Fluidity of natural membranes and phosphatidylserine and ganglioside dispersions: Effects of local anesthetics,cholesterol and protein

The microviscosity of artificial lipid membranes and natural membranes was measured by the fluorescence polarization technique employing perylene as the probe. Lipid dispersions composed of brain gangliosides exhibited greater microviscosity than phosphatidylserine (268 cP vs 173 cP, at 25 °C). Incorporation of cholesterol (30–50%) increased the microviscosity of lipid phases by 200–500 cP. Cholesterol's effect on membrane fluidity was completely reversed by digitonin but not by amphotericin B. Incorporation of membrane proteins into lipid vesicles gave varying results. Cytochrome b₅ did not alter membrane fluidity. However, myelin proteolipid produced an apparent increase in microviscosity, but this effect might be due to partitioning of perylene between lipid and protein binding sites since the latter have a higher fluorescence anisotropy than the lipid. The local anesthetics tetracaine and butacaine increased the fluidity of lipid dispersions, natural membranes and intact ascites tumor cell membranes. The effect of the anesthetics appears to be due to an increased disordering of lipid structure. The fluidity of natural membranes at the 25 °C varied as follows:polymorphonuclear leukocytes, 335 cP; bovine brain myelin, 270 cP; human erytherocyte, 180 cP; rat liver microsomes, 95 cP; rat liver mitochondria, 90 cP. In most cases the microviscosity of natural membranes reflects their cholesterol : phospholipid ratio. The natural variations in fluidity of cellular membranes probably reflect important fuctional requirements. Similarly, the effects of some drugs which alter membrane permeability may be the result of their effects on membrane fluidity.     

Fluidity of natural membranes and phosphatidylserine and ganglioside dispersions. Effect of local anesthetics, cholesterol and protein.

The microviscosity of artificial lipid membranes and natural membranes was measured by the fluorescence polarization technique employing perylene as the probe. Lipid dispersions composed of brain gangliosides exhibited greater microviscosity than phosphatidylserine (268 cP vs 173 cP, at 25 degrees C). Incorporation of cholesterol (30-50%) increased the microviscosity of lipid phases by 200-500 cP. Cholesterol's effect on membrane fluidity was completely reversed by digitonin but not by amphotericin B. Incorporation of membrane proteins into lipid vesicles gave varying results. Cytochrome b5 did not alter membrane fluidity. However, myelin proteolipid produced an apparent increase in microviscosity, but this effect might be due to partitioning of perylene between lipid and protein binding sites since tha latter have a higher fluorescence anisotropy than the lipid. The local anesthetics tetracain and butacaine increased the fluidity of lipid dispersions, natural membranes and intact ascites tumor cell membranes. The effect of anesthetics appears to be due to an increased disordering of lipid structure. The fluidity of natural membranes at 25 degrees C varied as follows: polymorphonuclear leukocytes, 335 cP; bovine brain myelin, 270 cP; human erythrocyte, 180 cP; rat liver microsomes, 95 cP; rat liver mitochondria, 90 cP. In most cases the microviscosity of natural membranes reflects their cholesterol: phospholipid ratio. The natural variations in fluidity of cellular membranes probably reflect important functional requirements. Similarly, the effects of some drugs which alter membrane permeability may be the result of their effects on membrane fluidity.     

Modulation of serotonin binding in rat brain by membrane fluidity

Preincubation of rat brain homogenates with increasing concentrations of n-hexanol reduced specific serotonin (5-HT) binding and increased membrane fluidity as measured by fluorescence depolarization using 1,6-diphenyl-1,3,5-hexatriene (DPH) as a probe. At 5 mM ascorbate maximal reductions of both membrane fluidity and specific 5-HT binding were observed. Both effects were enhanced in the presence of ferrous sulphate and oxygen. In the presence of ascorbate (5.7 mM) only one 5-HT binding site was observed in contrast with high and low affinity binding sites (K_D1 = 0.08 ± 0.04 nM, K_D2 = 28.8 ± 1.3 nM) found in the absence of ascorbate. The ascorbate induced decrease of specific 5-HT binding may be explained by lipid peroxidation, which decreases membrane fluidity, and by ascorbate's reducing properties. Since different correlations were found between membrane fluidity and specific 5-HT binding depending upon the presence of ascorbate or n-hexanol, the results suggest that membrane fluidity is a critical factor, however, just one of several determinants in 5-HT binding studies.     

Involvement of glycoconjugates in insulin-receptor interactions Studies in liver plasma membranes of control and diabetic mice

The involvement of glycoconjugates in the insulin-receptor interactions in mouse liver is tested by digestions of membranes with various enzymes. Trypsin decreased the binding of [¹²⁵I]insulin to liver membranes. After digestion with β-galactosidase no “high affinity” receptor sites could be detected. The effects observed with plant lectins confirm the involvement of galactoconjugates in the insulin binding process. Sophora japonica and Ricinus communis lectins (with galactose specificity) and concanavalin A largely inhibit the binding process of insulin and those effects concern the “high affinity” receptor sites. Other lectins (wheat germ agglutinin, Dolichos) and enzymes (α-l-fucosidase, $\text{β-N-}\text{acetyl-hexosaminidase and neuraminidase}$) are without effect on insulin binding.Comparative studies performed on diabetic mouse liver membrane (KK mice), previously characterized by decreased number of insulin receptors, are in good agreement with qualitatively similar receptor sites in both non-diabetic (control) and diabetic mice. Effects of enzymes and lectins yielded same results as compared to control membranes. Plasma membrane proteins and glycoproteins in both types of mouse are indistinguishable with respect to enzymic and chemical analysis. Sodium dodecyl sulphate acrylamide gel electrophoresis shows identical patterns. Moreover, the decrease in the number of insulin receptors is easily reversed with diet restriction. These data are consistent with the similarity of receptor sites in control and diabetic liver membrane.     

Differential effect of cholesterol on two types of 5-hydroxytryptamine binding sites

The specific binding of [3H]5-hydroxytryptamine ([3H]5-HT, [3H]serotonin) to rat cerebral cortex is increased approximately 1.5 to 2.0 fold by cholesterol hydrogen succinate (CHS) and is solubilized into the supernatant fraction by 12 mM CHS. [3H]5-HT binding sites can be constituted by incubating the supernatant fraction obtained from CHS-treated cerebral cortex with cerebellum in which no significant [3H]5-HT binding is detectable. The constituted [3H]5-HT binding could be displaced by unlabeled 5-HT, d-lysergic acid diethylamide (d-LSD) and spiperone as could the binding to cortex membranes. Unlabeled 5-HT, d-LSD and spiperone each inhibited specific [3H]5-HT binding to constituted binding sites by 50% at about 1 X 10(-9) M. Specific [3H]spiperone binding was not detectable in the constituted membranes. Stearic acid which is reported to have similar effects on membrane fluidity as cholesterol also increased specific [3H]5-HT binding in cortical membranes. Stearic acid does not affect specific [3H]spiperone binding. These results suggest that [3H]5-HT and [3H]spiperone binding sites are affected differently by membrane fluidity.     

Effects of lipid-phase separation on the filipin action on membranes of ergosterol-replaced Tetrahymena cells,as studied by freeze-fracture electron microscopy

The effects of lipid-phase separation on the filipin action on pellicle membranes of ergosterol-replaced Tetrahymena pyriformis cells were studied by freeze-fracture electron microscopy. The pellicle membranes with phase separations induced by chilling from 34°C (growth temperature) to lower temperatures (30, 22 and 15°C) were treated with filipin. This produced filipin-induced lesions (“pits”) only in the particulated (liquid) regions along the margin between solid and liquid domains, while they were produced in the particle-free (solid) areas when membranes were chilled to 15°C. The pellicle membranes with lesions induced by filipin at 34°C were chilled to 22°C. This chilling raised larger particle-free areas and more condensed particle-aggregations on the membranes than on the membranes without the filipin treatment. These results suggest that the membrane fluidity affects induction and development of the ergosterol-filipin complex in the membrane.     

Modulation of prolactin binding sites in vitro by membrane fluidizers. I. Effects on adult rat ventral prostatic membranes

The objective of this study was to determine if aliphatic alcohols, known fluidizers of certain membranes, could increase in vitro the apparent fluidity and prolactin binding capacity of membrane preparations obtained from ventral prostate glands of adult male rats. The degree of fluidization was monitored by a fluorescence polarization method using 1,6-diphenylhexatriene. Membrane preparations were either incubated with varying concentrations of ethanol, 1-propanol or 1-butanol and 125I-oPRL overnight at room temperature or were exposed to the alcohols for 15 min at room temperature and washed prior to the overnight incubation with ligand. Regardless of the conditions of incubation, alcohol exposure produced the same effects, a dose-dependent elevation and then decline in specific prolactin binding. Butanol produced a maximal 37-42% increase in prolactin binding at a concentration of 1.0%, propanol produced a maximal 40-56% increase in prolactin binding at a concentration of 3.8%, and ethanol produced a maximal 54-77% increase in prolactin binding at concentration of 4.8%. Scatchard analysis of the oPRL binding of ventral prostatic membranes indicated that the in vitro treatment of these membrane fluidizers increased the number of oPRL binding sites rather than the apparent affinity constant. The value of the microviscosity parameter decreased by 10-13%, 13-15% and 21-25%, after a 15 min exposure of prostatic membranes to 1.0% butanol, 3.8% propanol and 4.8% ethanol, respectively. These data suggest that in vitro fluidization of prostatic membrane modifies prolactin binding capacity and are consistent with in vivo prostatic prolactin receptor level-membrane fluidity relationships observed in earlier studies.     

Alterations in human red blood cell membrane properties induced by the lipopolysaccharide from Proteus mirabilis S1959

The effect of lipopolysaccharide (LPS, endotoxin), isolated from Proteus mirabilis S1959 strain, on red blood cell (RBC) membranes in whole cells as well as on isolated membranes was studied. Lipid membrane fluidity, conformational state of membrane proteins and the osmotic fragility of RBCs were examined using electron paramagnetic resonance spectroscopy and spectrophotometric method. Lipid membrane fluidity was determined using three spin-labeled fatty acids: 5-, 12- and 16-doxylstearic acid (5-, 12- and 16-DS). The addition of LPS S1959 to RBC suspension resulted in an increase in membrane fluidity, as indicated by 12-DS. At the concentrations of 0.5 and 1 mg/ml, LPS treatment led to a significant (P<0.05) increase in lipid membrane fluidity in the deeper region of lipid bilayer (determined by 12-DS). The conformational changes in membrane proteins were determined using two covalently bound spin labels, 4-maleimido-2,2,6,6-tetramethylpiperidine-1-oxyl and 4-iodoacetamido-2,2,6,6-tetramethylpiperidine-1-oxyl (ISL). The highest concentration of endotoxin significantly (P<0.05) decreased the relative rotational correlation time of ISL and significantly (P<0.05) increased the osmotic fragility of RBCs. The effect of endotoxin was much more profound in isolated membranes than in intact cells treated with LPS. At the concentrations 0.5 and 1 mg/ml, LPS led to a significant increase in h(w)/h(s) ratio. These results indicated increased membrane protein mobility, mainly in the spectrin-actin complex in membrane cytoskeleton. These data suggest that LPS-induced alterations in membrane lipids and cytoskeleton proteins of RBCs lead to loss of membrane integrity.     

S-100 protein-induced changes in the physical state of synaptosomal particulate fractions as monitored by spin labels

This report documents changes in the physical state of synaptosomal particulate fractions (SYN) upon binding of S-100 protein, as monitored by spin labels. Studies were conducted on SYN labeled with either 5-doxylstearic acid or 16-doxylstearic acid, which probe the polar region and the hydrophobic core of the lipid bilayer, respectively. S-100 perturbs to some extent both the polar surface and the hydrophobic core of SYN in a time- and temperature-dependent manner. Ca2+ is essential for S-100 to perturb the membranes. K+ almost completely inhibits the S-100 perturbing effect if present in the incubation medium, but fails to reverse the S-100-induced changes if added after S-100 has interacted with SYN. At room temperature and below, the overall S-100 effect registered after about 30 min of association of the protein with SYN is an increase in the fluidity of both the surface and the interior of the membranes. Spectra registered at intervals at room temperature indicate that the S-100 perturbing effect on the membrane surface is practically monophasic, consisting of an increase in fluidity, while that on the membrane interior is multiphasic, consisting of a decrease in fluidity during the first 10 min of association, followed by an increase in fluidity during the subsequent 20 min and a return to starting values during the second 30 min of association. Around 37 degrees C, on the contrary, a decrease in fluidity is registered in both regions. The data suggest that S-100 induces a spatial rearrangement of membrane components (proteins) involved in the specific binding and/or partially penetrates into the lipid bilayer.     

Biophysical properties of membrane lipids of anammox bacteria: II. Impact of temperature and bacteriohopanoids

Anammox bacteria possess unique membranes that are mainly comprised of phospholipids with extraordinary “ladderane” hydrocarbon chains containing 3 to 5 linearly concatenated cyclobutane moieties that have been postulated to form relatively impermeable membranes. In a previous study, we demonstrated that purified ladderane phospholipids form fluid-like mono- and bilayers that are tightly packed and relatively rigid. Here we studied the impact of temperature and the presence of bacteriohopanoids on the lipid density and acyl chain ordering in anammox membranes using Langmuir monolayer and fluorescence depolarization experiments on total lipid extracts. We showed that anammox membrane lipids of representatives of Candidatus “Kuenenia stuttgartiensis”, Candidatus “Brocadia fulgida” and Candidatus “Scalindua” were closely packed and formed membranes with a relatively high acyl chain ordering at the temperatures at which the cells were grown. Our findings suggest that bacteriohopanoids might play a role in maintaining the membrane fluidity in anammox cells.     

Does high affinity [3H] imipramine binding label serotonin reuptake sites in brain and platelet?

Recent studies have demonstrated the presence of high affinity binding sites for [³H] imipramine in membrane preparations derived from rat brain, human platelet, and human brain. Although initial reports concluded that there was no relationship between these binding sites and the reuptake sites for biogenic amines, subsequent studies in our laboratory suggested a close relationship between the high affinity imipramine binding site and the serotonin uptake or transport site (cf. ref. 9). To further establish whether these binding sites are associated with either platelet or neuronal uptake of serotonin, the relative potencies of a series of tricyclic antidepressants in inhibiting [³H] imipramine binding and serotonin uptake were determined under identical assay conditions. A close correlation between inhibition of serotonin uptake and [³H] imipramine binding was observed (r = 0.99, p < 0.001). In addition, electrolytic lesions of the midbrain raphe produced a decrease in [³H] imipramine binding in hypothalamic synaptosomes that paralleled the decrease in [³H] serotonin uptake. Finally incubation of synaptosomal membranes with 2,8-dinitroimipramine, an irreversible inhibitor of [³H] imipramine binding, produced a dose-dependent decrease in serotonin uptake, without altering the uptake of nonrepinephrine or dopamine. Taken together our results strongly suggest that high affinity binding of [³]] imipramine selectively labels serotonin uptake sites in brain and platelet.     

Modulation of prolactin binding sites in vitro by membrane fluidizers. Effects on male prostatic and female hepatic membranes in alcohol-fed rats

The objectives of this study were (i) to determine if in vivo administration of ethanol to rats produced changes in apparent lipid fluidity and prolactin binding capacity of male prostatic and female hepatic membranes and (ii) to compare the effects of membrane fluidizers (aliphatic alcohols) in vitro on prolactin binding of prostatic and hepatic membranes in control and alcohol-fed animals. In vitro ethanol has been shown by us previously to increase prolactin receptor levels presumably by unmasking cryptic prolactin receptors. The degree of fluidization was monitored by a fluorescence polarization method using 1,6-diphenylhexatriene. Adult male and female rats were given either water or 4% ethanol as the sole source of drinking fluid for a period of 6 weeks. No significant changes in plasma prolactin were observed between control and ethanol-treated groups of either sex. However, the microviscosity parameter, inversely related to lipid fluidity, was increased approx. 34% and 40%, respectively, in male prostatic and female rat hepatic membranes after ethanol feeding. Furthermore, 125I-prolactin binding capacity was decreased approx. 30% and 26%, respectively, in prostatic and hepatic membranes of alcohol fed animals. In vitro treatment with aliphatic alcohols had no effect on either microviscosity or prolactin binding in hepatic or prostatic membranes from ethanol-fed rats, but both fluidized and increased prolactin binding in the same membrane preparations from control rats. Our observations are consistent with the direct relationship between membrane fluidity and prolactin receptor levels. The changes in prostatic and hepatic membranes after alcohol feeding, namely decreased prolactin receptor levels, decreased fluidity and increased resistance to the fluidizing effects of in vitro aliphatic alcohols may reflect a fundamental membrane defect.     

Diadinoxanthin de‐epoxidation as important factor in the short‐term stabilization of diatom photosynthetic membranes exposed to different temperatures

The importance of diadinoxanthin (Ddx) de‐epoxidation in the short‐term modulation of the temperature effect on photosynthetic membranes of the diatom Phaeodactylum tricornutum was demonstrated by electron paramagnetic resonance (EPR), Laurdan fluorescence spectroscopy, and high‐performance liquid chromatography. The 5‐SASL spin probe employed for the EPR measurements and Laurdan provided information about the membrane area close to the polar head groups of the membrane lipids, whereas with the 16‐SASL spin probe, the hydrophobic core, where the fatty acid residues are located, was probed. The obtained results indicate that Ddx de‐epoxidation induces a two component mechanism in the short‐term regulation of the membrane fluidity of diatom thylakoids during changing temperatures. One component has been termed the “dynamic effect” and the second the “stable effect” of Ddx de‐epoxidation. The “dynamic effect” includes changes of the membrane during the time course of de‐epoxidation whereas the “stable effect” is based on the rigidifying properties of Dtx. The combination of both effects results in a temporary increase of the rigidity of both peripheral and internal parts of the membrane whereas the persistent increase of the rigidity of the hydrophobic core of the membrane is solely based on the “stable effect.”     

Decreased [3H]serotonin and [3H]spiperone binding consequent to lipid peroxidation in rat cortical membranes

The effect of lipid peroxidation on two types of serotonin binding sites was investigated. Incubation of rat cortical membranes with an ascobate-dependent peroxidizing system resulted in the formation of malonyldialdehyde and significant decreases in the specific binding of [³H]serotonin and [³H]spiperone to the treated membranes. When ascorbate concentrations were varied from 0.025 to 6.0 mM, malonyldialdehyde production increased to a maximum at 0.5 mM ascorbate and then declined. Conversely, the specific binding of [³H]serotonin and [³H]spiperone decreased to a minimum at 0.5 mM ascorbate and then increased. Regression analysis of the data revealed that the decrease in the two binding sites was linearly correlated to lipid peroxidation.     

Vitamin E: inhibition of retinol-induced hemolysis and membrane-stabilizing behavior.

For the elucidation of the mechanism of membrane stabilization by vitamin E, the effects of alpha-tocopherol and its model compounds on either retinol-induced hemolysis of rabbit erythrocytes or the permeability and fluidity of liposomal membranes have been studied. Retinol-induced rabbit erythrocyte hemolysis has been found not to be caused by the oxidative disruption of erythrocyte membrane lipids initiated by retinol oxidation, but rather to arise from physical damage of the membrane micelle induced by penetration of retinol molecules. In suppressing hemolysis, alpha-tocopherol was more effective than other naturally occurring tocopherols. alpha-Tocopheryl acetate, nicotinate, and 6-deoxy-alpha-tocopherol were more effective than alpha-tocopherol itself. The inhibitory effects of alpha-tocopherol model compounds having side chains with at least two isoprene units or a long straight chain instead of the isoprenoid side chain were similar to those of alpha-tocopherol. These data suggest that for protection of membranes against retinol-induced damage, the hydroxyl group of alpha-tocopherol is not critical, but rather the chroman ring, three methyl groups on the aromatic ring, and the long side chain are necessary. To verify the mechanism of the inhibitory effect on hemolysis, not only the effect of vitamin E and its model compounds on the membrane permeability and fluidity, but also the mobility of alpha-tocopherol molecule in membranes has been investigated using bilayer liposomes as the model membranes. Addition of alpha-tocopherol to membranes produced a greater decrease in the permeability and fluidity of rat liver phosphatidylcholine liposomes compared with egg yolk phosphatidylcholine liposomes. In dipalmitoylphosphatidylcholine liposomes, however, alpha-tocopherol was less effective, that is, the more unsaturated the lipids, the more they interact with alpha-tocopherol. 2,2,5,7,8-Pentamethyl-6-chromanol with no isoprenoid side chain and phytol without the chromanol moiety had no effect. The measurement of 13C NMR relaxation times revealed that the mobility of methyl groups on the aromatic ring of alpha-tocopherol in membranes is significantly restricted. In contrast, the methyl groups at positions 4'a and 8'a on the isoprenoid side chain have high degrees of motional freedom in the lipid core of membranes. Furthermore, it was found that alpha-tocopherol in membranes interacts with chromate ions added as potassium chromate outside the membranes, resulting in an increase in membrane fluidity. These results are compatible with those of the inhibitory effect on retinol-induced erythrocyte hemolysis. On the basis of the results obtained here, a possible mechanism for membrane stabilization by vitamin E is proposed.     

Mg-nucleotides induced dissociation of liposome-bound creatine kinase: reversible changes in its secondary structure and in the fluidity of the bilayer

MgADP binding to mitochondrial creatine kinase (mtCK) adsorbed on liposomes was induced by the photorelease of caged ADP. The nucleotide binding produced two types of structural changes. One was related to the well-established release of mtCK from the liposomes. The other corresponded to reversible structural changes induced by nucleotide binding to mtCK as demonstrated here. Infrared spectroscopy data show that the MgADP-induced desorption of mtCK from vesicles led to a slight increase in &#102 -helix structures in mtCK at the expense of a small decrease in &#103 -sheet structures and a concomitant increase in the fluidity of the membranes. The desorption of mtCK induced by MgADP and MgATP was almost complete, as shown by centrifugation and enzymatic activity measurements. The photorelease of MgADP in a reactive medium containing phosphocreatine and mtCK associated with liposomes led to nucleotide binding and to the formation of MgATP and creatine. Addition of phosphocreatine also desorbed mtCK from liposomes, while addition of creatine did not. Interpretation of these results would suggest that ADP, ATP or phosphocreatine induce the release of mtCK from membranes, increase the phospholipid bilayer fluidity, and may also decrease the number of contact sites between inner and outer mitochondrial membranes, thus affecting the activity of other mitochondrial enzymes. It is tempting to propose that membrane mtCK binding regulation by nucleotide and PCr concentrations may serve as a physiological adaptation for energy supply.     

Microviscosity parameters and protein mobility in biological membranes.

A fluorescence polarization technique with 1,6-diphenyl 1,3,5-hexatriene as a probe were employed to determine the microviscosity, n, in liposomes and biological membranes of different cholesterol to phospholipid mol ratio. From the temperature profile of n the flow activation energy, deltaE, and the unit flow volume, V, were derived. The increase of cholesterol/phospholipid ratio in liposomes is followed by a marked increase in n and a decrease in both deltaE and V. Liposomes of the same phospholipid composition as human erythrocyte membranes display in the extreme cases of cholesterol/phospholipid ratios 0 and 1.4 the values of n(25 degrees C) = 1.8 and 9.1 P, and deltaE = 15.0 and 6.5 kcal/mol, respectively. For most membranes studied the fluorescence polarization characteristics and the corresponding n values are similar to those obtained with these liposomes when the cholesterol/phospholipid level of the liposomes and the membranes were the same. However, unlike in liposomes deltaE of all membranes is in the narrow range of 6.5-8.5 kcal/mol, regardless of its cholesterol/phospholipid level. It is plausible that this is a general characteristic of biological membranes which originates from the vertical movement of membrane proteins to an equilibrium position which maintains constant deltaE and V values. This type of movement should affect the interrelation between lipid fluidity and protein mobility. Lipid microviscosity and the degree of rotational mobility of concanavalin A receptor sites in cell membranes were therefore determined. The examined cells were normal and malignant fibroblasts, as an example of cells that form solid tumours in vivo, and normal and malignant lymphocytes, as an example of cells that form ascites tumours in vivo. In both cell systems, opposite correlations between the lipid fluidity and the mobility of concanavalin A receptors were observed. In the fibroblasts the malignant cells possess a lower lipid fluidity but a higher receptor mobility, whereas in the lymphocytes the malignant cells possess a higher lipid fluidity but a lower receptor mobility. Thus, in these cell systems the degree of rotational mobility of concanavalin A receptors increases upon decreasing the lipid fluidity and decreases upon increasing the fluidity of the lipid core. This dynamic feature is in line with the above proposal according to which the concanavalin A receptor sites become more exposed to the aqueous surrounding upon increasing the microviscosity of the lipid layer and vice versa.     

Diazepam increases membrane fluidity of rat hippocampus synaptosomes

Diazepam in vitro produced a concentration-dependent increase of membrane fluidity in crude synaptic membranes from rat hippocampus, but not cerebellum. Similar effects were obtained with higher concentrations of Ro 15-1788 and PK 11195, while zopiclone was completely inactive. In vivo acute treatment with diazepam and Ro 15-1788 gave results similar to those in vitro. The specific benzodiazepine antagonist also significantly increased membrane fluidity and was not able to reverse diazepam's effect. The data are discussed in terms of a possible role of protein kinase inhibition by the drugs not mediated by the 'central' or 'peripheral' type of benzodiazepine receptors.     

Alterations in erythrocyte membrane lipid composition and fluidity in primary lipoprotein lipase deficiency.

Lipid composition of plasma lipoproteins and erythrocyte ghost membranes has been studied in 16 healthy normolipidaemic subjects and in 16 patients affected by primary lipoprotein lipase deficiency, resulting in severe chylomicronaemia and in cholesterol-depleted low-density lipoproteins and high-density lipoproteins. A significant decrease in membrane cholesterol/phospholipid ratio was observed in lipoprotein lipase deficient patients compared to controls (3.27 +/- 0.33 vs. 3.95 +/- 0.50, mean +/- S.D.; P less than 0.0001). There was also an increase in the erythrocyte membrane phosphatidylcholine/sphingomyelin ratio in lipoprotein lipase deficient patients compared to controls (1.53 +/- 0.10 vs. 1.05 +/- 0.13; P less than 0.0001) due to a concurrent increase in phosphatidylcholine and decrease in sphingomyelin relative concentrations in these patients. Erythrocyte ghost membrane fluidity was determined by fluorescence anisotropy and found to be higher in membranes from lipoprotein lipase deficient patients. This increase in membrane fluidity can be attributed in part to changes in membrane cholesterol and phospholipid concentrations in response to abnormal plasma lipoprotein composition.     

Mg-nucleotides induced dissociation of liposome-bound creatine kinase: reversible changes in its secondary structure and in the fluidity of the bilayer

MgADP binding to mitochondrial creatine kinase (mtCK) adsorbed on liposomes was induced by the photorelease of caged ADP. The nucleotide binding produced two types of structural changes. One was related to the well-established release of mtCK from the liposomes. The other corresponded to reversible structural changes induced by nucleotide binding to mtCK as demonstrated here. Infrared spectroscopy data show that the MgADP-induced desorption of mtCK from vesicles led to a slight increase in alpha-helix structures in mtCK at the expense of a small decrease in beta-sheet structures and a concomitant increase in the fluidity of the membranes. The desorption of mtCK induced by MgADP and MgATP was almost complete, as shown by centrifugation and enzymatic activity measurements. The photorelease of MgADP in a reactive medium containing phosphocreatine and mtCK associated with liposomes led to nucleotide binding and to the formation of MgATP and creatine. Addition of phosphocreatine also desorbed mtCK from liposomes, while addition of creatine did not. Interpretation of these results would suggest that ADP, ATP or phosphocreatine induce the release of mtCK from membranes, increase the phospholipid bilayer fluidity, and may also decrease the number of contact sites between inner and outer mitochondrial membranes, thus affecting the activity of other mitochondrial enzymes. It is tempting to propose that membrane mtCK binding regulation by nucleotide and PCr concentrations may serve as a physiological adaptation for energy supply.