Active amino acid transport in plasma membrane vesicles from Simian virus 40-transformed mouse fibroblasts. Characteristics of electrochemical Na+ gradient-stimulated uptake.

Selectively permeable membrane vesicles isolated from Simian virus 40-transformed mouse fibroblasts catalyzed Na+ gradient-coupled active transport of several neutral amino acids dissociated from intracellular metabolism. Na+-stimulated alanine transport activity accompanied plasma membrane material during centrifugation in discontinuous dextran 110 gradients. Carrier-mediated transport into the vesicle was demonstrated. When Na+ was equilibrated across the membrane, countertransport stimulation of L-[3H]alanine uptake occurred in the presence of accumulated unlabeled L-alanine, 2-aminoisobutyric acid, or L-methionine. Competitive interactions among neutral amino acids, pH profiles, and apparent Km values for Na+ gradient-stimulated transport into vesicles were similar to those previously described for amino acid uptake in Ehrlich ascites cells, which suggests that the transport activity assayed in vesicles is a component of the corresponding cellular uptake process. Both the initial rate and quasi-steady state of uptake were stimulated as a function of a Na+ gradient (external Na+ greater than internal Na+) applied artificially across the membrane and were independent of endogenous (Na+ + K+)-ATPase activity. Stimulation by Na+ was decreased when the Na+ gradient was dissipated by monensin, gramicidin D or Na+ preincubation. Na+ decreased the apparent Km for alanine, 2-aminoisobutyric acid, and glutamine transport. Na+ gradient-stimulated amino acid transport was electrogenic, stimulated by conditions expected to generate an interior-negative membrane potential, such as the presence of the permeant anions NO3- and SCN-. Na+-stimulated L-alanine transport was also stimulated by an electrogenic potassium diffusion potential (K+ internal greater than K+ external) catalyzed by valinomycin; this stimulation was blocked by nigericin. These observations provide support for a mechanism of active neutral amino acid transport via the "A system" of the plasma membrane in which both a Na+ gradient and membrane potential contribute to the total driving force.     

Highly permeant anions and glucose uptake as an alternative for quantitative generation and estimation of membrane potential differences in brush-border membrane vesicles

We have analyzed the combined utilization of highly permeant anions to induce membrane diffusion potentials and glucose uptake to probe the created potentials as a new approach to quantitative generation and estimation of membrane potential differences in vesicle studies. Rabbit jejunal brush-border membrane vesicles were used in our experiments so that membrane potential differences can be calculated from the Goldman-Hodgkin-Katz equation with the relative ion permeabilities recently reported for this preparation (Gunther, R.D., Schell, R.E. and Wright, E.M. (1984) J. Membrane Biol. 78, 119-127) or approximated by the Nernst potential for the anion. Iodide was selected as the highly permeant anion after showing its absence of effect on glucose uptake with equal concentrations of Na+ inside and outside the vesicles and the membrane potential clamped to zero with gramicidin D. Membrane potential was varied by altering the intra- and extravesicular iodide concentrations while keeping isosmolarity and isotonicity constant by chloride replacement. In these conditions, glucose uptake was sensitive and correlated to the expected membrane potentials. Moreover, a linear relationship between the log initial rate of glucose transport and membrane potential differences could be established. This linear relationship was quite insensitive to inside replacement of choline by potassium and to pH variations in the incubation medium, thus showing the reproducibility and the versatility of the method and the adequacy of glucose uptake as a probe for membrane potentials. However, no information can be gained on the stoichiometry of the Na+-glucose transporter as the slope of the straight line depends on both the charge carried by the fully loaded carrier and the point in the electric field at which the transition state of the carrier from cis to trans occurs. This new approach was compared with the more conventional one using valinomycin-induced K+-diffusion potentials and the Nernst potential for potassium as means for creating and estimating membrane potential differences. Both techniques were not equivalent, as linear relationships showing smaller slopes and sensitivity to pH were recorded with the latter. These differences are compatible with a potassium permeability in the presence of valinomycin that is lower than generally assumed, at least when compared to the permeability of the other ions present in the incubation medium.(ABSTRACT TRUNCATED AT 400 WORDS)     

Light-induced leucine transport in Halobacterium halobium envelope vesicles: a chemiosmotic system.

Halabacterium halobium cell envelope vesicles accumulate L-[14-C]leucine during illumination, against a large concentration gradient. Leucine uptake requires Na-+ and is optimal in KCl-loaded vesicles resuspended in KCl-NaCl solution (1.5 M:1.5 M). Half-maximal transport is seen at 1 X 10-minus 6 M leucine. In the dark the accumulated leucine is rapidly and exponentially lost from the vesicles. The action spectrum and the light-intensity dependence indicate that the transport is related to the extrusion of protons, mediated by bacteriorhodopsin. Since light gives rise to both a pH gradient and an opposing transmembrane potential (interior negative), it wass responsible for providing the energy for leucine transport. The following results were obtained under illumination: (1) membrane-permeant cations and valinomycin or gramicidin greatly inhibited leucine transport without altering the pH gradient; (2) buffering both inside and outside the vesicles eliminated the pH gradient while enhancing leucine transport; (3) dicyclohexylcarbodiimide increased the pH gradient without affecting leucine transport; (4) arsenate did not inhibit leucine uptake. A diffusion potential, established by adding valinomycin to KCl-loaded vesicles, caused leucine influx in the dark. These results suggest that the leucine transport system is not coupled to ATP hydrolysis, and responds to the membrane potential rather than to the pH gradient. The Na-+ dependence of the transport and the observation that a small NaCl pulse causes transient leucine influx in the dark in KCl-loaded vesicles, resuspended in KCl, even in the presence of p-trifluoromethoxycarbonylcyanide phenylhydrazone or with buffering, suggest that the translocation of leucine is facilitated by symport with Na-+.     

Control of the Ca2+-triggered bioluminescence of Veretillum cynomorium lumisomes.

Calcium ions can trigger an emission of light from Veretillum cynomorium lumisomes (bioluminescent vesicles) under conditions where they are not lysed. This process does not require a metabolically-linked source of energy, but is dependent upon the nature of the ions present inside and outside the vesicles. The Ca2+-triggered bioluminescence is stimulated by an asymmetrical distribution of cations or anions. Either high internal sodium or high external chloride is required for the maximal effect. When sodium is present outside the structure and potassium inside, the slow inward diffusion of calcium is decreased. Unbalanced diffusion of internal cations also stimulates the bioluminescence, suggesting control of the calcium influx by an electrochemical gradient. It is assumed that rapid outward diffusion of sodium or inward diffusion of chloride generates an electrical potential difference (inside negative) which drives the Ca2+-influx. With purified lumisomes it has been shown that Ca2+-triggered bioluminescence and calcium uptake (presumably net uptake) were correlated. In two instances uptake of the lipophilic cation dibenzyldimethylammonium has given direct evidence for the existence of a potential difference. With NaCl-loaded vesicles, it has not been possible to demonstrate an uptake of lipophilic cations but experiments with 22Na and 42D indicated a higher rate of sodium efflux, in accord with the proposed hypothesis.     

Control of the Ca2+-triggered bioluminescence of Veretillum cynomorium lumisomes

Calcium ions can trigger an emission of light from Veretillum cynomorium lumisomes (bioluminescent vesicles) under conditions where they are not lysed. This process does not require a metabolically-linked source of energy, but is dependent upon the nature of the ions present inside and outside the vesicles. The Ca²⁺-triggered bioluminescence is stimulated by an asymmetrical distribution of cations or anions. Either high internal sodium or high external chloride is required for the maximal effect. When sodium is present outside the structure and potassium inside, the slow inward diffusion of calcium is decreased. Unbalanced diffusion of internal cations also stimulates the bioluminescence, suggesting control of the calcium influx by an electrochemical gradient. It is assumed that rapid outward diffusion of sodium or inward diffusion of chloride generates an electrical potential difference (inside negative) which drives the Ca²⁺-influx. With purified lumisomes it has been shown that Ca²⁺-triggered bioluminescence and calcium uptake (presumably net uptake) were correlated. In two instances uptake of the lipophilic cation dibenzyldimethylammonium has given direct evidence for the existence of a potential difference. With NaCl-loaded vesicles, it has not been possible to demonstrate an uptake of lipophilic cations but experiments with ²²Na and ⁴²K indicated a higher rate of sodium efflux, in accord with the proposed hypothesis.     

Ion dependence of cystine and lysine uptake by rat renal brush-border membrane vesicles.

The shared transport system for uptake of L-cystine and L-lysine was examined in isolated rat renal brush-border membrane vesicles for the ionic requirements for activation of the system. No requirement for sodium was seen for either cystine or lysine influx. However, the efflux of lysine from the vesicle was stimulated by Na+. Therefore, the transport system appears to be asymmetric in its requirement for sodium. Two different divalent cations were used in the membrane isolations which resulted in different responses of cystine uptake to the electrogenic movement of K+ out of the vesicle. Membranes prepared by Mg-aggregation showed no stimulation of cystine influx by the imposition of a transient interior negative potential while vesicles prepared by Ca-aggregation did respond to electrogenic stimulation by an outwardly directed K-diffusion potential in the presence of valinomycin. Lysine influx was stimulated by electrogenic potassium efflux in both Mg-prepared and Ca-prepared membranes. No difference in sodium requirement for cystine influx was seen between the vesicles isolated by different cation-aggregation methods.     

Stimulation of cation transport in mitochondria by gramicidin and truncated derivatives

Gramicidin and the truncated derivatives desformylgramicidin (desfor) and des(formylvalyl)gramicidin (desval) stimulate monovalent cation transport in rat liver mitochondria. Cation fluxes were compared indirectly from the effect of cations on the membrane potential at steady state (state 4) or from the associated stimulation of electron transport. Rb+ transport was measured directly from the uptake of 86Rb. The truncated gramicidins show enhanced selectivity for K+ and Rb+ when compared to gramicidin. Moreover, the pattern of selectivity within the alkali cation series is altered, i.e., Rb+ greater than K+ greater than Cs+ greater than Na+ greater than Li+ for desfor and desval as compared to Cs+ greater than Rb+ greater than K+ = Na+ greater than Li+ for gramicidin. The cation fluxes through the truncated derivatives are more strongly dependent on the cation concentration. The presence of high concentrations of permeating cation enhances the transport of other cations through the truncated derivative channels, suggesting that cations are required for stabilizing the channel structure. In high concentrations of KCl, desfor and desval are nearly as effective as gramicidin in collapsing the mitochondrial membrane potential, and, consequently, in the uncoupling of oxidative phosphorylation and enhancement of ATP hydrolysis. Preliminary experiments with liposomes show that 86Rb exchange is stimulated by desfor and desval almost to the same extent as gramicidin. These results strongly suggest that the truncated gramicidins form a novel conducting channel which differs from the gramicidin head-to-head, single-stranded beta 6.3-helical dimer ("channel") in its conductance characteristic and its structure.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of Monovalent Cations on Na+/Ca2+ Exchange and ATP-Dependent Ca2+ Transport in Synaptic Plasma Membranes

Two Ca2+ transport systems were investigated in plasma membrane vesicles isolated from sheep brain cortex synaptosomes by hypotonic lysis and partial purification. Synaptic plasma membrane vesicles loaded with Na+ (Na+i) accumulate Ca2+ in exchange for Na+, provided that a Na+ gradient (in leads to out) is present. Agents that dissipate the Na+ gradient (monensin) prevent the Na+/Ca2+ exchange completely. Ca2+ accumulated by Na+/Ca2+ exchange can be released by A 23187, indicating that Ca2+ is accumulated intravesicularly. In the absence of any Na+ gradient (K+i-loaded vesicles), the membrane vesicles also accumulate Ca2+ owing to ATP hydrolysis. Monovalent cations stimulate Na+/Ca2+ exchange as well as the ATP-dependent Ca2+ uptake activity. Taking the value for Na+/Ca2+ exchange in the presence of choline chloride (external cation) as reference, other monovalent cations in the external media have the following effects: K+ or NH4+ stimulates Na+/Ca2+ exchange; Li+ or Cs+ inhibits Na+/Ca2+ exchange. The ATP-dependent Ca2+ transport system is stimulated by increasing K+ concentrations in the external medium (Km for K+ is 15 mM). Replacing K+ by Na+ in the external medium inhibits the ATP-dependent Ca2+ uptake, and this effect is due more to the reduction of K+ than to the elevation of Na+. The results suggest that synaptic membrane vesicles isolated from sheep brain cortex synaptosomes possess mechanisms for Na+/Ca2+ exchange and ATP-dependent Ca2+ uptake, whose activity may be regulated by monovalent cations, specifically K+, at physiological concentrations.     

Sodium-chloride transport in the medullary thick ascending limb of henle's loop: Evidence for a sodium-chloride cotransport system in plasma membrane vesicles

Sodium transport mechanisms were investigated in plasma membrane vesicles prepared from the medullary thick ascending limb of Henle's loop (TALH) of rabbit kidney. The uptake of 22Na into the plasma membrane vesicles was investigated by a rapid filtration technique. Sodium uptake was greatest in the presence of chloride; it was reduced when chloride was replaced by nitrate, gluconate or sulfate. The stimulation of sodium uptake by chloride was seen in the presence of a chloride gradient directed into the vesicle and when the vesicles were equilibrated with NaCl, KCl plus valinomycin so that no chemical or electrical gradients existed across the vesicle (tracer exchange experiments). Furosemide decreased sodium uptake into the vesicles in a dose-dependent manner only in the presence of chloride, with a Ki of around 5 X 10(-6) M. Amiloride, at 2 mM, had no effect on the chloride-dependent sodium uptake. Similarly, potassium removal had no effect on the chloride-dependent sodium uptake and furosemide was an effective inhibitor of sodium uptake in a potassium-free medium. The results show the presence of a furosemide-sensitive sodium-chloride cotransport system in the plasma membranes of the medullary TALH. There is no evidence for a Na+/H+ exchange mechanism or a Na+ -K+ -Cl- cotransport system. The sodium-chloride cotransport system would effect the uphill transport of chloride against its electrochemical potential gradient at the luminal membrane of the cell.     

Electrogenic behavior of synaptic vesicles from Torpedo californica.

Electrical potential changes in pure synaptic vesicles from Torpedo californica were monitored with the fluorescent dye 3,3'-dipropylthiadicarbocyanine iodide. Vesicles resuspended in variable external sodium ion in the presence of gramicidin established sodium ion membrane diffusion potentials. Vesicles resuspended in choline or acetylcholine chloride became hyperpolarized upon addition of gramicidin. Hyperpolarization was subsequently partially reversed spontaneously by choline or acetylcholine influx, which was confirmed by gel filtration, to yield a new, less negative, stable membrane potential. Thus, acetylcholine and choline are taken up electrogenically by synaptic vesicles.     

Purification and characterization of Ca2+/H+ antiporter from Bacillus subtilis

Ca2+ was accumulated in inside-out membrane vesicles of Bacillus subtilis when NADH was used as an energy source. A delta pH (acid interior) could also drive Ca2+ accumulation in the membrane vesicles and the accumulation was inhibited by carbonylcyanide p-trifluoromethoxyphenylhydrazone and nigericin plus K+. These results indicate the presence of a Ca2+/H+ antiporter (exchanger) in this organism. The antiporter was isolated and purified to homogeneity from the membrane proteins by chromatography on hydroxyapatite, diethylaminoethyl(DEAE)-Toyopearl 650 M and butyl-Toyopearl 650 M. The purified antiporter has a molecular mass of about 45 000 daltons and an isoelectric point of 5.0. The fluorescence quenching of a cyanine dye (3,3'-dipropylthiodicarbocyanine iodide [diS-C3-(5)] during Ca2+ accumulation in proteoliposomes by the purified antiporter showed the generation of a membrane potential (interior negative) suggesting a H+/Ca2+ stoichiometry above 2 in the transport. This was also supported by the result that the K+-diffusion potential, interior positive, stimulated the Ca2+ uptake in the presence of a delta pH. The apparent Km for Ca2+ of the antiporter was about 40 microM and La3+ inhibited the transport. Amino acid analysis of the purified antiporter indicated the presence of large amounts of glutamic and aspartic acids and small amounts of histidine, lysine and arginine. This is consistent with the low isoelectric point (about 5.0) of the protein.     

Na+/H+ exchange mechanism in the basolateral membrane of the rat enterocyte

Basolateral membrane vesicles from rat jejunal enterocytes, especially purified of brush-border contamination, were used for Na+ uptake. The basolateral membrane vesicles are osmotically active and under our experimental conditions Na+ binding is much lower than transport. An outwardly directed proton gradient stimulates Na+ uptake at both 5 microM and 5 mM concentrations. The proton gradient effect can be inhibited completely by 2 mM amiloride and partially by either FCCP or NH4Cl (NH3 diffusion). Membrane potential effects can be excluded by having valinomycin plus K+ on both sides of the vesicles. These results suggest that there is an Na+/H+ exchanger in the basolateral membrane of rat enterocytes.     

Characterization of amino acid transport in membrane vesicles from the thermophilic fermentative bacterium Clostridium fervidus.

Amino acid transport was studied in membrane vesicles of the thermophilic anaerobic bacterium Clostridium fervidus. Neutral, acidic, and basic as well as aromatic amino acids were transported at 40 degrees C upon the imposition of an artificial membrane potential (delta psi) and a chemical gradient of sodium ions (delta microNa+). The presence of sodium ions was essential for the uptake of amino acids, and imposition of a chemical gradient of sodium ions alone was sufficient to drive amino acid uptake, indicating that amino acids are symported with sodium ions instead of with protons. Lithium ions, but no other cations tested, could replace sodium ions in serine transport. The transient character of artificial membrane potentials, especially at higher temperatures, severely limits their applicability for more detailed studies of a specific transport system. To obtain a constant proton motive force, the thermostable and thermoactive primary proton pump cytochrome c oxidase from Bacillus stearothermophilus was incorporated into membrane vesicles of C. fervidus. Serine transport could be driven by a membrane potential generated by the proton pump. Interconversion of the pH gradient into a sodium gradient by the ionophore monensin stimulated serine uptake. The serine carrier had a high affinity for serine (Kt = 10 microM) and a low affinity for sodium ions (apparent Kt = 2.5 mM). The mechanistic Na+-serine stoichiometry was determined to be 1:1 from the steady-state levels of the proton motive force, sodium gradient, and serine uptake. A 1:1 stoichiometry was also found for Na+-glutamate transport, and uptake of glutamate appeared to be an electroneutral process.     

Voltage-dependence of Ca2+ uptake and ATP hydrolysis of reconstituted Ca2+-ATPase vesicles

Ca2+-ATPase from sarcoplasmic reticulum was reconstituted into phospholipid/cholesterol (9:1) vesicles (RO). Sucrose density gradient centrifugation of the RO vesicles separated a light layer (RL) with a high lipid/protein ratio and a heavy layer (RH). RH vesicles exhibited a high rate of Ca2+-dependent ATP hydrolysis but did not accumulate Ca2+. RL vesicles, on the other hand, showed an initial molar ratio of Ca2+ uptake to ATP hydrolysis of approximately 1.0. Internal trapping of transported Ca2+ facilitated studies over periods of several minutes. Ca2+ transport and ATP hydrolysis declined concomitantly, reaching levels near 0 with external Ca2+ concentrations less than or equal to 2 microM. Ca2+ uptake was inhibited by the Ca2+ ionophore A23187, the detergent Triton X-100, and the metabolic inhibitor quercetin. Ca2+ transport generated a transient electrical potential difference, inside positive. This finding is consistent with the hypothesis that the Ca2+ pump is electrogenic. Steady state electrical potentials across the membrane were clamped by using potassium gradients and valinomycin, and monitored with voltage-sensitive dyes. Over a range of +50 to -100 mV, there was an inverse relationship between the initial rate of Ca2+ uptake and voltage, but the rate of ATP hydrolysis was nearly constant. In contrast, lowering the external Ca2+ concentration depressed both transport and ATP hydrolysis. These findings suggest that the membrane voltage influences the coupling between Ca2+ transport and ATP hydrolysis.     

Mg2+-ATP-dependent sodium transport in inside-out basolateral plasma membrane vesicles from guinea-pig small intestinal epithelial cells

The transport of sodium into inside-out basolateral plasma membrane vesicles from small intestinal epithelial cells has been examined. It was found, under equilibrium conditions, that binding of 22Na represents approx. 55% of the total uptake during an equilibration period of 30 min; 45% of the total uptake correspond to passive sodium entry in the vesicle space. In addition to binding and to passive Na+ entry, two distinct mechanisms capable of accumulating sodium in the intravesicular space can be demonstrated when ATP is added to the incubation medium. One transports sodium actively in the absence of potassium, whereas the other requires the presence of potassium in the interior of the vesicles. The two mechanisms can also be differentiated by their affinities for sodium, their optimal pH and by their behaviour towards different inhibitors. Thus, the mechanism that transports sodium in the absence of potassium is refractory to ouabain, but is inhibited by ethacrynic acid and furosemide, whilst the mechanism that accumulates sodium inside the vesicles in the presence of internal potassium is strongly inhibited by ouabain, is weakly inhibited by ethacrynic acid and is insensitive of furosemide. ATP is a specific stimulator of both processes, and the requirement for magnesium is absolute in both cases.     

Driving forces in hepatocellular uptake of phalloidin and cholate

Active uptake of phalloidin and cholate in isolated rat liver cells depends upon both Na⁺ gradient and membrane potential. Omission of Na⁺ or inhibition of the (Na⁺ + K⁺)-ATPase diminished both phalloidin and cholate uptake. Dissipation of the sodium, potassium or proton gradient by monensin, nigericin, gramicidin and valinomycin blocked phalloidin uptake and also caused reduction of cholate transport. Chelation of Ca²⁺ and Mg²⁺ by EGTA or incubation of liver cells with NH₄Cl neither influenced phalloidin nor cholate uptake. Hyperpolarization of liver cells by the lipophilic anions NO₃⁻ or SCN⁻ enhanced phalloidin but reduced cholate uptake. Depolarization induced by a reversed K⁺ gradient reduced both kinds of transport. The results indicate that sodium ions and the membrane potential are driving forces for phalloidin and cholate uptake in hepatocytes.     

Evidence for an organic cation-proton antiport system in brush-border membranes isolated from the human term placenta

Uptake of guanidine, an endogenous organic cation, into brush-border membrane vesicles isolated from human term placentas was investigated. Initial uptake rates were manyfold greater in the presence of an outward-directed H+ gradient ([pH]o greater than [pH]i) than in the absence of a H+ gradient ([pH]o = [pH]i). Guanidine was transiently accumulated inside the vesicles against a concentration gradient in the presence of the H+ gradient. The H+ gradient-dependent stimulation of guanidine uptake was not due to a H+-diffusion potential because an ionophore (valinomycin or carbonylcyanide p-trifluoromethoxyphenylhydrazone)-induced inside-negative membrane potential failed to stimulate the uptake. In addition, uphill transport of guanidine could be demonstrated even in voltage-clamped membrane vesicles. The H+ gradient-dependent uptake of guanidine was inhibited by many exogenous as well as endogenous organic cations (cis-inhibition) but not by cationic amino acids. The presence of unlabeled guanidine inside the vesicles stimulated the uptake of labeled guanidine (trans-stimulation). These data provide evidence for the presence of an organic cation-proton antiporter in human placental brush-border membranes. Kinetic analysis of guanidine uptake demonstrated that the uptake occurred via two saturable, carrier-mediated transport systems, one being a high affinity, low capacity type and the other a low affinity, high capacity type. Studies on the effects of various cations on the organic cation-proton antiporter and the Na+-H+ exchanger revealed that these two transport systems are distinct.     

Mechanism of uncoupling of oxidative phosphorylation by gramicidin

The mechanism of the uncoupling of oxidative phosphorylation in rat liver mitochondria by gramicidin and truncated gramicidin derivatives was investigated. The derivatives desformylgramicidin and des(formylvalyl)gramicidin are not expected to form head to head, dimeric, ion-conducting channels, and thus allow an evaluation of the relevance of the stimulation of transmembrane cation conductance (and the resulting collapse of the proton electrochemical gradient) to the uncoupling of oxidative phosphorylation. When assayed for the enhancement of the passive diffusion of KSCN, gramicidin was 100-fold more potent than desformylgramicidin and 50-fold more potent than des(formylvalyl)gramicidin. Yet, in a medium devoid of alkalai cations, all three compounds were nearly equally potent uncouplers at low concentrations. Moreover, this uncoupling was not associated with stimulation of cation transport or a reduction of the magnitude of the proton electrochemical potential. In the same medium, gramicidin stimulated 86Rb uptake 50-fold more than desformylgramicidin and 10 times more than des(formylvalyl)gramicidin. At higher concentrations, gramicidin induced further uncoupling, which was associated with reduction of membrane potential (and presumably with transport of alkali cations), while the truncated derivatives were considerably less effective than gramicidin in this range. Thus, with the truncated derivatives, a better separation between decoupling (i.e., uncoupling not associated with reduction of delta mu H) and uncoupling is observed. In the same medium, gramicidin, but not the truncated derivatives, strongly inhibits the formation of both the membrane potential and delta pH by the H+-ATPase. This finding suggests direct interaction of gramicidin with the H+-ATPase. The truncated derivatives stimulated the ATPase without collapsing the membrane potential.(ABSTRACT TRUNCATED AT 250 WORDS)     

The modification of the unidirectional calcium fluxes of sarcoplasmic reticulum vesicles by monovlent cation ionophroes

Calcium uptake by rabbit skeletal muscle sarcoplasmic reticulum vesicles in phosphate-containing media exhibits time-dependent changes that arise from changing rates of calcium influx and efflux. The monovalent cation ionophore gramicidin, added before the start of the calcium uptake reaction, delayed the spontaneous calcium release that normally occurred after approx. 6 min in such reactions; the rate of calcium efflux was inhibited while calcium influx was little affected. Under these conditions, Ca2+-activated ATPase activity could remain unaltered. Gramicidin stimulated calcium uptake irrespective of the presence of a K+ gradient across the vesicle membrane. Valinomycin stimulated calcium uptake in a manner similar to that for gramicidin even in an NaCl-containing medium lacking potassium. Thus, dissipation of a transmembrane K+ gradient is unlikely to account for the effects of these ionophores on the spontaneous changes in calcium flux rates. Addition of gramicidin to partially calcium-filled vesicles inhibited the phase of spontaneous calcium reuptake because both calcium influx and efflux wre inhibited. Addition of gramicidin to partially calcium-filled vesicles in the presence of a water-soluble protein, such as bovine serum albumin, creatine kinase or pyruvate kinase, markedly stimulated calcium uptake. This stimulatory effect was due primarily to inhibition of calcium efflux, calcium influx being minimally influenced by the ionophore. After cleavage of the 100,000 dalton ATPase to 50,000 dalton fragments, which was not associated with changes in Ca2+-activated ATPase activity or initial calcium uptake rate, gramicidin increased rather than decreased calcium content when added to vesicles after the initial maximum in calcium content. Thus, the ability of monovalent cation ionophores to block calcium efflux from calcium-filled vesicles may reflect their interaction with a portion of the Ca2+-activated ATPase protein.     

Mechanism of L-glutamate transport in membrane vesicles from Bacillus stearothermophilus.

In the presence of electrochemical energy, several branched-chain neutral and acidic amino acids were found to accumulate in membrane vesicles of Bacillus stearothermophilus. The membrane vesicles contained a stereo-specific transport system for the acidic amino acids L-glutamate and L-aspartate, which could not translocate their respective amines, L-glutamine and L-asparagine. The transport system was thermostable (Ti = 70 degrees C) and showed highest activities at elevated temperatures (60 to 65 degrees C). The membrane potential or pH gradient could act as the driving force for L-glutamate uptake, which indicated that the transport process of L-glutamate is electrogenic and that protons are involved in the translocation process. The electrogenic character implies that the anionic L-glutamate is cotransported with at least two monovalent cations. To determine the mechanistic stoichiometry of L-glutamate transport and the nature of the cotranslocated cations, the relationship between the components of the proton motive force and the chemical gradient of L-glutamate was investigated at different external pH values in the absence and presence of ionophores. In the presence of either a membrane potential or a pH gradient, the chemical gradient of L-glutamate was equivalent to that specific gradient at different pH values. These results cannot be explained by cotransport of L-glutamate with two protons, assuming thermodynamic equilibrium between the driving force for uptake and the chemical gradient of the substrate. To determine the character of the cotranslocated cations, L-glutamate uptake was monitored with artificial gradients. It was established that either the membrane potential, pH gradient, or chemical gradient of sodium ions could act as the driving force for L-glutamate uptake, which indicated that L-glutamate most likely is cotranslocated in symport with one proton and on sodium ion.