Calcium permeability of synaptosomes induced by alpha-latrotoxin

The paper deals with characteristics of ionic alpha-latrotoxin-induced permeability of rat brain synaptosomes. It has been shown that the addition of alpha-latrotoxin to synaptosomes in the Ca2+-containing media resulted in an extensive and rapid uptake of 45Ca2+ in synaptosomes. alpha -Latrotoxin was not able to enhance the 22Na+ and 86Rb+ uptake or efflux in the Ca2+-containing and Ca2+- and Mg2+-free media. The dye di-O-C3 was used to monitor the membrane potential changes as a consequence of alpha-latrotoxin treatment of synaptosomes. It has been found that alpha-latrotoxin increased synaptosomal fluorescence in the Ca2+-containing media, but failed to induce any increase of fluorescence in Ca2+- and Mg2+-free media. It has been also shown that the calcium uptake induced by alpha-latrotoxin depends on free calcium concentration in synaptosomes. Toxin-induced calcium flows are shown to be of the vector character.     

Effect of chilling temperatures on osmotic water permeability and aquaporin activity in the plasma membranes from pea roots

The osmotic water permeability of plasma membrane vesicles was examined after isolation from the roots of 7-day-old etiolated pea ( Pisum sativum, cv. Orlovchanin) seedlings grown at optimal temperature and those exposed to 1-day chilling at 8°C in the end of the growth period. The homogenization medium for obtaining plasma membranes was supplemented with either SH-reagents or protein phosphatase inhibitors. The plasmalemma vesicles were purified from the microsome fraction by means of two-phase polymer system. The osmotic water permeability of membrane vesicles was evaluated from the rate of their osmotically induced shrinkage. The lowering of growth temperature was accompanied by the increase in osmotic water permeability of plasmalemma. These changes occurred without the corresponding increase in aquaporin content or permeability of membrane lipid matrix. The membranes from cooled seedlings were markedly depleted in the content of SH-groups. Furthermore, the treatment of membrane samples with a thiol-reducing agent, tributylphosphine did not raise the SH-group content in membranes from chilled plants, unlike such changes in membranes from warm-grown plants. When the homogenization medium contained dithiothreitol and phenylarsine oxide (an inhibitor of tyrosine protein phosphatases), the osmotic permeability of plasmalemma in preparations from warm-grown seedlings also increased. Based on these results, it is supposed that aquaporin-mediated water permeability of membranes is regulated through different pathways under optimal and adverse conditions for plant growth. Direct action of endogenous SH redox regulators on aquaporin activity is likely under optimal growth conditions, while protein phosphatase might mediate changes in aquaporin activity under unfavorable growth conditions.     

Bioenergetic response of isolated nerve terminals of rat brain to osmotic swelling

The swelling of nerve terminals of rat brain in a hypotonic medium (230 mOsm) induced the potential-independent entrance of 45Ca2+ into synaptosomes and intrasynaptosomal mitochondria that changed the energy status of synaptosomes, the rate of O2 consumption and the content of ATP being decreased. The ratio ATP/ADP decreased from 6.5 +/- 0.26 (310 mOsm medium) to 3.1 +/- 0.18 (the medium 230 mOsm). Studies on the equilibrium distribution of K+ (86Rb+) and [3H]TPP+ showed that contents of these cations in the nerve terminals were virtually the same on incubation in both iso- and hypotonic media. This indicated that the swelling did not damage intrasynaptosomal mitochondria and plasma membranes of the synaptosomes. The inhibition of oxidative phosphorylation increased twofold the rate of glycolysis. The incubation of synaptosomes in calcium-free medium (230 mOsm) in the presence of EGTA (1 mM) prevented the inhibition of oxidative phosphorylation and synthesis of ATP by the osmotic swelling. Ruthenium Red (10 microM) in the medium 230 mOsm inhibited the entrance of 45Ca2+ into the intrasynaptosomal mitochondria and normalized the oxidative phosphorylation to the control level (310 mOsm medium). The decrease in the energy potential of synaptosomes induced by the hypoosmotic shock is suggested to be associated with the increase in Ca2+ content in the cytoplasm, its transport into the mitochondria, and the inhibitory effect on oxidative phosphorylation.     

Variations in Membrane Sensitivity of Brain Region Synaptosomes to the Effects of Ethanol In Vitro and Chronic In Vivo Treatment

The effects of chronic ethanol treatment on the membrane order of synaptosomes from the cerebral cortex, striatum, cerebellum, brainstem, and hippocampus of rats were determined by measuring the fluorescence polarization of diphenylhexatriene (DPH) that had been incorporated into the synaptosomal membranes. Fischer-344 rats either were fed a nutritionally complete ethanol-containing liquid diet for 5 months or pair-fed with a diet that contained sucrose substituted isocalorically for ethanol. Polarization values for synaptosomes from all the brain regions studied were similar except for those from cerebral cortical synaptosomal membranes, which were significantly less ordered. Ethanol in vitro (30-500 mM) decreased the polarization values in synaptosomes from sucrose-control rats for all brain regions, although the sensitivity of cerebellar synaptosomes to the membrane disordering effects of ethanol in vitro was significantly greater that of synaptosomes from other brain regions. Chronic ethanol treatment did not alter baseline polarization for any brain region. Cerebellar and brainstem synaptosomes from the ethanol-fed rats were significantly less susceptible to the membrane disordering effects of ethanol in vitro compared to their sucrose controls, suggesting that chronic ethanol administration results in tolerance to ethanol's membrane effects. Striatal synaptosomes exhibited intermediate tolerance, whereas the sensitivities of cortical and hippocampal synaptosomes to membrane disordering by ethanol in vitro were not significantly affected by the chronic ethanol treatment. These results suggest that synaptosomal membranes have different membrane order requirements depending on the brain region from which they are prepared. Variations in brain regional neuronal membrane sensitivity to ethanol and differential tolerance development may contribute to some of the acute and chronic behavioral effects of ethanol.     

Binding constants of Li+, K+, and Tl+ in the gramicidin channel determined from water permeability measurements.

In an open circuit there can be no net cation flux through membranes containing only cation-selective channels, because electroneutrality must be maintained. If the channels are so narrow that water and cations cannot pass by each other, then the net water flux through those "single-file" channels that contain a cation is zero. It is therefore possible to determine the cation binding constants from the decrease in the average water permeability per channel as the cation concentration in the solution is increased. Three different methods were used to determine the osmotic water permeability of gramicidin channels in lipid bilayer membranes. The osmotic water permeability coefficient per gramicidin channel in the absence of cations was found to be 6 x 10(-14) cm3/s. As the cation concentration was raised, the water permeability decreased and a binding constant was determined from a quantitative fit to the data. When the data were fitted assuming a maximum of one ion per channel, the dissociation constant was 115 mM for Li+, 69 mM for K+, and 2 mM for Tl+.     

Permeability studies on red cell membranes of dog, cat, and beef

Water permeability coefficients of dog, cat, and beef red cell membranes have been measured under an osmotic pressure gradient. The measurements employed a rapid reaction stop flow apparatus with which cell shrinking was measured under a relative osmotic pressure gradient of 1.25 to 1.64 times the isosmolar concentration. For the dog red cell the osmotic permeability coefficient is 0.36 cm⁴/(sec, osmol). The water permeability coefficient for the dog red cell under a diffusion gradient was also measured (rate constant = 0.10/msec). The ratio between the two permeabilities was used to calculate an equivalent pore radius of 5.9 A. This value agrees welt with an equivalent pore radius of 6.2 A obtained from reflection coefficients of nonelectrolyte water-soluble molecules, and is consistent with data on the permeability of the dog red cell membrane to glucose. These data provide evidence supporting the existence of equivalent pores in single biological membranes.     

Permeability properties of peroxisomal membranes from yeasts

We have studied the permeability properties of intact peroxisomes and purified peroxisomal membranes from two methylotrophic yeasts. After incorporation of sucrose and dextran in proteoliposomes composed of asolectin and peroxisomal membranes isolated from the yeasts Hansenula polymorpha and Candida boidinii a selective leakage of sucrose occurred indicating that the peroxisomal membranes were permeable to small molecules. Since the permeability of yeast peroxisomal membranes in vitro may be due to the isolation procedure employed, the osmotic stability of peroxisomes was tested during incubations of intact protoplasts in hypotonic media. Mild osmotic swelling of the protoplasts also resulted in swelling of the peroxisomes present in these cells but not in a release of their matrix proteins. The latter was only observed when the integrity of the cells was disturbed due to disruption of the cell membrane during further lowering of the concentration of the osmotic stabilizer. Stability tests with purified peroxisomes indicated that this leak of matrix proteins was not associated with the permeability to sucrose. Various attempts to mimic the in vivo situation and generate a proton motive force across the peroxisomal membranes in order to influence the permeability properties failed. Two different proton pumps were used for this purpose namely bacteriorhodopsin (BR) and reaction center-light-harvesting complex I (RCLH_I complex). After introduction of BR into the membrane of intact peroxisomes generation of a pH-gradient was not or barely detectable. Since this pump readily generated a pH-gradient in pure liposomes, these results strengthened the initial observations on the leakiness of the peroxisomal membrane fragments. Generation of a membrane potential () was also not observed when RCLH_I complex was introduced into vesicles of purified peroxisomal membranes. The significance of the observed permeability of isolated yeast peroxisomal membranes to small molecules with respect to current and future in vitro import studies is discussed.Abbreviations CL cardiolinin - PE phosphatidylethanolamine - PC phosphatidylcholine - MES 2-(N-Morpholino)ethanesulfonic acid - R₁₈ octadecyl Rhodamine B Chloride - SUVs small unilamellar vesicles - RCLH_I-complex reaction center-light-harvesting complex I - BR bacteriorhodopsin - DCCD N,N-dicyclohexylcarbodiimide     

Permeability properties of the membrane of vesicular stomatitis virions

Observations of the light-scattering properties of several enveloped viruses indicate that virions (vesicular stomatitis, SV5 and influenza), in common with other membrane systems, are osmotically active, responding to NaCl gradients by swelling in hypo-osmolar solutions and shrinking in hyperosmolar solutions. The permeability barrier responsible for this osmotic response in vesicular stomatitis virions was modified both by protease treatment to remove the viral glycoprotein and by treatment with the polyene antibiotic filipin, an agent known to interact with cholesterol in liposomes and membranes. Filipin altered the kinetic and equilibrium permeability behavior of virions but the extent of leakage of osmotic shocking agent was less than that in lecithin/cholesterol and lecithin/ergosterol liposomes and in ergosterol-containing ciliary membranes. Negative-staining electron microscopy revealed that filipin treatment caused structural changes in the viral membrane. Intact virions exhibited appreciably larger responses to osmotic change than did protease-treated virus particles. Thus, the osmotic barrier in intact vesicular stomatitis virions may not be exclusively lipid in nature.     

PIP1 aquaporins,sterols, and osmotic water permeability of plasma membranes from etiolated pea seedlings

Plasma membrane isolated from microsomal membranes of pea seedling root and shoot cells by means of aqueous two-phase polymer system was separated by flotation in discontinuous OptiPrep gradient into “light” (≤1.146 g/cm³) and “heavy” (≥1.146 g/cm³) fractions. Osmotic water permeability of plasma membrane and its two fractions was investigated by inducing transmembrane osmotic gradient on the vesicle membrane and recording the kinetics of vesicle osmotic shrinkage by the stopped-flow method. Rate constants of osmotic shrinkage and coefficients of osmotic water permeability of the membranes were estimated on the basis of the kinetic curve approximation by exponential dependencies and using electron microscope data on vesicles sizes. In plasma membrane and its fractions the content of sterols and PIP1 aquaporins was determined. It was found that in “light” PM fractions from both roots and shoots the content of PIP1 aquaporins and sterols was higher and the osmotic water permeability coefficient was lower than in “heavy” fractions of plasma membrane. The results indicate that plasma membrane of roots and shoots is heterogeneous in osmotic water permeability. This heterogeneity may be related with the presence of microdomains with different content of aquaporins and sterols in the membrane.     

Ultrastructure and permeability characteristics of the membranes of mucous granules of the hagfish

Mucous granules secreted by the slime glands of the hagfish. Eptatretus stouti, were studied after ultrarapid cryofixation and freeze substitution in diverse media (osmium tetroxide in acetone; several aqueous glutaraldehyde-based media with or without osmium). Only freeze substitution with osmium tetroxide-acetone preserved the granules intact, allowing visualization of its single unit membrane. Tests of the rupture or stability of freshly secreted mucous granules in sea water and other aqueous media showed the membranes of the granules are permeable to all inorganic cations tested, ranging in relative mass from ammonium to barium. They are permeable to the univalent anions chloride, nitrate, and bicarbonate, but not to the di- or trivalent anions sulfate, phosphate, and citrate. Moreover, in solutions where nonpenetrant anions were present, rupture occurred if the osmotic pressure was below a critical level (about 800 mOsmol/l). The structural and permeability characteristics of the granules account for the explosive speed with which they rupture, releasing their mucous contents, on contact with sea water.     

Permeability properties of the membrane of vesicular stomatitis virions.

Observations of the light-scattering properties of several enveloped viruses indicate that virions (vesicular stomatitis, SV5 and influenza), in common with other membrane systems, are osmotically active, responding to NaCl gradients by swelling in hypo-osmolar solutions and shrinking in hyperosmolar solutions. The permeability barrier responsible for this osmotic response in vesicular stomatitis virions was modified both by protease treatment to remove the viral glycoprotein and by treatment with the polyene antibiotic filipin, an agent known to interact with cholesterol in liposomes and membranes. Filipin altered the kinetic and equilibrium permeability behavior of virions but the extent of leakage of osmotic shocking agent was less than that in lecithin/cholesterol and lecithin/ergosterol liposomes and in ergosterol-containing ciliary membranes. Negative-staining electron microscopy revealed that filipin treatment caused structural changes in the viral membrane. Intact virions exhibited appreciably larger responses to osmotic change than did protease-treated virus particles. Thus, the osmotic barrier in intact vesicular stomatitis virions may not be exclusively lipid in nature.     

Aquaporin Content in Cell Membranes of Mesembryanthemum crystallinum as Affected by Plant Transition from C3 to CAM Type of Photosynthesis

Western-blot analysis was used to determine the contents of aquaporin isoforms MIP A, MIP B, and MIP C in cell membranes isolated from roots and leaves of Mesembryanthemum crystallinum plants with C₃ and Crassulacean acid metabolism (CAM) types of photosynthesis. These membrane preparations were also used to assess osmotic water permeability; to this end, the rate of osmotic vesicle shrinking was registered as the light scattering intensity by the method of stopped flow. The cell membranes represented by the plasmalemma and the tonoplast-enriched fraction were obtained by separating the microsomes in a two-phase polymer system. Plant transition from C₃ to CAM-photosynthesis occurred in the course of plant development or was induced by salinization. All three isoforms under study were found in the plasma membranes of roots and leaves of the C₃ plants, whereas in the CAM plants, independent of the transition-inducing factor, the aquaporin contents notably decreased in the leaf membranes and remained unchanged in the roots. In the membranes isolated from roots and leaves of the C₃ plants, the values of osmotic water permeability exceeded two–threefold the corresponding indices characteristic of the CAM plants. The authors believe that aquaporin isoforms in M. crystallinum are under the organ- and tissue-specific control.     

Monitoring of potassium-stimulated catecholamine changes in striatal synaptosomal preparations and in corpus striatum of rats: a comparative voltammetric study.

Voltammetric techniques were used to compare the effects of K(+)-induced depolarization on catecholamine levels in in vitro synaptosomal preparations of the corpus striatum with those in the in vivo corpus striatum of anaesthetized animals. In vitro, the catechol-oxidation currents could be recorded only in dopamine-preloaded synaptosomes. In isolated synaptosomes prepared in the presence of elevated concentrations of Ca2+ (1 mmol.l-1) and Na+ (135 mmol.l-1), K(+)-induced depolarization had variable effects on catechol-oxidation current. The stimulatory effect of K(+)-induced depolarization (a short transient increase of catechol-oxidation current lasting for 30 s) could be observed after the addition of dopamine loaded synaptosomes in EGTA into the medium with elevated K+ concentration (90 mmol.l-1) and decreased concentrations of Na+ (75 mmol.l-1) and Ca2+ (0.75 mmol.l-1). These results suggest that experimental procedures and parameters of ionic composition of incubation media have to be carefully controlled, owing to an enhanced in vitro permeability of membranes of isolated synaptosomes for Ca2+ and Na+. In in vivo experiments, microinjection of KCl (3 microliters of 0.5 mol.l-1 KCl in 10 mmol.l-1 HEPES, pH 7.4) resulted in the appearance of several phases of catechol-oxidation current: the current increased (to severalfold of the control values) followed by a decrease or even total disappearance, with a gradual return to control values. Under conditions of depletion of extracellular calcium by EGTA (5 microliters of 0.5 mol.l-1 KCl + 0.25 mol.l-1 EGTA in 10 mmol.l-1 HEPES, pH 7.4) K(+)-induced depolarization confirmed the key role of calcium in the release of catecholamine transmitters as well as that in processes regulating the uptake and metabolism of these transmitters. The voltammetric techniques used in the present study may be a useful tool in extending of our knowledge about the cellular mechanisms of stimulus-response coupling in nerve cells.     

Topographic studies of gangliosides of intact synaptosomes from rat brain cortex

Gangliosides in the external surface of intact synaptosomes from rat brain cortex have been studied by oxidation of exposed galactose and galactosamine groups with galactose oxidase followed by reduction with labeled sodium borohydride. Purified synaptosomes were labeled, disrupted by osmotic shock, and the particulate components fractionated on diatrizoate to give four synaptosomal membrane fractions (A-D) and a mitochondrial pellet (E). Fractions A and B represent synaptosomal plasma membranes. When intact synaptosomes were labeled, the major portion of the total radioactivity incorporated into ganglioside fraction was found to be in G _M1 ³ species. With isolated membrane fractions little selectivity was seen: (1) more label was present compared to intact synaptosomes, and (2) zones corresponding to G_M2, G_M1, G_D1a, G_D1b were the major gangliosides labeled. The results confirm the conclusion that membrane fractions A and B are derived from the exposed synaptosome surface and also show that G_M1 is the major ganglioside species available for enzyme oxidation at the surface.     

Modulation of brain mitochondrial function by deprenyl

The present study shows that deprenyl, a known inhibitor of monoamine oxidase B (MAO B), may generate changes in mitochondrial function. Brain submitochondrial membranes (SMP), synaptosomes and cytosolic fractions were incubated with different deprenyl concentrations and nitric oxide synthase (NOS) activity was measured. The effect of deprenyl on oxygen consumption, calcium-induced permeability transition and hydrogen peroxide (H(2)O(2)) production rates was studied in intact mitochondria. Respiratory complexes and monoamine oxidase activities were also measured in submitochondrial membranes. Incubation of brain submitochondrial membranes with deprenyl 10, 25 and 50 microM inhibited nitric oxide synthase activity in a concentration-dependent manner. The same effect was observed in cytosolic fractions and synaptosomes. Monoamine oxidase activity was inhibited at lower deprenyl concentrations (from 0.5 microM). Cytochrome oxidase (complex IV) activity was found 42% increased in the presence of 25 microM deprenyl in a condition of maximal nitric oxide synthase activity. Incubation of brain mitochondria with deprenyl 25 microM produced a 60% increase in oxygen uptake in state 3, but no significant changes were observed in state 4. Pre-incubation of brain mitochondria with deprenyl 0.5 and 1 microM inhibited calcium-induced mitochondrial permeability transition and decreased hydrogen peroxide production rates. Our results suggest that in vitro effects of deprenyl on mitochondrial function can occur through two different mechanisms, involving nitric oxide synthase inhibition and decreased hydrogen peroxide production.     

Effect of osmotic shock on the viability, optical density and permeability of heated Escherichia coli cells

The object of this work was to study the effect of a short incubation in 0.01 M tris buffer, pH 7.0, with a different NaCl content (0-10%) on the viability, optic density and permeability of intact and heated at 52 degrees C Escherichia coli B/r cells. In contrast to the intact cells, the viability of the heated cells depended on osmotic pressure in the medium into which they were transferred after heating. The survival rate was highest when the cells were transferred into an isotonic buffer. In the case of hypotonic and hypertonic media, the survival rate of the cells decreased owing to the death of cells which were responsible for the formation of small colonies under the isotonic conditions. This was accompanied with a more intensive drop in the optic density of bacterial suspensions while their permeability increased (when the cells were transferred into the hypotonic conditions). The role of membranes in the processes of bacterial heat inactivation is discussed on the basis of the results obtained.     

Osmotic tolerance limits and membrane permeability characteristics of stallion spermatozoa treated with cholesterol

Stallion spermatozoa exhibit osmotic damage during the cryopreservation process. Recent studies have shown that the addition of cholesterol to spermatozoal membranes increases the cryosurvival of bull, ram and stallion spermatozoa, but the exact mechanism by which added cholesterol improves cryosurvival is not understood. The objectives of this study were to determine if adding cholesterol to stallion sperm membranes alters the osmotic tolerance limits and membrane permeability characteristics of the spermatozoa. In experiment one, stallion spermatozoa were treated with cholesterol-loaded cyclodextrin (CLC), subjected to anisotonic solutions and spermatozoal motility analyzed. The spermatozoa were then returned to isotonic conditions and the percentages of motile spermatozoa again determined. CLC treatment increased the osmotic tolerance limit of stallion spermatozoa in anisotonic solutions and when returned to isotonic conditions. The second and third experiments utilized an electronic particle counter to determine the plasma membrane characteristics of stallion spermatozoa. In experiment two, stallion spermatozoa were determined to behave as linear osmometers. In experiment three, spermatozoa were treated with CLC, incubated with different cryoprotectants (glycerol, ethylene glycol or dimethyl formamide) and their volume excursions measured during cryoprotectant removal at 5° and 22 °C. Stallion spermatozoa were less permeable to the cryoprotectants at 5 °C than 22 °C. Glycerol was the least permeable cryoprotectant in control cells. The addition of CLC’s to spermatozoa increased the permeability of stallion spermatozoa to the cryoprotectants. Therefore, adding cholesterol to spermatozoal membranes reduces the amount of osmotic stress endured by stallion spermatozoa during cryopreservation.     

EFFECT OF TEMPERATURE AND IONOPHORES ON THE PERMEABILITY OF SYNAPTOSOMES

Synaptosomes swell rapidly in isosmotic solutions of glycerol or urea, but the swelling in solutions of larger non-electrolytes, such as erythritol, glucose or sucrose is slower. The permeability of synaptosomes to non-electrolytes is temperature dependent, and the low activation energies for the permeation of urea (13 kcal/mol) and erythritol (9.5 kcal/mol) indicate that the penetration of non-electrolytes into the synaptosomes does not imply complete dehydration of the molecules. The relative permeability of synaptosomes to cations, as measured by the rate of swelling in isosmotic solutions of acetate salts is in the order: NH⁺₄ > Na⁺ > Li⁺ > K⁺ > Ca²⁺. The ionophores, X-537A and nigericin, or valinomycin + FCCP, which promote exchange of cations for H⁺, cause swelling of synaptosomes in solutions of potassium salts of acetate or propionate, but not in KCI, whereas H⁺ release is higher in KCI medium. This suggests that the organic unions cross the membrane after combining with H⁺ to form the respective weak acids. The relative permeability to anions is in the order: acetate ? propionate > Cl^? > SO^2-₄? maleate ? succinate. The energies of activation for the permeability of synaptosomes to potassium acetate in the presence of X-537A or gramicidin D are 13 kcal/mol and 7.5 kcal/mol, respectively, which reflects different mechanisms of action for the two ionophores in the membranes.     

High-affinity, sodium-gradient-dependent transport of choline into vesiculated presynaptic plasma membrane fragments from the electric organ of Torpedo marmorata and reconstitution of the solubilized transporter into liposomes

Vesiculated fragments of presynaptic plasma membranes have been isolated from the purely cholinergic electromotor nerve terminals of Torpedo marmorata. Synaptosomes, generated from the terminals by homogenization, were separated on a discontinuous Ficoll gradient and then lysed by osmotic shock at 2 degrees C, pH 8.5 in the presence of 0.1 mM MgCl2. These conditions for lysis were optimal for choline transport. Electron micrographs of lysed synaptosomes showed vesiculated membranes with diameters smaller than those of synaptosomes; occasionally, synaptic vesicles were observed attached to them. Intact mitochondria or synaptosomes and basal laminae were not present. High-affinity (KT = 1.7 microM) uptake of choline into these vesiculated membrane fragments showed: an absolute dependence on the Na+ gradient (outside greater than inside), a transient Na+-gradient-dependent accumulation of choline over the equilibrium concentration (over-shoot), electrogenicity and rheogenicity, since the uptake was further stimulated in the presence of a Na+ gradient by valinomycin, dependence on the presence of external Cl-, and partial dependence on a Cl- gradient (outside greater than inside), high-affinity (Ki = 25 nM) inhibition by hemicholinium-3 and temperature sensitivity. The plasma membranes were further purified by centrifugal density gradient fractionation on a 4-12% Ficoll gradient. Several enzymes and polypeptides copurified with the specific binding sites for choline present in the membranes. The fraction with the most binding sites was one denser than 12% Ficoll. This was also the fraction richest in acetylcholinesterase, 5'-nucleotidase and polypeptides of relative molecular mass, Mr (X 10(-3)) of greater than 200, 140, 68 (doublet), 57, 54 and 28. Acetylcholinesterase was positively identified as a Mr 68 000 component by immune blot. By contrast the ouabain-sensitive ATPase showed a negative correlation with choline binding sites. When the solubilized proteins of the vesiculated membranes were transferred to liposomes, they conferred on the latter the capacity to take up choline in a manner closely resembling its transport in natural membranes but with an initial (one minute) rate of uptake approximately 10-times greater per mg of protein. Several proteins were selectively transferred to the liposomes including ones of Mr (X 10(-3)) 34, 42, 47, 54, 60, 68, 92, 160 and greater than 200. The polypeptides of Mr (X 10(-3)) 140, 57 and 28 were lost in the transfer.(ABSTRACT TRUNCATED AT 400 WORDS)     

Inhibitory effect of cholesterol on interaction between cytoplasmic actin and liposomes, and restorative effect of high osmotic pressure

Although cholesterol is one of the major components of plasma membranes in eukaryotic cells, very little is known about its role in biological membranes. We reported previously (Okimasu et al., Cell Struct. Funct. 11, 273-283, 1986) that introduction of cholesterol into the liposomal membrane caused a decrease in membrane permeability, especially by the binding of cytoplasmic proteins to the liposomal membrane. The present study was carried out to further clarify the biochemical function of cholesterol in the membrane-protein interactions, especially under high osmotic pressure. The association of membranes with cytoplasmic proteins and their permeability were decreased by the introduction of cholesterol, but its effects were diminished in a hypertonic medium. The protein species associated with cholesterol-containing liposomes vary depending on the sort of hypertonic condition. It was suggested that since the degree of lipid packing by the cholesterol was reduced by the locally increased curvature in the lipid bilayer under high osmotic pressure, some cytoplasmic proteins can penetrate into the liposomal membrane.