Osmotically induced removal of water from fungal cells as determined by a spin probe technique

Effects of physical environment on plasma membrane semipermeability and osmotic induction of changes in aqueous cytoplasmic volume were studied in vegetative and spore cells of a plant pathogenic fungus, Fusarium sulphureum. A direct method, employing a spin probe molecule that partitioned between intracellular aqueous and hydrophobic phases, allowed measurement of reversible water movement out of macroconidial cells and chlamydospores exposed to solutions of high osmolarity. Equilibrium distribution of the spin probe between intracellular aqueous and lipid phases was more rapid than movement of water in and out of the cells. The extent of water removal was exponentially dependent on osmotic strength. Some cells became irreversibly permeable to divalent cations on treatment with sodium chloride above 1.5 osmolar but addition of sucrose to the suspension medium at equivalent osmolar concentrations caused water removal without adversely affecting the viability. Sucrose also protected the plasma membrane against damage during freeze-drying. Induction of plasma membrane damage by osmotic shock or freeze-drying permitted rapid permeation of nickel ions. Neither slow equilibration of intracellular components with divalent paramagnetic cations nor partial permeability of damaged plasma membranes to these ions was observed.     

Membrane damage by hemolytic viruses, toxins, complement, and other cytotoxic agents. A common mechanism blocked by divalent cations

Hemolytic viruses, bacterial and animal toxins, the components of activated complement, cationic proteins, and detergents induce a sequence of permeability changes at the plasma membrane that are in every case sensitive to changes in ionic strength and to divalent cations. Individually, each agent exhibits positive cooperativity; when two agents are present together, they show synergy. It is concluded that such cytotoxic agents damage membranes by a common mechanism. Hence permeability changes are unlikely to depend on the formation of specific, protein-lined channels, as previously envisaged in the case of activated complement or certain bacterial toxins.     

Studies on the cation permeability of human red cell ghosts. Characterization and biological significance of two membrane sites with high affinities for Ca.

Net K movements in reconstituted human red cell ghosts and the resealing of ghosts to cations after osmotic hemolysis of red cells have been studied as functions of the free Ca ion concentration. The Ca-dependent specific increase in K permeability was shown to be mediated by a site close to the internal surface of the membrane with an apparent dissociation constant ap pH 7.2 for Ca (K'p1) of 3-5 X 10(-7) M, for Sr of 7 X 10(-6) M. Ba and Mg did not increase the K-permeability of the membrane but inhibited the Ca-mediated permeability changes. K'D1 decreased in a nonlinear fashion when the pH was increased from 6.0 to 8.5. Two different pK' values of this membrane site were found at pH 8.3 and 6.3. The Ca-activated net K efflux into a K-free medium was almost completely inhibited by an increase in intracellular Na from 4 to 70mM. Extracellular K antagonized this Na effect. Changes in the extracellular Na (0.1-140 mM) or K (0.1-6 mM) concentrations had little effect and did not change K'p1. The Ca-stimulated recovery of a low cation permeability in ghost cells appeared to be mediated by a second membrane site which was accessible to divalent cations only during the process of hemolysis in media of low ionic strength. The apparent dissociation constant for Ca at this site (K'p2) varied between 6 X 10(-7) and 4 X 10(-6) M at pH 7.2 Mg, Sr, and Ba could replace Ca functionally. The selectivity sequence was Ca greater than Sr greater than Ba greater than Mg. K'p2 was independent on the pH value in the range between 6.0 and 8.0 Hill coefficients of 2 were observed for the interaction of Ca with both membrane sites suggesting that more than one Ca ion is bound per site. The Hill cofficients were affected neither by the ion composition nor by the Ph values of the intra-and extracellular media. It is concluded that two different pathways for the permeation of cations across the membrane are controlled by membrane sites with high affinities for Ca: One specific for K, one unspecific with respect to cations. The K-specific "channel" has properties similar to the K channel in excitable tissues.     

Effect of surface-active agents on the electrical properties of bacterial cells

The work was aimed at studying the effect of cationic, anionic and non-ionogenic surfactants on the frequency dependence of the electroorientation effect (EOE) and on the electrophoretic mobility (EPM) of rod-like bacteria. The character of concentration dependences was found to differ for EOE and EPM at a low frequency of the electric field (20 to 10(4) Hz). Analysis of EOE changes at a high frequency (4 X 10(5) to 3 X 10(7) Hz) showed that anionic and non-ionogenic surfactants at a concentration up to 10(-3) M did not damage Escherichia coli, a Gram-negative bacterium, in contrast to Bacillus cereus, a Gram-positive bacterium. Cationic surfactants affected the cells of the both species. The optical properties of bacterial cells were found to change under the action of cationic surfactants.     

Inhibition of anion and glucose permeabilities by anesthetics in erythrocytes. The mechanisms of action of positively and negatively charged drugs

(1) The mode of action of anesthetics as inhibitors of Cl- and glucose transports in human red cells was studied. The term anesthetic is taken in its broad meaning as defined by Seeman (Seeman, P. (1972) Pharmacol. Rev. 24, 583-655) and covers anionic and cationic liposoluble compounds which reversibly block the rising phase of the action potential, without effect on the resting membrane potential. (2) Phenothiazine derivatives were chosen as prototypes of anesthetics because they represent a set of compounds having the same basic chemical structure, the phenothiazine ring, but with either a positive or a negative charge. (3) The Cl- self-exchange is inhibited by both cationic and anionic derivatives. However, to obtain the same level of inhibition, it is necessary to use a concentration 10-100 times higher with cationic than with anionic drugs. (4) At a concentration which inhibits Cl- permeability, cationic derivatives induce a very strong morphological change (cup-shaped cells: stomatocytes or spherostomatocytes) and protect erythrocytes against osmotic hemolysis, signifying that the membrane is fully expanded. Conversely, with anionic derivatives, inhibition occurs at a concentration which does not induce any apparent shape change or protect against osmotic hemolysis: there is no significant membrane expansion. (5) Glucose permeability, measured by glucose exit, is inhibited by cationic and anionic phenothiazine, but always at a concentration which fully expands the membrane as indicated by morphological changes and anti-hemolytic effects. It is interesting to point out that whilst glucose exit shows inhibition by cationic derivatives, glucose exchange flux is scarcely altered. (6) It is concluded that cationic and anionic anesthetics are general inhibitors of transmembrane solute movements involving a facilitated-diffusion process. However, the mechanism of inhibition is not identical for all: inhibition of glucose permeability by anionic and cationic anesthetics, as well as inhibition of Cl- permeability by cationic anesthetics may be of a non-specific nature and result from their interaction with the bilayer (this indirect effect is discussed); on the other hand, inhibition of Cl- permeability by anionic anesthetics may result from a specific perturbation of the transport mechanism according to recent evidence in some cases (Cousin, J.L. and Motais, R. (1979) J. Membrane Biol. 46, 125-153; Zaki, L., Ruffing, W. G?rtner, E.M., Fasold, H., Motais, R. and Passow, H. (1977) 11th FEBS Meeting, Copenhagen, A4 17-671.     

Proton-linked transport systems as sensors of changes in the membrane surface potential

The kinetic properties of proton linked transport systems and their relation to the membrane surface potential were studied in yeast cells. (1) The negative surface potential of cells rich in anionic phospholipids was found to be 2-times higher than that of control cells; in agreement with their 2-fold increase in the anionic/zwitterionic phospholipid ratio (A/Z). (2) At low external concentration of substrates (high-affinity systems), higher uptake activities were observed for the anions, glutamate, aspartate and phosphate; the zwitterion glycine and the cations lysine and arginine, in both phosphatidylserine and phosphatidylinositol rich cells when compared to control cells. (3) On the other hand, at high external concentration of substrates (low-affinity systems), lower uptake activities were observed for glutamate, aspartate, phosphate and glycine in the cells rich in anionic phospholipids. (4) A decrease in Km without significant alteration in Vmax was found in the high-affinity transport systems that can be explained by the increase in proton concentration at the interface caused by the enhancement in negative surface charge of the cells rich in anionic phospholipids. (5) The mechanisms of the high-affinity proton linked transport systems are compatible with a model which is necessarily ordered, protons before anions. The low-affinity transport systems, on the other hand, follow a random order of binding. The transport systems studied behave as sensors of the changes in surface potential. The reduction of the surface potential reversed the transport alterations with the following sequence: monovalent cations less than divalent cations less than cationic local anesthetics.     

Sensitivity of Escherichia coli cell membranes to various classes of detergents

The mechanisms of interaction between non-ionic or cationic surfactants with Escherichia coli K-12 cell membranes were studied using an approach based on the registration of changes in the membrane permeability to ethidium bromide, a fluorescent dye for nucleic acids. Triton X-100, a non-ionic detergent, was shown to exert no effect on the permeability of intact cell membranes. Triton X-100 interacted with the bacteria only after treatment with EDTA, a complexing agent for bivalent cations. Cetyltrimethyl ammonium bromide increased the permeability to ethidium bromide and the action of this cationic detergent did not require the pretreatment with the complexing agent. SDS, an anionic detergent, damaged E. coli K-12 and this could be registered by the lowering of intensity of light scattering by the bacterial suspension. The surface charge of E. coli K-12 cells was shown to influence the interaction of ionic detergents with bacterial cell membranes. Its variation by changing the pH of the incubation medium did not make E. coli K-12 sensitive to Triton X-100.     

Distinct permeation profiles of the connexin 30 and 43 hemichannels

Connexin 43 (Cx43) hemichannels may form open channels in the plasma membrane when exposed to specific stimuli, e.g. reduced extracellular concentration of divalent cations, and allow passage of fluorescent molecules and presumably a range of smaller physiologically relevant molecules. However, the permeability profile of Cx43 hemichannels remains unresolved. Exposure of Cx43-expressing Xenopus laevis oocytes to divalent cation free solution induced a gadolinium-sensitive uptake of the fluorescent dye ethidium. In spite thereof, a range of biological molecules smaller than ethidium, such as glutamate, lactate, and glucose, did not permeate the pore whereas ATP did. In contrast, permeability of glutamate, glucose and ATP was observed in oocytes expressing Cx30. Exposure to divalent cation free solutions induced a robust membrane conductance in Cx30-expressing oocytes but none in Cx43-expressing oocytes. C-terminally truncated Cx43 (M257) displayed increased dye uptake and, unlike wild type Cx43 channels, conducted current. Neither Cx30 nor Cx43 acted as water channels in their hemichannel configuration. Our results demonstrate that connexin hemichannels have isoform-specific permeability profiles and that dye uptake cannot be equaled to permeability of smaller physiologically relevant molecules in given settings.     

Fibrinogen binding to human platelet plasma membranes. Identification of two steps requiring divalent cations

Fibrinogen binding to platelet plasma membranes, which is a prerequisite for platelet aggregation, was determined by incubating 125I-labeled fibrinogen with isolated membranes and measuring the amount of radioactivity sedimenting with the membranes through 15% sucrose. Fibrinogen binding was optimal at 10(-3) M Ca2+. Scatchard analyses of the fibrinogen binding showed that the membrane capacity for fibrinogen was 1.6 X 10(-12) mol/mg of membrane protein, with a dissociation constant (Kd) = 1.2 X 10(-8) M. When Ca2+ levels were manipulated by the addition of varying amounts of EGTA at a fixed Mg2+ concentration of 3 X 10(-3) M, specific binding of fibrinogen to platelet membranes occurred only at Ca2+ concentrations greater than or equal to 10(-6) M. Membranes isolated from platelets of an individual with Glanzmann's thrombasthenia bound only 12% as much fibrinogen as control platelets. The data in the present study suggest that there are two divalent cation binding sites that must be occupied for fibrinogen to bind: one site is specific for calcium and is saturated at 10(-6) M Ca2+; the other site is less specific and is saturated at a 10(-3) M concentration of either Ca2+ or Mg2+. Fibrinogen binding to intact platelets and, consequently, platelet aggregation only required 10(-3) M extracellular divalent cation and was not specific for Ca2+. These data indicate that the cytoplasm is a potential source for the requirement of 10(-6) M Ca2+, and that changes in the intracellular concentration of Ca2+ may cause the expression of fibrinogen receptors during ADP-induced platelet activation.     

A role for anionic sites in epithelial architecture. Effects of cationic polymers on cell membrane structure

The effects of several cationic polymers (poly-L-lysines, protamine, and histone) on rabbit gall bladder epithelial cells were studied to explore possible roles for negative sites in the membrane. The tissue was bathed for 30 min at 37°C in Ringer''s solutions containing from 0.1 to 100.0 µg/ml of cationic polymers, and subsequently was fixed with 1% OsO₄ and examined with the electron microscope. All cationic polymers, at appropriate concentrations, produced similar changes in membrane structure. Adjacent membranes frequently were fused. Membrane structures such as microvilli lost rigidity. Cell membranes showed an apparent increase in permeability as judged by osmotically traumatized cells. These results indicate that fixed anionic sites play significant roles in stabilizing epithelial membrane structures.     

Charged sieving by the basal lamina and the distribution of anionic sites on the external surfaces of fat body cells

The distribution of anionic sites on the basal lamina has been examined with highly cationic ferritin. The penetration of ferritins, with a range of charges from anionic to highly cationic, through the basal lamina into the spaces between fat body cells in an insect is correlated with the charge of the tracer. The anionic sites of the basal lamina may therefore affect the composition of the lymph that bathes the fat body cells. There was more cationic ferritin bound to the plasma membrane reticular reticular system than to the lateral plasma membranes, suggesting that there may be regional differences in surface charge.     

The effect of charged lipids on bacteriorhodopsin membrane reconstitution and its photochemical activities

Bacteriorhodopsin (BR) was reconstituted into artificial lipid membrane containing various charged lipid compositions. The proton pumping activity of BR under flash and continuous illumination, proton permeability across membrane, as well as the decay kinetics of the photocycle intermediate M₄₁₂ were studied. The results showed that lipid charges would significantly affect the orientation of BR inserted into lipid membranes. In liposomes containing anionic lipids, BRs were more likely to take natural orientation as in living cells. In neutral or positively charged liposomes, most BRs were reversely assembled, assuming an inside out orientation. Moreover, the lipids charges also affect BR’s M intermediate kinetics, especially the slow component in M intermediate decay. The half-life M_412s increased significantly in BRs in liposomes containing cationic lipids, while decreased in those in anionic liposomes.     

The mechanism of the hemolytic activity of polyene antibiotics

The kinetics of the filipin-, amphotericin B- and nystatin-induced hemolysis of human erythrocytes were investigated. Filipin-induced hemolysis is of the damage type. It is an all-or-none process, partly inhibited by Ca2+ or Ba2+ but not by Mg2+, Na+ or SO42-. The hemolytic activity of filipin is explained by the formation of large aggregates within the erythrocyte membrane in the form of large perforations, permeable to substances of low molecular weight as well as to macromolecules, including hemoglobin. In isotonic KCl solution, both amphotericin B and nystatin, at low concentrations, form smaller aggregates within the membranes. As a result, the permeability of the membranes to KCl increases and hemolysis occurs. However, the kinetics of the hemolysis induced by the two polyenes is complex. The process shows some features of the permeability type and some of the damage type. It is suggested that amphotericin B and nystatin may simultaneously form a number of transport systems, differing in their molecular organisation and hemolytic activity. Their participation in erythrocyte membrane permeability can be modified by small changes in membrane organisation and the chemical composition of the incubation medium. In isotonic solutions of divalent cation chlorides, and at higher antibiotic concentration, additional aggregates, allowing divalent cations to permeate, appear. These structures do not permit SO4(2-) to permeate.     

Stability of Model Membranes in Extreme Environments

The first forms of cellular life required a source of amphiphilic compounds capable of assembling into stable boundary structures. Membranes composed of fatty acids have been proposed as model systems of primitive membranes, but their bilayer structure is stable only within a narrow pH range and low ionic strength. They are particularly sensitive to aggregating effects of divalent cations (Mg+2, Ca+2, Fe+2) that would be present in Archaean sea water. Here we report that mixtures of alkyl amines and fatty acids form vesicles at strongly basic and acidic pH ranges which are resistant to the effects of divalent cations up to 0.1 M. Vesicles formed by mixtures of decylamine and decanoic acid (1:1 mole ratio) are relatively permeable to pyranine, a fluorescent anionic dye, but permeability could be reduced by adding 2 mol% of a polycyclic aromatic hydrocarbon such as pyrene. Permeability to the dye was also reduced by increasing the chain length of the amphiphiles. For instance, 1:1 mole ratio mixtures of dodecylamine and dodecanoic acid were able to retain pyranine dye during and following gel filtration. We conclude that primitive cell membranes were likely to be composed of mixtures of amphiphilic and hydrophobic molecules that manifested increased stability over pure fatty acid membranes.     

The water permeability of Arabidopsis plasma membrane is regulated by divalent cations and pH

Mechanisms that regulate water channels in the plant plasma membrane (PM) were investigated in Arabidopsis suspension cells. Cell hydraulic conductivity was measured with a cell pressure probe and was reduced 4-fold as compared to control values when calcium was added in the pipette and in bathing solution. To assess the significance of these effects in vitro, PM vesicles were isolated by aqueous two-phase partitioning and their water transport properties were characterized by stopped-flow spectrophotometry. Membrane vesicles isolated in standard conditions exhibited reduced water permeability (P(f)) together with a lack of active water channels. In contrast, when prepared in the presence of chelators of divalent cations, PM vesicles showed a 2.3-fold higher P(f) and active water channels. Furthermore, equilibration of purified PM vesicles with divalent cations reduced their P(f ) and water channel activity down to the basal level of membranes isolated in standard conditions. Ca2+ was the most efficient with a half-inhibition of P(f) at 50-100 microM free Ca2+. Water transport in purified PM vesicles was also reversibly blocked by H+, with a half-inhibition of P(f )at pH 7.2-7.5. Thus, both Ca2+ and H+ contribute to a membrane-delimited switch from active to inactive water channels that may allow coupling of water transport to cell signalling and metabolism.     

X-ray diffraction measurement of the monolayer spontaneous curvature of dioleoylphosphatidylglycerol

Phosphatidylglycerol (PG) is an anionic lipid commonly found in large proportions in the cell membranes of bacteria and plants and, to a lesser extent, in animal cells. PG plays an important role in the regulation and determination of the elastic properties of the membrane. Using small angle X-ray scattering experiments, we obtain that the monolayer spontaneous curvature of dioleoylphosphatidylglycerol (DOPG) is -1/150+/-0.021 nm(-1) when measured in 150 mM NaCl. When the experiments are carried out in 150 mM NaCl and 20mM MgCl(2), the value obtained for the monolayer spontaneous curvature is -1/8.7+/-0.037 nm(-1). These values are of importance in modelling the effects of curvature elastic stress in membrane lipid homeostasis in the bacterium Acholeplasma laidlawii [Alley, S.H., Barahona, M., Ces, O., Templer, R.H., in press. Biophysical regulation of lipid biosynthesis in the plasma membrane. Biophys. J.] and indicate that divalent cations can play a significant role in altering curvature elastic stress.     

Water transport across red-cell membranes following reductive methylation of the major transmembrane protein, Band 3: implications to increased divalent cation membrane permeability

1H-T-NMR methods in conjunction with normally membrane-impermeable Mn2+ were used to study the effect of reductive methylation of specific lysine residues of Band 3, the major transmembrane protein, on water permeability. At 21 degrees C, the water apparent transverse relaxation time (T2) was decreased by nearly 16% (p less than .00001) for cells with modified Band 3. Atomic absorption measurements of control and methylated cells showed an increased level of Mn2+ in the erythrocyte cytosol following methylation. This increased level of this paramagnetic relaxation agent is sufficient to relax interior water protein to the values obtained. Thus, following specific methylation of band 3, increased membrane permeability to divalent cations is observed. The results are discussed with reference to possible conformation changes of Band 3 following methylation, and the findings are interpreted be mean that the conformation of Band 3 has influence on cation permeability to erythrocyte membranes.     

Activation of the ADP/ATP carrier from mitochondria by cationic effectors

The ADP/ATP carrier from the mitochondrial inner membrane was found to be influenced by cationic substances from the hydrophilic surroundings. Under low-ionic-strength conditions, addition of these cationic effectors fully activated the reconstituted adenine nucleotide translocator. The list of activators included divalent cations, polyamines, peptides and cationic proteins. The minimum requirement for an activator to be effective was the presence of at least two positive net charges, regardless of the size of the molecule. Cationic molecules were not activating when an intramolecular charge compensation was possible or when the two charges were too far apart from one another. The affinity of these activators varied from several hundred microM (diaminoalkanes, divalent cations) to 1 microM (cytochrome c, spermine) and even down to a few nM (polylysine). The activation by cations was fully reversible and was not due to fusion processes. It was not mediated by an interaction with the anionic substrates ADP and ATP, nor by interaction with the liposomes. The stimulation could directly and functionally be correlated to the reconstituted carrier protein. Activation was not observed in intact mitochondria, but could be demonstrated when the outer mitochondrial membrane had been removed by treatment with digitonin. These mitoplasts were stimulated by polycations similar to the ADP/ATP carrier in the reconstituted system.     

Binding of polycationic antibiotics and polyamines to lipopolysaccharides of Pseudomonas aeruginosa.

Polycations, such as aminoglycoside and peptide antibiotics, and naturally occurring polyamines were found to bind to the lipopolysaccharide of Pseudomonas aeruginosa and alter its packing arrangement. Binding of cations was measured by the displacement of a cationic spin probe from lipopolysaccharide into the aqueous environment upon addition of competitive cations. The level of probe displacement was dependent on the concentration and charge of the competing cation, with the more highly charged cations being more effective at displacing probe. The relative affinity of several antibiotics for lipopolysaccharide correlated with their ability to increase outer membrane permeability, while the relative affinity of several polyamines correlated with their ability to stabilize the outer membrane. Probe mobility within the lipopolysaccharide head group was shown to be decreased by cationic antibiotics and unaltered or increased by polyamines. We propose that antibiotic permeability and disruption of outer membrane integrity by polycationic antibiotics results from binding of the antibiotic to anionic groups on lipopolysaccharide with a consequent change in the conformation of lipopolysaccharide aggregate structure.     

Transformation of Escherichia coli with plasmid deoxyribonucleic acid: calcium-induced binding of deoxyribonucleic acid to whole cells and to isolated membrane fractions.

Plasmid deoxyribonucleic acid (DNA) was tightly bound to cells of Escherichia coli at 0 degrees C in the presence of divalent cations. During incubation at 42 degrees C, 0.1 to 1% of this DNA became resistant to deoxyribonuclease. Deoxyribonuclease-resistant DNA binding and the ability to produce transformants became saturated when transformation mixtures contained 1 to 2 micrograms of plasmid NTP16 DNA and about 5 X 10(8) viable cells. Under optimum conditions, between 1 and 2 molecule equivalents of 3H-labeled NTP16 DNA per viable cell became deoxyribonuclease resistant. Despite this, only 0.1 to 1% of viable cells became transformed by saturating amounts of the plasmid. The results suggest that transport of DNA across the inner membrane is a limiting step in transformation. After transformation the bulk of labeled plasmid DNA remained associated with outer membranes. However, in vitro assays indicated that plasmid DNA would bind equally well to preparations of inner or outer membranes provided divalent cations were present to preparations of inner or outer membranes provided divalent cations were present. Divalent cations promoted differing levels of binding to isolated inner and outer membranes in the order Ca2+ much greater than Ba2+ greater than Sr2+ greater than Mg2+. This parallels their relative efficiencies in promoting transformation. Binding of plasmid DNA was greatly reduced when outer membranes were treated with trypsin; this suggests that protein components may be required for the binding or transport of DNA (or both) during transformation.