from Halobacterium halobium R1M1. The light-induced Na+ efflux was inhibited at the same DCCD concentration (greater than 40 nmol/mg protein) as required for inhibition of the Na+-dependent membrane potential (delta phi) formation. This supports our previous indication that the DCCD-sensitive, Na+-dependent transformation of pH-gradient (delta pH) into delta phi is mediated by Na+/H+-antiporter (Murakami, N. and Konishi, T. (1985) J. Biochem. 98, 897-907). FCCP or a combination of valinomycin and triphenyltin (TPT) inhibits the light-induced Na+ efflux in accordance with the notion of protonmotive force (delta mu H+)-driven antiporter. However, a marked lag in initiation of the Na+ efflux occurred in the presence of valinomycin, TPMP+, or a small amount of FCCP, suggesting that a gating step is involved in the Na+ efflux. On the other hand, the delta pH-dissipating ionophore TPT did not cause the lag. A simultaneous determination of delta phi, delta pH, and Na+ efflux rate at the initial stage of illumination revealed that the antiporter is gated by delta phi rather than delta mu H+.

The effect of ionophores on the production of extracellular dextransucrase byLeuconostoc mesenteroides

The effects of several ionophores on the production of extracellular dextransucrase by Leuconostoc mesenteroides were examined at different pH values. Valinomycin severely inhibited extracellular dextransucrase production at culture pH values greater than 6.3 in complex medium containing 250 mM K+. The inhibitory action of valinomycin was a function of the culture pH as no effect on dextransucrase production could be demonstrated at pH 5.5. The ΔpH dissipating agent nigericin did not inhibit dextransucrase production at a culture pH of 6.4 but affected it severely as the pH decreased. Extracellular dextransucrase was not detected in cultures treated with a combination of valinomycin and nigericin. The results suggest a role for the total protonmotive force in the production of extracellular dextransucrase by L. mesenteroides.

Isolation of pepsinogen granules from rabbit gastric mucosa.

Pepsinogen granules were isolated from rabbit stomachs by a combination of differential and isopycnic gradient centrifugation. The isolation procedure utilized 1 M sucrose and alkaline pH to stabilize the granules. The isolated granules were shown to be 8.4-fold enriched in pepsinogen and free of mitochondria and microsome enzyme markers. In addition to pepsinogen, a cation-insensitive but anion-sensitive Mg2+-ATPase co-purified with the zymogen. The enzyme was unaffected by aurovertin, oligomycin, and ouabain, but inhibited by high concentrations of vanadate, N,N'-dicyclohexylcarbodiimide, and azide. The enzyme activity was stimulated by tetrachlorosalicylanilide and the combination of valinomycin and nigericin in K+-containing media. The similarities between this enzyme and other secretory granule ATPases are discussed.

Surface charge on thylakoid membranes: regulation of photosynthetic electron transfer in cyanobacteria

The establishment of the steady-state rate of photosynthetic O2 evolution by cells of Anabaena variabilis and other cyanobacteria was found to be preceded by a lag-phase the duration of which depended on the time of cell preincubation in the dark. Electron acceptors (benzoquinone, N,N,N′,N′-tetramethyl-p-phenylenediamine, 2,3,5,6-tetramethyl-p-phenylenediamine or 2,6-dichlorophenolindophenol) abolished the lag-phase as well as the inhibitory effect of cyanide on electron transfer. Mono-, di-and trivalent cations added to the cell suspension markedly reduced the lag-phase. As cation concentrations were increased, acceleration and subsequent deceleration of the O2 evolution rate were observed. The efficient concentrations of cations decreased as their valency increased. The lag-phase and the rate of photosynthetic O2 evolution by the blue-green algae are suggested to depend on the value of the membrane surface charge governing the electrostatic interaction between unidentified membrane-bound redox components. The combination of valinomycin and nigericin as well as gramicidin D enhanced the duration of the lagphase by deenergization of thylakoid membrane.

Subcellular localization of H+-ATPase from pumpkin hypocotyls (Cucurbita maxima L.) by membrane fractionation

A new method of preparing sealed vesicles from membrane fractions of pumpkin hypocotyls in ethanolamine-containing buffers was used to investigate the subcellular localization of H(+)-ATPase measured as nigericin-stimulated ATPase. In a fluorescence-quench assay, the H(+) pump was directly demonstrated. The H(+) pump was substrate-specific for Mg·ATP and 0.1 mM diethylstilbestrol completely prevented the development of a Δ pH. The presence of unsupecific phosphatase hampered the detection of nigericin-stimulated ATPase. Unspecific phosphatases could be demonstrated by comparing the broad substrate specificity of the hydrolytic activities of the fractions with the clear preference for Mg·ATP as the substrate for the proton pump. Inhibitor studies showed that neither orthovanadate nor molybdate are absolutely specific for ATPase or acid phosphatase, respectively. Diethylstilbestrol seemed to be a specific inhibitor of ATPase activity in fractions containing nigericin-stimulated ATPase, but it stimulated acid phosphatase which tended to obscure its effect on ATPase activity. Nigericin-stimulated ATPase had its optimum at pH 6.0 and the nigericin effect was K(+)-dependent. The combination of valinomycin and carbonylcyanide m-chlorophenylhydrazone had a similar effect to nigericin, but singly these ionophores were much less stimulatory. After prolonged centrifugation on linear sucrose gradients, nigericin-stimulated ATPase correlated in dense fractions with plasma membrane markers but a part of it remained at the interphase. This lessdense part of the nigericin-stimulated ATPase could be derived from tonoplast vesicles because α-mannosidase, an enzyme of the vacuolar sap, remained in the upper part of the gradient. Nigericinstimulated ATPase did not correlate with the mitochondrial marker, cytochrome c oxidase, whereas azide inhibition of ATPase activity did.

Na+-driven flagellar motors of an alkalophilic Bacillus strain YN-1.

Flagellar motors of some alkalophilic Bacillus strains have been suggested to be powered by the electrochemical potential gradient of Na+, namely the (formula: see text) (Hirota, N., Kitada, M., and Imae, Y. (1981) FEBS Lett. 132, 278-280). In the present study, we quantitatively measured the (formula: see text) and motility of one of the strains, YN-1. Swimming speed of YN-1 cells increased linearly with a logarithmic increase of Na+ concentration in the medium up to 100 mM. The intracellular Na+ concentration and the membrane potential of the cell were about 30 mM and -170 mV, respectively, and stayed constant irrespective of Na+ concentration in the medium. Thus, the swimming speed changed as a function of the chemical potential difference of Na+ across the cell membrane. When the membrane potential of YN-1 cells was decreased by a combination of valinomycin and various concentrations of K+ in the medium, the swimming speed of the cells decreased linearly and reached zero at around -90 mV. Under the condition, the intracellular Na+ concentration stayed constant. Thus, the membrane potential was also a determinant of the swimming speed. Furthermore, the chemical potential of Na+ and the membrane potential were found to be equivalent as the energy source for motility. Therefore, it is concluded that the (formula: see text) is the energy source for the flagellar motors of YN-1 cells. Threshold value of the (formula: see text) for motility was about -100 mV.

Effects of ionophores and dicyclohexylcarbodiimide on Mycoplasma gallisepticum adherence to erythrocytes.

To test the influence of the electrochemical ion gradient across mycoplasma membranes on the capacity of organisms to adhere to host cells, Mycoplasma gallisepticum cells were treated with valinomycin, carbonylcyanide m-chlorophenylhydrazone, and N,N'-dicyclohexylcarbodiimide (DCCD) singly or in combination. Uptake of [3H]tetraphenylphosphonium by the treated cells was employed as a measure of the effects of the ionophores on membrane potential. In the absence of K+, valinomycin increased, whereas carbonylcyanide m-chlorophenylhydrazone, and DCCD decreased [3H]tetraphenylphosphonium uptake. However, with a high level of K+ or with DCCD, uptake of [3H]tetraphenylphosphonium in the presence of valinomycin decreased below control levels, indicating that, generally, the ionophores affected membrane potential in the expected manner. The treated organisms were tested for their capacity to attach to glutaraldehyde-fixed human erythrocytes. DCCD was the best inhibitor of mycoplasma attachment, and in combination with valinomycin attachment, capacity decreased by about 40%. The combination of valinomycin plus carbonylcyanide m-chlorophenylhydrazone was less effective; it decreased attachment by about 15 to 25%. It was concluded that the dissipation of ion gradients across cell membranes decreases only partially mycoplasma adherence, in line with previous findings that isolated mycoplasma membranes retain the major part of the attachment capacity of intact cells.

Respiratory control and the basis of light-induced inhibition of respiration in chromatophores from Rhodopseudomonas capsulata

A preparation of chromatophores from Rhodopseudomonas capsulata is described in which NADH oxidase activity is stimulated by uncouplers and during ATP synthesis, and is strongly inhibited upon illumination. The inhibition is completely reversed by addition of a protonophore or the combination of valinomycin, nigericin and K + , thus demonstrating that the proton electro-chemical gradient generated by light-driven cyclic electron flow is responsible for the inhibition of respiration. By analogy it is suggested that light-induced inhibition of respiration in intact cells of photosynthetic bacteria is also controlled by the proton electrochemical gradient. The results show that all chromatophores that possess oxidase activity must also have light-driven cyclic electron flow pathways. This has implications for experiments in which carotenoid band shifts and distributions of permeant anions have been compared as indicators of membrane potentials in chromatophores.

The internal pH and membrane potential of the insulin-secretory granule.

The membrane potential (DeltaPsi) and the pH gradient (DeltapH) across the membrane of the insulin-secretory granule were determined in studies in vitro from the uptake of the permeant anion thio[(14)C]cyanate or the permeant base [(14)C]methylamine. Freshly prepared granules incubated in iso-osmotic medium containing sucrose and low concentrations of buffer salts exhibited an acidic internal pH and a DeltaPsi positive inside. Addition of MgATP(2-) under these conditions did not alter the DeltapH, but produced a marked increase in the DeltaPsi. Conversely, when a permeant anion was also included, ATP produced a marked increase in the DeltapH and a lesser increment in the DeltaPsi. NH(4) (+) salts reduced the DeltapH across granule membranes. In the presence of ATP this effect was accompanied by a reciprocal increase in the DeltaPsi. A similar reciprocity was evident when nigericin was added together with K(+) or on decreasing the medium pH, suggesting that these gradients were linked by a common electrogenic process. The effects of ATP were reversed by the protonophore carbonyl cyanide p-trifluoromethoxyphenylhydrazone, the combination of valinomycin, nigericin and K(+), and by the Mg(2+)-dependent ATPase inhibitor tributyltin. Uptakes of (14)C-labelled tracer molecules were also markedly reduced by cryogenic disruption of the granule membrane or hypo-osmotic incubation conditions. These results were readily interpreted within a chemiosmotic hypothesis, which proposed that the insulin granules possess an inwardly-directed electrogenic proton-translocating Mg(2+)-dependent ATPase with the additional postulate that the membrane has a low proton permeability. The intragranular pH was estimated as being between 5 and 6 in vivo. Such a value corresponds to optimal conditions for the crystallization of zinc-insulin hexamers. Several other functions related to chemiosmotic processes within insulin granules, however, may be envisaged.

Proton-translocating Mg2+-dependent ATPase activity in insulin-secretory granules.

Insulin-secretory granules isolated from a pancreatic islet-cell tumour by centrifugation on Percoll density gradients exhibited a membrane-associated Mg(2+)-dependent ATPase activity. In granule suspensions incubated in iso-osmotic media, activity was increased 2-3-fold by carbonyl cyanide p-trifluoromethoxyphenylhydrazone, the combination of valinomycin, nigericin and K(2)SO(4) or by the addition of a detergent. Permeant anions also increased Mg(2+)-dependent ATPase activity under iso-osmotic conditions when combined with K(+) and nigericin, or NH(4) (+). It was deduced that a major component of the activity was coupled to the translocation of protons into the granule interior. The granule membrane appeared poorly permeable to H(+), K(+), NH(4) (+) and SO(4) (2-) but permeable, in increasing order, to phosphate or acetate, Cl(-), I(-) and SCN(-). Like the proton-translocating ATPase of mammalian mitochondria the granule enzyme when membrane-bound was inhibited by up to 85% by tributyltin or NN'-dicyclohexylcarbodi-imide and was solubilized in a tributyltin-insensitive form after extraction with dichloromethane. It was clearly not a mitochondrial contaminant as evidence by the distribution of marker proteins on density gradients. Unlike mitochondrial activity it was insensitive to oligomycin, efrapeptin, atractyloside, azide and oxyanions. Its properties, however, were indistinguishable from those of the proton-translocating ATPase found in the chromaffin granules of the adrenal medulla. Moreover, insulin granules and chromaffin granules exhibited similar levels of activity. This indicated that in spite of the differences in their internal composition, granules from tissues involved in polypeptide and amine hormone secretion possess catalytic components in common. Only a minor role for the ATPase in amine transport in insulin granules was apparent. Rather, its presence here may relate to the process of secretory vesicle morphogenesis or to the exocytotic mechanism.

The Relation between Membrane Potential and the Transport Activity of Systems a and L in Plasma Membrane Vesicles of the Ehrlich Cell

Plasma membrane vesicles isolated from Ehrlich ascites tumor cells have been used to investigate the role of the transmembrane potential in the energetics of Systems A and L. As expected, Na+ -dependent System A was responsive to changes in membrane potential. System L activity, as measured by transport of 2-amino-norbornane-2-carboxylic acid (BCH), was shown to be Na+ -independent and was not altered by changes in the membrane potential. The combination of valinomycin and nigericin decreased accumulation of MeAIB but not that of BCH. The presence of nigericin alone caused a significant decrease in uptake by System A and a decrease in uptake by System L to a lesser degree. The inhibitory action of nigericin might reflect its ability to dissipate the Na+ gradient rather than an effect on K+ or H+ flows. The results indicate that modes of energization not produced through the transmembrane potential must account for any uphill operation of System L.

An acid transporting enzyme in human gastric mucosa.

Isolation of a microsomal fraction from human gastric mucosa followed by density gradient centrifugation yielded a vesicular membrane preparation free of mitochondrial markers, containing a K+-activated, ouabain-insensitive ATPase with an activity of 20.7 mumol P1 released/mg protein per h. Sodium dodecyl sulfate gel electrophoresis showed that the human gastric membrane vesicles contained a major polypeptide of 110,000 daltons, which accounted for approximately or equal to 30% of the total protein stained and was phosphorylated by [gamma-32P]ATP and dephosphorylated in the presence of K+. Electron microscopy revealed the presence of vesicles with an average size of 0.13 micrometer in diameter. Addition of 0.65 microM ATP to this vesicular preparation resulted in the uptake of 17 nmol H+/mg protein which was dependent on the presence of K+. The gradient was dissipated by a combination of valinomycin and protonophore after consumption of the ATP. Incubation of fixed human fundic sections or human gastric biopsy with monospecific hog gastric membrane antibody followed by fluorescein-conjugated goat anti-rabbit gamma-globulin, showed fluorescent staining in the middle portion of the gastric glands. These data indicate that human stomach contains a H+ transport ATPase with characteristics similar to those established for lower species.

Protonmotive force and motility of Bacillus subtilis.

Motility of Bacillus subtilis was inhibited within a few minutes by a combination of valinomycin and a high concentration of potassium ions in the medium at neutral pH. Motility was restored by lowering the concentration of valinomycin or potassium ions. The valinomycin concentration necessary for motility inhibition was determined at various concentrations of potassium ions and various pH's. At pH 7.5, valinomycin of any concentration did not inhibit the motility, when the potassium ion concentration was lower than 9 mM. In the presence of 230 mM potassium ion, the motility inhibition by valinomycin was not detected at pH lower than 6.1. These results are easily explained by the idea that the motility of B. subtilis is supported by the electrochemical potential difference of the proton across the membrane, or the protonmotive force. The electrochemical potential difference necessary for motility was estimated to be about -90 mV.

Coupling in cytochrome c oxidase

Cytochrome c oxidase (ferrocytochrome c: oxygen oxidoreductase; EC 1.9.3.1) can be resolved into an electron transfer complex (ETC) and an ionophore transfer complex (ITC). Coupling requires an interaction between the moving electron in the ETC and a moving, positively charged ionophore-cation adduct in the ITC. The duplex character of cytochrome oxidase facilitates this interaction. The ITC mediates cyclical cation transport. It can be replaced as the coupling partner by the combination of valinomycin and nigericin in the presence of K(+) when cytochrome oxidase is incorporated into liposomes containing acidic phospholipids or by the combination of lipid cytochrome c and bile acids in an ITC-resolved preparation of the ETC. Respiratory control can be induced by incorporating cytochrome oxidase into vesicles of unfractionated whole mitochondrial lipid. The activity of the ITC is suppressed by such incorporation and this suppression leads to the emergence of respiratory control. The ionophoroproteins of the ITC can be extracted into organic solvents; some 50% of the total protein of cytochrome oxidase is extractable. The release of free ionophore is achieved by tryptic digestion of the ionophoroprotein. Preliminary to this release the ionophoroprotein is degraded to an ionophoropeptide. Electrogenic ionophores, as well as uncoupler, are liberated by such proteolysis. The ITC contains a set of ionophoroproteins imbedded in a matrix of phospholipid.

Effects of ionophores on renomedullary prostaglandin output [formula omitted]: [formula omitted] Vanderbilt University, Nashville, TN

1.1. The effect of ionophores on the intralysosomal pH (as estimated from the distribution of a weak acid or base), on the distribution of 42K+ across the lysosomal membrane, and on the intralysosomal degradation of 125I-labelled bovine serum albumin has been studied.2.2. Nigericin and X537A equilibrate both 42K+ and H+ across the lysosomal membrane. Gramicidin equilibrates H+ across the lysosomal membrane, this equilibration being more effective in a NaCl than in a KCl medium. Thus all three ionophores exhibit the same ion specificity as in other membranes.3.3. The effect of the exchange-diffusion ionophores cannot be imitated by the combination of valinomycin with an uncoupler. Valinomycin by itself also has no effect.4.4. X537A and nigericin inhibit the intralysosomal degradation of 125I-labelled albumin only when potassium is present. In a sucrose-containing medium no effect is found. Similar results were obtained with gramicidin.5.5. These data suggest that the lysosomal membrane is impermeable to monovalent cations at 25 or 37°C, and that the transport of protons is organised in such a way that electroneutrality is maintained.

Effect of ionophores on intralysosomal pH

1. 1. The effect of ionophores on the intralysosomal pH (as estimated from the distribution of a weak acid or base), on the distribution of 42K + across the lysosomal membrane, and on the intralysosomal degradation of 125I-labelled bovine serum albumin has been studied. 2. 2. Nigericin and X537A equilibrate both 42K + and H + across the lysosomal membrane. Gramicidin equilibrates H + across the lysosomal membrane, this equilibration being more effective in a NaCl than in a KCl medium. Thus all three ionophores exhibit the same ion specificity as in other membranes. 3. 3. The effect of the exchange-diffusion ionophores cannot be imitated by the combination of valinomycin with an uncoupler. Valinomycin by itself also has no effect. 4. 4. X537A and nigericin inhibit the intralysosomal degradation of 125I-labelled albumin only when potassium is present. In a sucrose-containing medium no effect is found. Similar results were obtained with gramicidin. 5. 5. These data suggest that the lysosomal membrane is impermeable to monovalent cations at 25 or 37°C, and that the transport of protons is organised in such a way that electroneutrality is maintained.