Evidence for an efflux carrier system involved in the secretion of glutamate by Corynebacterium glutamicum

Corynebacterium glutamicum effectively secretes L-glutamate when growing under biotin limitation. The secretion of glutamate was studied with respect to kinetic and energetic parameters: rate of glutamate uptake and efflux, specificity of transport, dependence of efflux on the energy state of the cell, concentration gradient of glutamate and ions, and membrane potential. By comparing these parameters when measured in biotin-limited, i.e. producer cells, and biotin-supplemented, i.e. non-producer cells, respectively, the following conclusions could be drawn: 1. The efflux of L-glutamate in C. glutamicum cannot be explained by passive permeation of this amino acid through the plasma membrane, as it has been assumed in the generally accepted model of glutamate secretion in biotin-limited cells. 2. It is unlikely that the efflux of glutamate occurs via an inversion of the glutamate uptake system. 3. Based on our results concerning the specificity and the kinetics of glutamate transport as well as the observed regulation phenomena, we conclude that secretion of glutamate in C. glutamicum occurs by a special efflux carrier system.Abbreviations dw dry weight - OD optical density - TPP tetraphenyl phosphonium bromide     

Cell Death Induced by Mild Physical Perturbations Could Be Related to Transient Plasma Membrane Modifications

An understanding of membrane destabilization induced by osmotic treatments is important to better control cell survival during biotechnological processes. The effects on the membranes of the yeast Saccharomyces cerevisiae of perturbations similar in intensity (same amount of energy) but differing in the source type (heat, compression and osmotic gradient) were investigated. The anisotropy of the fluorescent probe 1,6-diphenyl-1,3,5-hexatriene was measured before and after each treatment to assess the reversibility of the membrane changes related to each treatment. Except for heat shock at 75°C, changes in membrane fluidity were reversible after the return to initial conditions, showing that two kinds of physical stress can be distinguished regarding the reversibility of membrane changes: high and mild energy stresses. With the application of osmotic gradients, anisotropy was assessed during treatment with five osmotic pressure levels from 30.7 to 95.4 MPa with two different yeast strains and related to the rate of cell death caused by each stress. The exposure of cells to increasing osmotic pressures involved a progressive lowering of membrane anisotropy during lethal perturbations. Osmotic stresses associated with reversible fluidity changes of increasing intensity in the membrane led to proportional death rates and time-dependant cell death of increasing rapidity during the application of the stress. Finally, a hypothesis relating the extent of membrane structural changes to the kinetic of cell death is proposed.     

Rates of chilling to 0°C: implications for the survival of microorganisms and relationship with membrane fluidity modifications

The effects of slow chilling (2°C min⁻¹) and rapid chilling (2,000°C min⁻¹) were investigated on the survival and membrane fluidity of Escherichia coli, of Bacillus subtilis, and of Saccharomyces cerevisiae. Cell death was found to be dependent on the physiological state of cell cultures and on the rate of temperature downshift. Slow temperature decrease allowed cell stabilization, whereas the rapid chilling induced an immediate loss of viability of up to more than 90 and 70% for the exponentially growing cells of E. coli and B. subtilis, respectively. To relate the results of viability with changes in membrane physical state, membrane anisotropy variation was monitored during thermal stress using the fluorescence probe 1,6-diphenyl-1,3,5-hexatriene. No variation in the membrane fluidity of all the three microorganisms was found after the slow chilling. It is interesting to note that fluorescence measurements showed an irreversible rigidification of the membrane of exponentially growing cells of E. coli and B. subtilis after the instantaneous cold shock, which was not observed with S. cerevisiae. This irreversible effect of the rapid cold shock on the membrane correlated well with high rates of cell inactivation. Thus, membrane alteration seems to be the principal cause of the cold shock injury.     

Membrane fluidity of halophilic ectoine-secreting bacteria related to osmotic and thermal treatment

In response to sudden decrease in osmotic pressure, halophilic microorganisms secrete their accumulated osmolytes. This specific stress response, combined with physiochemical responses to the altered environment, influence the membrane properties and integrity of cells, with consequent effects on growth and yields in bioprocesses, such as bacterial milking. The aim of this study was to investigate changes in membrane fluidity and integrity induced by environmental stress in ectoine-secreting organisms. The halophilic ectoine-producing strains Alkalibacillus haloalkaliphilus and Chromohalobacter salexigens were treated hypo- and hyper-osmotically at several temperatures. The steady-state anisotropy of fluorescently labeled cells was measured, and membrane integrity assessed by flow cytometry and ectoine distribution. Strong osmotic downshocks slightly increased the fluidity of the bacterial membranes. As the temperature increased, the increasing membrane fluidity encouraged more ectoine release under the same osmotic shock conditions. On the other hand, combined shock treatments increased the number of disintegrated cells. From the ectoine release and membrane integrity measurements under coupled thermal and osmotic shock conditions, we could optimize the secretion conditions for both bacteria.     

Psychrophilic <Emphasis Type="Italic">Pseudomonas syringae</Emphasis> requires <Emphasis Type="Italic">trans</Emphasis>-monounsaturated fatty acid for growth at higher temperature

A psychrophilic bacterium, Pseudomonas syringae (Lz4W) from Antarctica, was used as a model system to establish a correlation, if any, between thermal adaptation, trans-fatty acid content and membrane fluidity. In addition, attempts were made to clone and sequence the cti gene of P. syringae (Lz4W) so as to establish its characteristics with respect to the cti of other Pseudomonas spp. and also to in vitro mutagenize the cti gene so as to generate a cti null mutant. The bacterium showed increased proportion of saturated and trans-monounsaturated fatty acids when grown at 28°C compared to cells grown at 5°C, and the membrane fluidity decreased with growth temperature. In the mutant, the trans-fatty acid was not synthesized, and the membrane fluidity also decreased with growth temperature, but the decrease was not to the extent that was observed in the wild-type cells. Thus, it would appear that synthesis of trans-fatty acid and modulation of membrane fluidity to levels comparable to the wild-type cells is essential for growth at higher temperatures since the mutant exhibits growth arrest at 28°C. In fact, the cti null mutant-complemented strain of P. syringae (Lz4W-C30b) that was capable of synthesizing the trans-fatty acid was indeed capable of growth at 28°C, thus confirming the above contention. The cti gene of P. syringae (Lz4W) that was cloned and sequenced exhibited high sequence identity with the cti of other Pseudomonas spp. and exhibited all the conserved features.     

Biochemical changes accompanying the long-term starvation of Micrococcus luteus cells in spent growth medium

Changes in the biochemical properties of Micrococcus luteus cells were studied during the transition to a dormant state after incubation in an extended stationary phase. The overall DNA content after 150 days of starvation was similar to its initial level, while the RNA content decreased by 50%. Total lipids and protein, phospholipids and membrane proteins declined rapidly within the first 1–10 days of starvation. After 180 days of starvation, cells contained 43% of the protein and 35% of the lipid initially present. Starvation for 120 days resulted in the loss of phosphatidylglycerol and, to some extent, of phosphatidylinositol, giving a membrane whose phospholipids consisted mainly of cardiolipin. The membrane fluidity declined during starvation, as judged by diphenyl hexatriene fluorescence anisotropy measurements. Oxidase activities declined to zero within the first 20–30 days of starvation, while the dehydrogenases and cytochromes were more stable. The activities of some cytoplasmic enzymes were lost very rapidly, while NADPH-linked isocitrate dehydrogenase had 30% of its initial activity after 120 days of starvation. For all parameters tested there were significant fluctuations during the first 10–20 days of starvation, which may reflect cryptic growth in the culture.Abbreviations MPN Most probable number - DPH Diphenyl hexatriene     

Membrane fluidity determines sensitivity of filamentous fungi to chitosan

The antifungal mode of action of chitosan has been studied for the last 30 years, but is still little understood. We have found that the plasma membrane forms a barrier to chitosan in chitosan‐resistant but not chitosan‐sensitive fungi. The plasma membranes of chitosan‐sensitive fungi were shown to have more polyunsaturated fatty acids than chitosan‐resistant fungi, suggesting that their permeabilization by chitosan may be dependent on membrane fluidity. A fatty acid desaturase mutant of Neurospora crassa with reduced plasma membrane fluidity exhibited increased resistance to chitosan. Steady‐state fluorescence anisotropy measurements on artificial membranes showed that chitosan binds to negatively charged phospholipids that alter plasma membrane fluidity and induces membrane permeabilization, which was greatest in membranes containing more polyunsaturated lipids. Phylogenetic analysis of fungi with known sensitivity to chitosan suggests that chitosan resistance may have evolved in nematophagous and entomopathogenic fungi, which naturally encounter chitosan during infection of arthropods and nematodes. Our findings provide a method to predict the sensitivity of a fungus to chitosan based on its plasma membrane composition, and suggests a new strategy for antifungal therapy, which involves treatments that increase plasma membrane fluidity to make fungi more sensitive to fungicides such as chitosan.     

Cultured human skin fibroblasts modify their plasma membrane lipid composition and fluidity according to growth temperature suggesting homeoviscous adaptation at hypothermic (30 degrees C) but not at hyperthermic (40 degrees C) temperatures.

Mammalian cell metabolism is responding to changes in temperature. Body temperature is regulated around 37 degrees C, but temperatures of exposed skin areas may vary between 20 degrees C and 40 degrees C for extended periods of time without apparent disturbance of adequate cellular functions. Cellular membrane functions are depending from temperatures but also from their lipid environment, which is a major component of membrane fluidity. Temperature-induced changes of membrane fluidity may be counterbalanced by adaptive modification of membrane lipids. Temperature-dependent changes of whole cell- and of purified membrane lipids and possible homeoviscous adaptation of membrane fluidity have been studied in human skin fibroblasts cultured at 30 degrees C, 37 degrees C, and 40 degrees C for ten days. Membrane anisotropy was measured by polarized fluorescence spectroscopy using TMA-DPH for superficial and DPH for deeper membrane layers. Human fibroblasts were able to adapt themselves to hypothermic temperatures (30 degrees C) by modifying the fluidity of the deeper apolar regions of the plasma membranes as reported by changes of fluorescence anisotropy due to appropriate changes of their plasma membrane lipid composition. This could not be shown for the whole cells. At 40 degrees C growth temperature, adaptive changes of the membrane lipid composition, except for some changes in fatty acid compositions, were not seen. Independent from the changes of the membrane lipid composition, the fluorescence anisotropy of the more superficial membrane layers (TMA-DPH) increased in cells growing at 30 degrees C and decreased in cells growing at 40 degrees C.     

Analysis of composition and structure of <Emphasis Type="Italic">Clostridium thermocellum</Emphasis> membranes from wild-type and ethanol-adapted strains

Clostridium thermocellum is a candidate organism for consolidated bioprocessing of lignocellulosic biomass into ethanol. However, commercial use is limited due to growth inhibition at modest ethanol concentrations. Recently, an ethanol-adapted strain of C. thermocellum was produced. Since ethanol adaptation in microorganisms has been linked to modification of membrane lipids, we tested the hypothesis that ethanol adaptation in C. thermocellum involves lipid modification by comparing the fatty acid composition and membrane anisotropy of wild-type and ethanol-adapted strains. Derivatization to fatty acid methyl esters provided quantitative lipid analysis. Compared to wild-type, the ethanol-adapted strain had a larger percentage of fatty acids with chain lengths >16:0 and showed a significant increase in the percentage of 16:0 plasmalogens. Structural identification of fatty acids was confirmed through mass spectral fragmentation patterns of picolinyl esters. Ethanol adaptation did not involve modification at sites of methyl branching or the unsaturation index. Comparison of steady-state fluorescence anisotropy experiments, in the absence and presence of ethanol, provided evidence for the effects of ethanol on membrane fluidity. In the presence of ethanol, both strains displayed increased fluidity by approximately 12%. These data support the model that ethanol adaptation was the result of fatty acid changes that increased membrane rigidity that counter-acted the fluidizing effect of ethanol.     

Evidence for a correlation between swimming velocity and membrane fluidity of Tetrahymena cells

The influence of the physical state of the membrane on the swimming behaviour of Tetrahymena pyriformis was studied in cells with lipid-modified membranes. When the growth temperature of Tetrahymena cells was increased from 15°C to 34°C or decreased from 39°C to 15°C, their swimming velocity changed gradually in a similar to the adaptive change in membrane lipid composition. Therefore, such adaptive changes in swimming velocity were not observed during short exposures to a different environment. Tetrahymena cells adapted to 34°C swam at 570 μm/s. On incubation at 15°C these cells swam at 100 μm/s. When the temperature was increased to 34°C after a 90-min incubation at 15°C, the initial velocity was immediately recovered. On replacement of tetrahymanol with ergosterol, the swimming velocity of 34°C-grown cells decreased to 210 μm/s, and the cells ceased to move when the temperature was decreased to 15°C. To investigate the influence of the physical state of the membrane on the swimming velocity, total phospholipids were prepared from Tetrahymena cells grown under these different conditions. The fluidities of liposomes of these phospholipid were measured using stearate spin probe. The membrane fluidity of the cells cooled to 15°C increased gradually during incubation at 15°C. On the other hand, the fluidity of the heated cell decreased during incubation at 34°C. Replacement of tetrahymanol with ergosterol decreased the membrane fluidity markedly. Consequently, a good correlation was observed between swimming velocity and membrane fluidity; as the membrane fluidity increased, the swimming velocity increased linearly up to 600 μm/s. These results provide evidence for the regulation of the swimming behaviour by physical properties of the membrane.     

Loss of carotenoids from membranes of Pantoea sp. YR343 results in altered lipid composition and changes in membrane biophysical properties

Bacterial membranes are complex mixtures of lipids and proteins, the combination of which confers biophysical properties that allows cells to respond to environmental conditions. Carotenoids are sterol analogs that are important for regulating membrane dynamics. The membrane of Pantoea sp. YR343 is characterized by the presence of the carotenoid zeaxanthin, and a carotenoid-deficient mutant, ΔcrtB, displays defects in root colonization, reduced secretion of indole-3-acetic acid, and defects in biofilm formation. Here we demonstrate that the loss of carotenoids results in changes to the membrane lipid composition in Pantoea sp. YR343, including increased amounts of unsaturated fatty acids in the ΔcrtB mutant membranes. These mutant cells displayed less fluid membranes in comparison to wild type cells as measured by fluorescence anisotropy of whole cells. Studies with artificial systems, however, have shown that carotenoids impart membrane rigidifying properties. Thus, we examined membrane fluidity using spheroplasts and vesicles composed of lipids extracted from either wild type or mutant cells. Interestingly, with the removal of the cell wall and membrane proteins, ΔcrtB vesicles were more fluid than vesicles made from lipids extracted from wild type cells. In addition, carotenoids appeared to stabilize membrane fluidity during rapidly changing temperatures. Taken together, these results suggest that Pantoea sp. YR343 compensates for the loss of carotenoids by changing lipid composition, which together with membrane proteins, results in reduced membrane fluidity. These changes may influence the abundance or function of membrane proteins that are responsible for the physiological changes observed in the ΔcrtB mutant cells.     

HDL derived from the different phases of conjugated diene formation reduces membrane fluidity and contributes to a decrease in free cholesterol efflux from human THP-1 macrophages

Oxidized HDL (ox-HDL) has been reported to reduce free cholesterol efflux from cells. In this study we investigate the effect of different stages of ox-HDL on macrophage membrane fluidity and its effect on free cholesterol efflux from macrophages as a cell function influenced by ox-HDL. HDL was oxidized by means of conjugated diene production using copper as a prooxidant. Fluidity of HDL and human THP-1 macrophage membranes was evaluated by changes in fluorescence anisotropy (r) by DPH probe where lower (r) values give higher fluidity. We found that ox-HDL derived from the propagation phase (PP-HDL) and the decomposition phase (DP-HDL) became less fluid ((r): 0.263+/-0.001, 0.279+/-0.002, respectively) than HDL from the lag phase (LP-HDL) and native HDL (nat-HDL) ((r): 0.206+/-0.001) (P<0.05). Macrophages incubated with PP-HDL and DP-HDL had less fluid membranes ((r): 0.231+/-0.001, 0.243+/-0.002, respectively) than those incubated with LP-HDL and nat-HDL ((r): 0.223+/-0.001) (P<0.05). Consequently, fluidity was reduced not only in ox-HDL but also in the cell membranes exposed to ox-HDL. A significant negative correlation was observed between macrophage membrane fluorescence anisotropy (r) and free cholesterol efflux from these cells (-0.876; P<0.05). Thus, lower membrane fluidity was associated with lower free cholesterol efflux from cells. In conclusion, the increase in the HDL oxidation process leads to a lost of macrophage membrane fluidity that could contribute to an explanation of the reduction of free cholesterol efflux from cells by ox-HDL.     

Membrane oxidative damage induced by ionizing radiation detected by fluorescence polarization

Effects of ionizing radiation on biological membranes include alterations in membrane proteins, peroxidation of unsaturated lipids accompanied by perturbations of the lipid bilayer polarity. We have measured radiation-induced membrane modifications using two fluorescent lipophilic membrane probes (TMA-DPH and DPH) by the technique of fluorescence polarization on two different cell lines (Chinese hamster ovary CHO-K1 and lymphoblastic RPMI 1788 cell lines). γ-Irradiation was performed using a ⁶⁰Co source with dose rates of 0.1 and 1 Gy/min for final doses of 4 and 8 Gy. Irradiation induced a decrease of fluorescence intensity and anisotropy of DPH and TMA-DPH in both cell lines, which was dose-dependent but varied inversely with the dose rate. Moreover, the fluorescence anisotropy measured in lymphoblastic cells using TMA-DPH was found to decrease as early as 1 h after irradiation, and remained significantly lower 24 h after irradiation. This study indicates that some alterations of membrane fluidity are observed after low irradiation doses and for some time thereafter. The changes in membrane fluidity might reflect oxidative damage, thus confirming a radiation-induced fluidization of biological membranes. The use of membrane fluidity changes as a potential biological indicator of radiation injury is discussed. Received: 14 May 1996 / Accepted in revised form: 30 September 1996     

Heptanol-induced decrease in cardiac gap junctional conductance is mediated by a decrease in the fluidity of membranous cholesterol-rich domains

To assess whether alterations in membrane fluidity of neonatal rat heart cells modulate gap junctional conductance (g _j ), we compared the effects of 2mm 1-heptanol and 20 μm 2-(methoxyethoxy)ethyl 8-(cis-2-n-octylcyclopropyl)-octanoate (A₂C) in a combined fluorescence anisotropy and electrophysiological study. Both substances decreased fluorescence steady-state anisotropy (r_ss), as assessed with the fluorescent probe 1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene (TMA-DPH) by 9.6±1.1% (mean ±sem,n=5) and 9.8±0.6% (n=5), respectively, i.e., both substances increased bulk membrane fluidity. Double whole-cell voltage-clamp experiments showed that 2mm heptanol uncoupled cell pairs completely (n=6), whereas 20 μm A₂C, which increased bulk membrane fluidity to the same extent, did not affect coupling at all (n=5). Since gap junction channels are embedded in relatively cholesterol-rich domains of the membrane, we specifically assessed the fluidity of the cholesterol-rich domains with dehydroergosterol (DHE). Using DHE, heptanol increased r_ss by 14.9±3.0% (n=5), i.e., decreased cholesterol domain fluidity, whereas A₂C had no effect on r_ss (−0.4±6.7%,n=5). Following an increase of cellular “cholesterol” content (by loading the cells with DHE), 2mm heptanol did not uncouple cell pairs completely:g _j decreased by 80±20% (range 41–95%,n=5). The decrease ing _j was most probably due to a decrease in the open probability of the gap junction channels, because the unitary conductances of the channels were not changed nor was the number of channels comprising the gap junction. The sensitivity of non-junctional membrane channels to heptanol was unaltered in cholesterol-enriched myocytes. These results indicate that the fluidity of cholesterol-rich domains is of importance to gap junctional coupling, and that heptanol decreasesg _j by decreasing the fluidity of cholesterol-rich domains, rather than by increasing the bulk membrane fluidity.     

Thermal adaptation in biological membranes: interacting effects of temperature and pH

Summary The interacting effects of pH and temperature on membrane fluidity were studied in plasma membranes isolated from liver of rainbow trout (Oncorhynchus mykiss) acclimated to 5 and 20°C. Fluidity was determined as a function of temperature under conditions of both constant (in potassium phosphate buffer) and variable pH (in imidazole buffer, consistent with imidazole alphastat regulation) from the fluorescence anisotropy of two probes: 1,6-diphenyl-1,3,5-hexatriene, which intercalates into the bilayer interior, and 1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene which is anchored at the membrane/water interface. The temperature dependence of the anisotropy parameter for 1,6-diphenyl-1,3,5-hexatriene in plasma membranes of 20°C-acclimated trout was greater when determined in phosphate (AP per °C=-0.047) than in imidazole buffer (AP per °C=-0.022); similar, but less significant, trends were noted with 1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene. In contrast, the temperature dependence of fluidity (AP/°C in the range-0.0222 to-0.027) did not vary with buffer composition in membranes of 5°C-acclimated trout. In phosphate buffer, anisotropy parameter values for 1,6-diphenyl-1,3,5-hexatriene were significantly lower in 5°C-than 20°C-acclimated trout, indicating a less restricted probe environment following cold acclimation and nearly perfect compensation (91%) of fluidity. Temperature-dependent patterns of acid-base regulation were estimated to account for 11–40% of the fluidization evident in membranes of 5°C-trout, but a period of cold acclimation was required for complete fluidity compensation. In contrast, no homeoviscous adaptation was evident in imidazole buffer, indicating that membrane fluidity is sensitive to buffer composition. Accordingly, vesicles of bovine brain phosphatidylcholine, suspensions of triolein, and plasma membranes of 5°C-acclimated trout were consistently more fluid in imidazole than phosphate buffer. Membranes of 5°C-acclimated trout were enriched in molecular species of phosphatidylcholine containing 22:6n3 (at the expense of species containing 18:1n9 and 18:2n6) compared to membranes of 20°C-trout; consequently, the unsaturation index was significantly higher (3.29 versus 2.73) in trout maintained at 5 as opposed to 20°C. It is concluded that: 1) the chemical composition of the internal milieu can significantly influence the physical properties of membrane lipids; 2) temperature-dependent patterns of intracellular pH regulation may partially offset the ordering effect of low temperature on membrane fluidity in 20°C-acclimated trout transferred to 5°C, but not in 5°C-acclimated trout transferred to warmer temperatures; 3) the majority of the thermal compensation of plasma membrane fluidity resulting from a period of temperature acclimation most likely reflects differences in membrane composition between acclimation groups; 4) imidazole apparently interacts with trout hepatocyte plasma membranes in a unique way.Abbreviations _im netcharge stateofproteins - AP anisotropyparameter - bw body weight - DPH 1,6-diphenyl-1,3,5-hexatriene - HEPES N-2-hydroxyethylpiperazine-N-2-ethanesulphonicacid - PC phosphatidylcholine - pH_e pHofarterial blood - pH_i intracellular pH - TMA-DPH 1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene - TRIS tris(hydroxymethyl)aminomethane     

Instructions for authors

Sex differences in the kinetic parameters of p-aminohippuric acid (PAH) transport in brush border (BBMV) and basolateral (BLMV) membrane vesicles from kidney cortex have been observed. Membrane fluidity of BBMV was higher in females as compared with male rats as indicated by anisotropy values (0.1897 ± 0.0010 vs. 0.2003 ± 0.0014, p < 0.05, for females and males respectively). Membrane fluidity of BLMV were similar in both sexes. Western blot studies revealed that OAT1 protein in female BLMV was present at only 40% of level found in BLMV from male rats. The lower expression of OAT1 in BLMV in association with the higher BBMV fluidity (which may affect the affinity of PAH transporter in this membrane domain) observed in females may be responsible, at least in part, for the gender difference described in renal PAH secretion.     

Effect of temperature on membrane fluidity and calcium conductance of the excitable ciliary membrane from Paramecium

Fluorescence anisotropy and average fluorescence lifetime of diphenylhexatriene were measured in artificial lipid membrane vesicles. Within the temperature range investigated (15–52°C) both parameters correlate and can be used interchangeably to measure membrane fluidity. Fluorescence anisotropy of DPH in membrane vesicles of cilia from the protozoan Paramecium tetraurelia decreased slightly from 5 to 37°C, yet, no phase transition was observed. An estimated flow activation energy of approx. 2 kcal/mol indicated that the ciliary membrane is very rigid and not readily susceptible to environmental stimuli. The ciliary membrane contains two domains of different membrane fluidity as indicated by two distinct fluorescence lifetimes of diphenylhexatriene of 7.9 and 12.4 ns, respectively. Ca²⁺ flux into ciliary membrane vesicles of Paramecium as measured with the Ca²⁺ indicator dye arsenazo III showed a nonlinear temperature dependency from 5 to 35°C with a minimum around 15°C and increasing flux rates at higher and lower temperatures. The fraction of vesicles permeable for Ca²⁺ remained unaffected by temperature. The differences in temperature dependency of Ca²⁺ conductance and membrane fluidity indicate that the Ca²⁺ permeability of the ciliary membrane is a membrane property which is not directly affected by the fluidity of its lipid environment.     

Triggering Glutamate Excretion in Corynebacterium glutamicum by Modulating the Membrane State with Local Anesthetics and Osmotic Gradients

Corynebacterium glutamicum can be triggered to excrete glutamate by the addition of local anesthetics, particularly tetracaine. Glutamate efflux is a carrier-mediated process and not due to unspecific membrane permeabilization. The concentration of local anesthetics triggering optimum excretion depended on the type of anesthetic and varied, ranging from 0.1 (chlorpromazine), 1.3 (tetracaine), and 2.6 mM (butacaine) to 15 mM (benzocaine), in close resemblance to the order of efficiency in anesthetic effect. The onset of glutamate excretion was not correlated to a change in the viscosity or fluidity of the membrane, as measured by electron spin resonance spectroscopy, nor was it related to an action of the anesthetic as an uncoupler. Tetracaine-triggered glutamate excretion was sensitive to changes in the transmembrane osmotic gradient, although an osmotic gradient alone could not trigger glutamate excretion. Tetracaine-triggered glutamate efflux was inhibited by an external rise in osmolality and stimulated by a corresponding decrease. The effects of osmotic gradients and the addition of local anesthetics on glutamate excretion were mutually exchangeable, indicating similar modes of action. We suggest that this common principle is a change in the membrane strain. C. glutamicum cells which excrete glutamate without manipulation of the membrane, e.g., biotin-limited cells or glutamate production mutants, were not stimulated by the addition of tetracaine.     

Influence of Membrane Physical State on the Lysosomal Proton Permeability

Influence of membrane physical state on the proton permeability of isolated lysosomes was assessed by measuring the membrane potential with 3,3′-dipropylthiadicarbocyanine iodide and monitoring their proton leakage with p-nitrophenol. Changes in the membrane order were examined by the steady-state fluorescence anisotropy of 1,6-diphenyl-1,3,5-hexatriene. Both the membrane potential and proton leakage increased with fluidizing the lysosomal membranes by benzyl alcohol and decreased with rigidifying the membranes by cholesteryl hemisuccinate. The proton permeability increased to the maximum of 42% by the benzyl alcohol treatment and decreased to the minimum of 38.1% by the cholesteryl hemisuccinate treatment. Treating the lysosomes with protonophore CCCP increased the proton permeability by 58%. The effects of the membrane fluidization and rigidification can be reversed by rigidifying the fluidized membranes and fluidizing the rigidified membranes, respectively. The results indicate that the proton permeability of lysosomes increased and decreased with increasing and decreasing their membrane fluidity, respectively. Moreover, the lysosomal proton permeability did not alter further if the changes, either an increase or a decrease, in the fluidity exceeded some amount. The results suggest that the proton permeability of lysosomes can be modulated finitely by the alterations in their membrane physical state. Received: 27 September 1999 / Revised: 27 December 1999     

Use of laurdan fluorescence intensity and polarization to distinguish between changes in membrane fluidity and phospholipid order

Laurdan is a fluorescent probe that detects changes in membrane phase properties through its sensitivity to the polarity of its environment in the bilayer. Variations in membrane water content cause shifts in the laurdan emission spectrum, which are quantified by calculating the generalized polarization (GP). We tested whether laurdan fluorescence could be used to distinguish differences in phospholipid order from changes in membrane fluidity by examining the temperature dependence of laurdan GP and fluorescence anisotropy in dipalmitoylphosphatidylcholine (DPPC) vesicles. The phase transition from the solid ordered phase to the liquid disordered phase was observed as a decrease in laurdan GP values from 0.7 to −0.14 and a reduction in anisotropy from 0.25 to 0.12. Inclusion of various amounts of cholesterol in the membranes to generate a liquid ordered phase caused an increase in the apparent melting temperature detected by laurdan GP. In contrast, cholesterol decreased the apparent melting temperature estimated from anisotropy measurements. Based on these results, it appeared that laurdan anisotropy detected changes in membrane fluidity while laurdan GP sensed changes in phospholipid order. Thus, the same fluorescent probe can be used to distinguish effects of perturbations on membrane order and fluidity by comparing the results of fluorescence emission and anisotropy measurements.