Cholesterol stimulation of penetration of unilamellar liposomes by hydrophobic compounds

Summary The incorporation of cholesterol into unilamellar liposomes greatly increased the transmembranous movement of hydrophobic ionophores such as nigericin. In reconstituted liposomes containing rhodopsin as the only protein, the presence of cholesterol lowers by 10-fold or more the amount of nigericin required to eliminate the light-driven proton gradient. These effects are seen both above and below the transition temperature of the phospholipid used for reconstitution.Cholesterol similarly increases the ability of A-23187, 1799, or NH₄SCN to collapse the proton gradient of bacteriorhodopsin vesicles. Cholesterol also lowers the concentration of nigericin or valinomycin required for a rapid translocation of Rb⁺ into protein-free liposomes. It also lowers the concentration of A-23187 required for the release of Ca⁴⁵ trapped in protein-free liposomes. In contrast to these observations and in confirmation of previous findings, we observed that cholesterol decreased the permeability of liposomes for glucose. Thus the effects of cholesterol on the permeability of the membrane vary with the chemical nature of the permeating compounds. We have also confirmed that in multilamellar liposomes cholesterol decreases the permeability of Rb⁺ in the presence of valinomycin. It therefore appears that the effect of cholesterol changes with the size and structural features of the model membranes.     

Proton electrochemical gradient and rate of controlled respiration in mitochondria

The correlation between deltamuH, the proton electrochemical potential difference, and the rate of controlled respiration is analyzed. deltamuH (the proton concentration gradient) is measured on the distribution of [3H]acetate, and deltapsi (the membrane potential) on the distribution of 86Rb+, 45Ca2+ and [3H]triphenylmethylphosphonium used either alone or simultaneously. The effects of the addition of ADP + hexokinase (state-3 ADP) and of carbonylcyanide trifluoromethoxyphenylhydrazone (state-3 uncoupler) on respiration and deltamuH are not equivalent: the uncoupler depresses deltamuH more than ADP at equivalent respiratory rates. The effects of the additions of nigericin-valinomycin and of ionophore A23187 (state-3 cation transport) and of carbonylcyanide trifluoromethoxy-phenylhydrazone (state 3-uncoupler) on respiration and deltamuH are also not equivalent: the uncoupler depresses deltamuH more than A23187 and nigericin + valinomycin at equivalent respiratory rate. A23187 is very efficient in stimulating respiration with negligible deltamuH changes.     

Functional reconstitution of the gamma-aminobutyric acid transporter from synaptic vesicles using artificial ion gradients.

The gamma-aminobutyric acid transporter of rat brain synaptic vesicles was reconstituted in proteoliposomes, and its activity was studied in response to artificially created membrane potentials or proton gradients. Changes of the membrane potential were monitored using the dyes oxonol VI and 3,3'-diisopropylthiodicarbocyanine iodide, and changes of the H+ gradient were followed using acridine orange. An inside positive membrane potential was generated by the creation of an inwardly directed K+ gradient and the subsequent addition of valinomycin. Under these conditions, valinomycin evoked uptake of [3H]GABA which was saturable. Similarly, [3H]glutamate uptake was stimulated by valinomycin, indicating that both transporters can be driven by the membrane potential. Proton gradients were generated by the incubation of K(+)-loaded proteoliposomes in a buffer free of K+ or Na+ ions and the subsequent addition of nigericin. Proton gradients were also generated via the endogenous H+ ATPase by incubation of K(+)-loaded proteoliposomes in equimolar K+ buffer in the presence of valinomycin. These proton gradients evoked nonspecific, nonsaturable uptake of GABA and beta-alanine but not of glycine in proteoliposomes as well as protein-free liposomes. Therefore, transporter activity was monitored using glycine as an alternative substrate. Proton gradients generated by both methods elicited saturable glycine uptake in proteoliposomes. Together, our data confirm that the vesicular GABA transporter can be energized by both the membrane potential and the pH gradient and show that transport can be achieved by artificial gradients independently of the endogenous proton ATPase.     

The lysosomal proton pump is electrogenic

Lysosomes were purified approximately 40-fold from rat kidney cortex by differential and Percoll density gradient centrifugation. In a sucrose medium, the lysosomes quenched the fluorescence of the potential sensitive dye diS-C3-(5) (3,3'-dipropylthiocarbo-cyanine iodide) in a time-dependent manner, indicating that the dye accumulates within the lysosomal interior. After treatment of the lysosomes with valinomycin, the dye fluorescence displayed a logarithmic dependence upon the external K+ concentration; thus, the fluorescence signal provides a semiquantitative measure of the lysosomal membrane potential (delta psi). In the absence of valinomycin, lysosomal quenching of diS-C3-(5) fluorescence was partially reversed by agents which collapse the lysosomal pH gradient (ammonium sulfate, chloroquine, and K nigericin), suggesting that the proton gradient across the lysosomal membrane contributes to delta psi. A rapid increase in diS-C3-(5) fluorescence, indicative of an increase in delta psi, was observed upon the addition of Mg-ATP to the lysosomes. The ATP-dependent fluorescence change was inhibited by protonophores, K valinomycin, permeable anions, and N-ethylmaleimide, but was unaffected by ammonium sulfate, K nigericin, or sodium vanadate. Oligomycin had no effect at concentrations below 2 micrograms/ml; at higher concentrations, oligomycin partially inhibited the fluorescence response to Mg-ATP, but it also inhibited the fluorescence response to K valinomycin, suggesting that it had modified the permeability of the lysosomal membrane. Dicylohexylcarbodiimide behaved similarly to oligomycin. Mg-ATP also altered the lysosomal distribution of 86Rb+ (in the presence of valinomycin) and S[14C]CN-, consistent with an increase in the potential of the lysosomal interior of 40-50 mV. The results demonstrate that the lysosomal proton pump is electrogenic.     

Methanogenesis and the K+ transport system are activated by divalent cations in ammonia-treated cells of Methanospirillum hungatei

We describe a K+ transport system in Methanospirillum hungatei cells depleted of cytoplasmic K+ via an ammonia/K+ exchange reaction (Sprott, G. D., Shaw, K. M., and Jarrell, K. F. (1984) J. Biol. Chem. 259, 12602-12608). Ammonia-treated cells contained low concentrations of ATP and were unable to make CH4 or to transport 86Rb+. All of these properties were restored by CaCl2, MgCl2, or MnCl2, and not by CoCl2 or NiCl2. The Rb+ transport system had a Km of 0.42 and Vmax of 29 nmol/min X mg; K+ inhibited competitively. Both H2 and CO2 were required for appreciable transport, whereas air, valinomycin, or nigericin were potent inhibitors. The influx of Rb+ was electrogenic and associated with proton efflux, producing a delta pH (alkaline inside) in acidic media. In the absence of K+ (or Rb+), the activation of CH4 synthesis by Mg2+ produced little change in the cytoplasmic pH, showing that methanogenesis did not elicit a net efflux of protons. The pH optimum for transport was in the range 6.0-7.3 where the transmembrane pH gradient would contribute minimally to the proton motive force. Protonophores at pH 6.3 caused a partial decline in CH4 synthesis and the ATP content and dramatically collapsed Rb+ transport. These and other inhibitor experiments, coupled with the fact that the Rb+ gradient was too large to be in equilibrium with the proton motive force alone, suggest a role for both ATP and the proton motive force in Rb+ transport. Also, a role for K+ in osmoregulation is indicated.     

Regulation of gastric acid secretion via modulation of a chloride conductance

When gastric microsomes were purified from resting and stimulated rabbit mucosae, they were found to be generally similar in (H+ + K+)-ATPase activity, peptide composition in single-dimension sodium dodecyl sulfate-gel electrophoresis, and in size. In the stimulated vesicles, optimal proton transport activity was found at pH 7.4, 20-50 mM KCl, and 1 mM ATP-Mg. However, in the case of resting vesicles, the presence of valinomycin and an inward Cl-gradient was also necessary for Mg-ATP-dependent proton transport. Measurement of K+ and Cl-diffusion potentials using 3,3-dipropylthiadicarboxocyanine iodide as a potential sensitive dye showed that both resting and stimulated vesicles developed K+ gradient-dependent potentials in the presence of an impermeant anion, but that Cl- gradient-dependent potentials were observed only in the stimulated preparation. 86Rb+ self-exchange was found in both types of vesicles, but Cl- self-exchange was confined to vesicles derived from stimulated mucosae. Putative inhibitors of anion conductance such as furosemide and anthracene 9-carboxylic acid blocked proton transport, Cl- conductance, 36Cl- uptake, and Cl- exchange. The inhibition of proton transport was overcome by valinomycin. ATPase activity in the presence of nigericin, an H+:K+ exchanger, was unaffected by these inhibitors. K+ conductance, Rb+ uptake, and Rb+ exchange were insensitive to these inhibitors. Thus, activation of acid secretion by the stimulated parietal cell appears to involve at least the appearance of a discrete Cl- conductance in the pump-associated membrane.     

Passive ion permeability of the chromaffin-granule membrane.

'Ghosts' of bovine chromaffin granules, in which the complex mixture of proteins and solutes normally found in the granule matrix is replaced by buffered sucrose are osmotically sensitive. They shrink when the osmotic pressure of the suspension medium is increased, and swell if solute entry is facilitated by the addition of ionophores. Swelling in the presence of ionophores has been used to investigate the passive ion permeability of these membranes. They have a very low permeability to K+ ions (of the order of 10(-10) cm/s); their permeability to protons, Na+ and choline ions is too low to be detected by these methods. Their passive permeability to anions decreases in the order: CNS- greater than I- greater than CCl3CO2- greater than Br- greater than Cl- greater than SO4(2)- greater than CH3CO2-, HCO3-, F-, PO4(3)- the permeability to hiocyanate being of the order of 10(-7) cm/s. Coupled proton and anion entry is extremely slow, except for weak acids. Fluoride, unexpectedly, also appears to enter rapidly when proton/K+ exchange is facilitated by nigericin. In the presence of K+ salts, nigericin, like valinomycin, induces lysis of intact granules, an effect that is not dependent on the presence of a permeant anion, but is dependent on the pH gradient across the membrane.     

Use of 1-anilino-8-naphthalene-sulfonate as a probe of gastric vesicle transport.

The interaction of 1-anilino-8-naphthalene-sulfonate (ANS) with vesicles derived from hog fundic mucosa was studied in the presence of valinomycin and with the addition of ATP. Evidence was found for two classes of sites, those rapidly accessible to ANS with a KD of 7.5 micronM and those slowly accessible, but rapidly accessed in the presence of valinomycin with a KD of 2.5 micronM. ATP transiently increases the quantum yield of the latter ANS binding sites only in the presence of valinomycin, but does not alter the number of KD of those sites. The time course of this increase correlates with H+ uptake and Rb+ extrusion by those vesicles and H+ carries such as tetrachlorsalicylanilide or nigericin abolish the ATP response. With ATP addition in the presence of SC14N and valinomycin there is transient uptake of SCN-. It is concluded that ANS is acting as a probe of a structural change dependent on a potential and H+ gradient.     

Bile salt-induced calcium fluxes in artificial phospholipid vesicles

The ionic permeability of selected biological membranes is increased by bile salts. To examine changes in calcium permeability during the exposure of artificial membranes to bile salts, we investigated calcium uptake by unilamellar and multilamellar phospholipid vesicles. In the presence of 750 microM taurodeoxycholate, uptake of radiolabelled calcium by unilamellar vesicles increased 2.5-fold over control values. Calcium uptake by multilamellar vesicles as measured with a free calcium indicator, arsenazo III, increased 2.2- or 21-fold in the presence of 60 microM lithocholate or 3 beta-hydroxy-5-cholenoate, respectively. Results were directly influenced by experimental variables such as bile salt hydrophobicity, external calcium concentration, and the bile salt/lipid molar ratio. Observed membrane solubilization was minimal despite increased calcium permeability. Comparison of radiolabelled calcium uptake with radiolabelled sodium or radiolabelled rubidium uptake indicated that bile salt-dependent calcium uptake was 60-140-times greater than bile salt-dependent uptake of either monovalent cation. In an effort to delineate forces affecting calcium translocation, vesicles were exposed either to valinomycin, which induced an electrochemical gradient across the membrane, or to nigericin, which induced a proton gradient. Exposure to valinomycin minimally influenced bile salt-induced calcium uptake while exposure to nigericin significantly promoted uptake by 40-70%. The results suggest that bile salts promote calcium uptake by a mechanism which may be similar to those of other carboxylic ionophores.     

Histone inhibition of mitochondrial proton transport.

Histone blocks proton uptake by mitochondria incubated in the presence of valinomycin or DNP. In the presence of DNP valinomycin-induced H+ uptake is not affected by histone. H+ uptake induced by nigericin is not affected by histone as well. Postulated mechanism of histone action involves the immobilization of proton translocation in mitochondrial membrane and induction of local change in H+ concentration, the prevention of the interaction between H+ and natural K+-carrier and Mg2+ transport system or valinomycin.     

The use of phospholipid-impregnated millipore filters for recording nonelectrogenic cation flows in the presence of Men+/nH+ exchangers

It has been shown that with a cation (K+, Na+, Ca2+) concentration gradient on a Millipore filter impregnated with a decane solution of phospholipid, in the presence of a Men+/nH+ exchanger (nigericin, monensin, A23187), addition of a protonophore induces the formation of an electric potential positively charged on the side where the concentration of the cation is lower. The formation of the potential is induced by the hydrogen ion concentration gradient in the filter and in the unstirred layers as a result of the Men+/nH+ exchange. In such a system, with a pH gradient on the filter in the presence of monensin and valinomycin, a potential is generated with the plus on the side of the lower concentration of hydrogen. The effect is the result of the formation of a potassium ion concentration gradient in the unstirred layers in the course of the K+/H+ exchange. It is concluded that phospholipid-impregnated filters can be used for search and identification of electroneutral membrane ionophores of the Men+/nH+ exchanger type.     

Acetylcholine Release from Isolated Synaptic Vesicles Related to Ionic Permeability Changes: Continuous Detection with a Chemiluminescent Method

The effect of ionic permeability changes on acetylcholine (ACh) release from isolated cholinergic synaptic vesicles of Torpedo was studied using a chemiluminescent method for continuous ACh detection. Vesicles rendered freely permeable to potassium by valinomycin lost most of their ACh content in K+ media, if the accompanying anion was permeant; it thus appeared that ACh leakage occurred as the result of internal osmotic changes. Upon addition of ionophores that catalyse monovalent cation/H+ exchange (gramicidin D or a mixture of valinomycin plus protonophore FCCP), a rapid but transient ACh release was observed. Surprisingly, nigericin which also catalyses K+/H+ exchange, had no effect on ACh release. The divalent cation ionophore A23187 promoted ACh release only when calcium (and not magnesium) was introduced into the external medium in a millimolar concentration range. As the simultaneous addition of the protonophore FCCP and A23187 decreased this calcium-dependent ACh leakage, a releasing effect of A23187 through Ca2+/H+ exchange is suspected. The present results emphasise the role of internal protons for ACh retention inside synaptic vesicles.     

Membrane potential in liposomes measured by the transmembrane distribution of 86Rb+, tetraphenylphosphonium or triphenylmethylphosphonium: effect of cholesterol in the lipid bilayer

Valinomycin-induced potassium diffusion potential (delta psi, inside negative) in the liposomes made of phosphatidylcholine and various amounts of cholesterol was measured by uptake of 86Rb+, tetraphenylphosphonium (TPP+) or triphenylmethylphosphonium (TPMP+). In any liposome, the values of membrane potential obtained by 86Rb+ uptake (delta psi Rb) agreed well with those calculated from the imposed potassium concentration gradient using the Nernst equation, and were not affected by the presence of cholesterol. However, both delta psi TPP and delta psi TPMP showed smaller values than delta psi Rb when the cholesterol content in liposomes increased. delta psi TPMP at a stationary state was much smaller than delta psi TPP. The orientational order parameter of the lipids' bilayer with various cholesterol content was estimated from fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene. The results indicated that the permeation of TPP+ or TPMP+ into liposomes containing a large amount of cholesterol is strongly restricted by the high ordering of phosphatidylcholine acyl chains.     

In vitro translocation of protein across Escherichia coli membrane vesicles requires both the proton motive force and ATP

The energy requirement for protein translocation across membrane was studied with inverted membrane vesicles from an Escherichia coli strain that lacks all components of F1F0-ATPase. An ompF-lpp chimeric protein was used as a model secretory protein. Translocation of the chimeric protein into membrane vesicles was totally inhibited in the presence of carbonyl cyanide m-chlorophenylhydrazone (CCCP) or valinomycin and nigericin and partially inhibited when either valinomycin or nigericin alone was added. Depletion of ATP with glucose and hexokinase resulted in the complete inhibition of the translocation process, and the inhibition was suppressed by the addition of ATP-generating systems such as phosphoenolpyruvate-pyruvate kinase or creatine phosphate-creatine kinase. These results indicate that both the proton motive force and ATP are required for the translocation process. The results further suggest that both the membrane potential and the chemical gradient of protons (delta pH), of which the proton motive force is composed, participate in the translocation process.     

Dependence of mitochondrial coenzyme A uptake on the membrane electrical gradient

Coenzyme A (CoA) transport was studied in isolated rat heart mitochondria. Uptake of CoA was assayed by determining [3H]CoA associated with mitochondria under various conditions. Various oxidizable substrates including alpha-ketoglutarate, succinate, or malate stimulated CoA uptake. The membrane proton (delta pH) and electrical (delta psi) gradients, which dissipated with time in the absence of substrate, were maintained at their initial levels throughout the incubation in the presence of substrate. Addition of phosphate caused a concentration-dependent decrease of both delta pH and CoA uptake. Nigericin also dissipated the proton gradient and prevented CoA uptake. Valinomycin also prevented CoA uptake into mitochondria. Although the proton gradient was unaffected, the electrical gradient was completely abolished in the presence of valinomycin. Addition of 5 mM phosphate 10 min after the start of incubation prevented further uptake of CoA into mitochondria. A rapid dissipation of the proton gradient upon addition of phosphate was observed. Addition of nigericin or valinomycin 10 min after the start of incubation also resulted in no further uptake of CoA into with mitochondria; valinomycin caused an apparent efflux of CoA from mitochondria. Uptake was found to be sensitive to external pH displaying a pH optimum at pHext 8.0. Although nigericin significantly inhibited CoA uptake over the pHext range of 6.75-8, maximal transport was observed around pHext 8.0-8.25. Valinomycin, on the other hand, abolished transport over the entire pH range. The results suggest that mitochondrial CoA transport is determined by the membrane electrical gradient. The apparent dependence of CoA uptake on an intact membrane pH gradient is probably the result of modulation of CoA transport by matrix pH.     

Effect of ionophores A23187 and nigericin on the light-induced redistribution of Mg2+, K+ and H+ across the thylakoid membrane.

Passive redistributions of Mg2+ and K+ ions across the thylakoid membranes, occurring in association with the light-driven electrogenic influx of hydrogen ions have been examined in suspensions of broken spinach chloroplasts under a variety of conditions. (i) In accord with results of Hind el al. (Proc. Natl. Acad. Sci. U.S. (1974) 71, 1484), it was found that at a low K/Mg concentration ratio in the medium, the K-efflux is negligibly small, whereas a substantial Mg-efflux is observed. The converse is true when the K/Mg concentration ratio in the medium is high. (ii) In the presence of A23187, which was found to cause approximately a 60% inhibition of the light-induced pH-gradient, a significant influx of Mg2+ was observed in the light at a high K/Mg concentration ratio. Conversely the Mg-influx was small in the presence of A23187 when the K/Mg concentration ratio in the medium was low. Under these conditions, the Mg-influx was considerably increased upon the addition of valinomycin. A23187 was found not to affect the K-efflux in the light. (iii) The light-induced K-influx observed in the presence of nigericin also was found to be dependent on the concentration ratio of the monovalent and divalent cation. Its magnitude increased upon an increase in the K/Mg ratio. The results are interpreted in terms of a simplified model in which the total passive efflux of cations, driven by the potential set by the electrogenic proton pump, is considered to be a constant fraction of the proton influx. According to this, an increase in the flux of an ion species, induced either by raising its concentration, or by increasing its permeability through the membrane, will cause a decrease in the flux of the other cations. The relevance of the results is discussed with respect to conclusions about the involvement and relative magnitudes of the passive K and Mg effluxes across the thylakoid membrane during energization of intact chloroplasts and chloroplasts in situ.     

Measurements of the proton motive force generated by cytochrome c oxidase from Bacillus subtilis in proteoliposomes and membrane vesicles

Cytochrome c oxidase from Bacillus subtilis was reconstituted in liposomes and its energy-transducing properties were studied. The reconstitution procedure used included Ca2+-induced fusion of pre-formed membranes. The orientation of the enzyme in liposomes is influenced by the phospholipid composition of the membrane. Negatively charged phospholipids are essential for high oxidase activity and respiratory control. Analyses of the proteoliposomes by gel filtration, density gradient centrifugation and electron microscopy indicated a heterogeneity of the proteoliposomes with respect to size and respiratory control. Cytochrome c oxidase activity in the proteoliposomes resulted in the generation of a proton motive force, internally negative and alkaline. In the presence of the electron donor, ascorbate/N,N,N',N'-tetramethyl-p-phenylenediamine/cytochrome c or ascorbate/phenazine methosulphate, the reconstituted enzyme generated an electrical potential of 84 mV which was increased by the addition of nigericin to 95 mV and a pH gradient of 32 mV which was increased by the addition of valinomycin to 39 mV. Similar results were obtained with beef-heart cytochrome c oxidase reconstituted in liposomes. The maximal proton motive force which could be generated, assuming no endogenous ion leakage, varied over 110-140 mV. From this the efficiency of energy transduction by cytochrome c oxidase was calculated to be 18-23%, indicating that the oxidase is an efficient proton-motive-force-generating system.     

The effect of trypsin treatment on the incorporation and energy-transducing properties of bacteriorhodopsin in liposomes

Bacteriorhodopsin and trypsin-modified bacteriorhodopsin have been reconstituted into liposomes by means of a low pH-sonication procedure. The incorporation of bacteriorhodopsin in these proteoliposomes is predominantly in the same direction as in vivo and the direction of proton pumping is from inside to outside the liposomes. The direction of proton translocation and electrical potential generation was studied as a function of the reconstitution pH. Light-dependent proton extrusion and generation of a Δp, interior negative and alkaline was observed at a reconstitution pH below 3.0 using bacteriorhodopsin, and at a pH below 3.5 using trypsin-modified bacteriorhodopsin. The shift in inflection point is explained in terms of differences between bacteriorhodopsin and trypsin-modified bacteriorhodopsin in a specific protein-phospholipid interaction which depends on the surface charge density of the cytoplasmic side of bacteriorhodopsin. The magnitude of the protonmotive force (Δp) generated by trypsin-modified bacteriorhodopsin in liposomes was quantitated. Illumination of the proteoliposomes resulted in the generation of a high Δp (135 mV, inside negative and alkaline), with a major contribution of the pH gradient. The ionophores nigericin and valinomycin induced, respectively, a compensatory interconversion of ΔpH into Δψ and vice versa. If no endogenous proton permeability of the membrane would exist, a protonmotive force could be generated of − 143 mV as electrical potential alone or − 162 mV as pH gradient alone.     

Proton transport by gastric membrane vesicles.

A highly purified membrane fraction was derived from hog gastric mucosa by a combination of differential and density gradient centrifugation and free flow electrophoresis. This final fraction was 35-fold enriched with respect to cation activated ouabain-insensitive ATPase. Antibody against this fraction was shown to be bound to the luminal surface of the gastric glands. The addition of ATP to this fraction or the density gradient fraction resulted in H+ uptake into an osmotically sensitive space. The apparent Km for ATP was 1.7-10(-4) M in the absence of a K+ gradient similar to that found for ATPase activity. The reaction is specific for ATP and requires cation in the sequence K+ greater than Rb+ greater than Cs+ greater than Na+ greater than Li+ and inhibited by ATPase inhibitors such as N,N'-dicylclohexyl-carbodiimide. Maximal H+ uptake occurs with an outward K+ gradient but the minimal apparent KA is found in the absence of a K+ gradient. The pH optimum for H+ uptake is between 5.8 and 6.2 which corresponds to the pH range for phosphroylation of the enzyme, but is considerably less than the pH maximum of the K+ dependent dephosphorylation. In the presence of an inward K+ gradient, protonophores such as tetrachlorsalicylanilide only partially abolish the H+ gradient but valinomycin dissipates 75% of the gradient, and nigericin abolishes the gradient. The vesicles therefore have a low K+ conductance but a measurable H+ conductance, hence a K+ gradient can produce an H+ gradient in the presence of valinomycin. The uptake and spontaneous leak of H+ are temperature sensitive with a similar transition temperature. Ultraviolet irradiation inactivates ATPase and proton transport at the same rate, approximately at twice the rate of p-nitrophenylphosphatase inactivation. It is concluded that H+ uptake by these vesicles is probably due to a dimeric (H+ + K+)-ATPase and is probably non-electrogenic.     

Effect of metabolic inhibitors on entry of exogenous deoxyribonucleic acid into Ca2+-treated Escherichia coli cells.

The effect of various metabolic inhibitors (carbonylcyanid-m-chlorophenylhydrazone, nigericin, valinomycin, dicyclocarbodiimide, arsenate, NaF, etc.) and lipid-soluble synthetic ions (tetraphenylphosphonium bromide and tetraphenylboron sodium) on deoxyribonucleic acid (DNA) entry during transformation of Ca2+-treated Escherichia coli cells with plasmid DNA and on cell viability was investigated. In contrast to intact cells, Ca2+-treated E. coli cells were permeable to nigericin, valinomycin, and the other drugs tested. The inhibitors differentially affected [14C]proline active transport, and whereas some drugs inhibited transformation, the effects did not correlate with the effects on transport. The most potent inhibitors of transformation were nigericin, dicyclocarbodiimide, and tetraphenylboron sodium. Carbonylcyanid-m-chlorophenylhydrazone, tetraphenylphosphonium bromide, and valinomycin were relatively inactive. Tetraphenylboron sodium- and nigericin-treated cells bound were plasmid [14C]DNA in the deoxyribonuclease-resistant form than the control and other sample cells. Nevertheless, te penetration of exogenous plasmid DNA into the cell was greatly reduced, at least in case of nigericin. Unlike the other drugs, nigericin and dicyclocarbodiimide drastically affected the cell viability, the former within very short times of interaction. It is concluded that proton motive force does not play any significant role in DNA entry into Ca2+-treated E. coli cells. The results also suggest that adenosine 5'-triphosphate is not required for DNA entry either. The inhibitory effect of certain drugs is discussed in terms of structural perturbations induced by the drugs in cell envelope membranes.