Effects of 2-hydroxyoleic acid on the structural properties of biological and model plasma membranes

Genetic hypertension is associated with alterations in lipid metabolism, membrane lipid composition and membrane-protein function. 2-Hydroxyoleic acid (2OHOA) is a new antihypertensive molecule that regulates the structure of model membranes and their interaction with certain peripheral signalling proteins in vitro. While the effect of 2OHOA on elevated blood pressure is thought to arise through its influence on signalling proteins, its effects on membrane lipid composition remain to be assessed. 2OHOA administration altered the lipid membrane composition of hypertensive and normotensive rat plasma membranes, and increased the fluidity of reconstituted liver membranes from hypertensive rats. In spontaneously hypertensive rats (SHR), treatment with 2OHOA increased the cholesterol and sphingomyelin content while decreasing that of phosphatidylserine-phosphatidylinositol lipids. In addition, monounsaturated fatty acid levels increased as well as the propensity of reconstituted membranes to form HII-phases. These data suggest that 2OHOA regulates lipid metabolism that is altered in hypertensive animals, and that it affects the structural properties of liver plasma membranes in SHR. These changes in the structural properties of the plasma membrane may modulate the activity of signalling proteins that associate with the cell membrane such as the Galphaq/11 protein and hence, signal transduction.     

The hypotensive drug 2-hydroxyoleic acid modifies the structural properties of model membranes

We studied the interactions of the hypotensive drug, 2-hydroxyoleic acid (2OHOA), with model membranes using the techniques of DSC, ³¹P NMR and X-ray diffraction. We demonstrate that 2OHOA alters the thermotropic behaviour of 1,2-dielaidoyl-sn-glycero-3-phosphoethanolamine (DEPE), thereby promoting the formation of hexagonal phases (H_II), despite stabilizing the lamellar phase (L_α). The lattice parameters of lamellar and non-lamellar structures were not altered by the presence of 2OHOA. The molecular bases underlying the alterations in membrane structure provoked by 2OHOA were analysed by comparing the effects produced by 2OHOA with the closely related fatty acids (FAs), oleic acid (OA) and elaidic acid (EA). The capacity of C-18 FAs to induce H_II-phase formation followed the order OA>2OHOA>EA. Furthermore, while 2OHOA stabilized the L_α phase, OA destabilized it. The net negative charge of 2OHOA at physiological pH (～7.4) influenced its effect on membrane structure. By analysing the molecular architecture of 2OHOA in DEPE monolayers, interactions between the carboxylate groups of 2OHOA and the amine groups of DEPE were observed, as well as between the 2-hydroxyl group of the FA and the carbonyl oxygen of the phospholipid acyl chain. These structural characteristics provoked an increase in the P-to-N and P-to-P distances of neighbouring phospholipid headgroups in the presence of 2OHOA, with respect to those observed with OA and EA. The higher headgroup area at the lipid–water interface in presence of 2OHOA could account for the differential effect of this drug on the phase behaviour of DEPE membranes.     

The hypotensive drug 2-hydroxyoleic acid modifies the structural properties of model membranes

We studied the interactions of the hypotensive drug, 2-hydroxyoleic acid (2OHOA), with model membranes using the techniques of DSC, 31P NMR and X-ray diffraction. We demonstrate that 2OHOA alters the thermotropic behaviour of 1,2-dielaidoyl-sn-glycero-3-phosphoethanolamine (DEPE), thereby promoting the formation of hexagonal phases (H(II)), despite stabilizing the lamellar phase (Lalpha). The lattice parameters of lamellar and non-lamellar structures were not altered by the presence of 2OHOA. The molecular bases underlying the alterations in membrane structure provoked by 2OHOA were analysed by comparing the effects produced by 2OHOA with the closely related fatty acids (FAs), oleic acid (OA) and elaidic acid (EA). The capacity of C-18 FAs to induce H(II)-phase formation followed the order OA > 2OHOA > EA. Furthermore, while 2OHOA stabilized the Lalpha phase, OA destabilized it. The net negative charge of 2OHOA at physiological pH (approximately 7.4) influenced its effect on membrane structure. By analysing the molecular architecture of 2OHOA in DEPE monolayers, interactions between the carboxylate groups of 2OHOA and the amine groups of DEPE were observed, as well as between the 2-hydroxyl group of the FA and the carbonyl oxygen of the phospholipid acyl chain. These structural characteristics provoked an increase in the P-to-N and P-to-P distances of neighbouring phospholipid headgroups in the presence of 2OHOA, with respect to those observed with OA and EA. The higher headgroup area at the lipid-water interface in presence of 2OHOA could account for the differential effect of this drug on the phase behaviour of DEPE membranes.     

Phospholipase A2 induced effects on the structural organization and physical properties of pea chloroplast membranes

Phospholipase A₂ (PLA₂)-induced effects on the membrane organization, fluidity properties and surface charge density of pea chloroplasts were investigated. It was observed that lipolytic treatment with PLA₂ altered the chloroplast structure having as a result a swelling of thylakoids and a total destruction of normal granal structure. In spite of this, the thylakoid membranes remained in close contact. At the same time, a slight decrease of surface charge density was registered, thus explaining the adhesion of swelled membranes. Fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene (DPH) was measured during PLA₂ treatment. A pronounced decrease of DPH fluorescence polarization was found, indicating that phospholipase treatment resulted in considerable disordering and/or fluidization of the thylakoid membranes. The increased fluidity could be attributed to the destabilizing effect of the products of enzymatic hydrolysis of the phospholipids (free fatty acids, lysophospholipids) on the bilayer structure of thylakoids membranes.Abbreviations 9-AA 9-aminoacridine - BSA bovine serium albumin - DCMU 3-/3,4-dichlorophenyl-1,1-dimethyl/urea - DPH 1,6-diphenyl-1,3,5-hexatriene - EDTA ethylenediaminetetraacetic acid - HEPES N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid - LHC light harvesting chlorophyll a/b-protein complex of PS II - MES 2/N-morpholine/ethanesulfonic acid - PLA₂ phospholipase A₂ - PS I, PS II photosystem I and photosystem II, respectively - S lipid structural order parameter - THF tetrahydrofuran - TRICINE N-/tris/hydroxymethyl/methyl/glicine     

Hormonal effects on fatty acid binding and physical properties of rat liver plasma membranes

Summary The fluorescent fatty acids,trans-parimaric andcis-parinaric acid, were used as analogs of saturated and unsaturated fatty acids in order to evaluate binding of fatty acids to liver plasma membranes isolated from normal fed rats. Insulin (10^–8 to 10^–6 m) decreasedtrans-parinaric acid binding 7 to 26% whilecis-parinaric acid binding was unaffected. Glucagon (10^–6 m) increasedtrans-parinaric acid binding 44%. The fluorescence polarization oftrans-parinarate,cis-parinarate and 1,6-diphenyl-1,3,5-hexatriene was used to investigate effects of triiodothyronine, insulin and glucagon on the structure of liver plasma membranes from normal fed rats or from rats treated with triiodothyronine or propylthiouracil. The fluorescence polarization oftrans-parinarate,cis-parinarate, and 1,6-diphenyl-1,3,5-hexatriene was 0.300±0.004, 0.251±0.003, and 0.302±0.003, respectively, in liver plasma membranes from control rats and 0.316±0.003, 0.276±0.003 and 0.316±0.003, respectively, in liver plasma membranes from hyperthyroid rats (p<0.025,n=5). Propylthiouracil treatment did not significantly alter the fluorescence polarization of these probe molecules in the liver plasma membranes. Thus, liver plasma membranes from hyperthyroid animals appear to be more rigid than those of control animals. The effects of triiodothyronine, insulin and glucagon addedin vitro to isolated liver plasma membrane preparations were also evaluated as follows: insulin (10^–10 m) and triiodothyronine (10^–10 m) increased fluorescence polarization oftrans-parinaric acid,cis-parinaric acid and 1,6-diphenyl-1,3,5-hexatriene in liver plasma membranes while glucagon (10^–10 m) had no effects. These hormonal effects on probe fluorescence polarization in liver plasma membranes were abolished by pretreatment of the rats for 7 days with triiodothyronine. Administration of triiodothyronine (10^–10 m)in vitro increased the fluorescence polarization of trans-parinaric acid in liver plasma membranes from propylthiouracil-treated rats. Thus, hyperthyroidism appeared to abolish thein vitro increase in polarization of probe molecules in the liver plasma membranes. Temperature dependencies in Arrhenius plots of absorption-corrected fluorescence and fluorescence polarization oftrans-parinaric acid,cis-parinaric acid and 1,6-diphenyl-1,3,5-hexatriene were noted near 25°C in liver plasma membranes from triiodothyronine-treated rats and near 18°C in liver plasma membranes from propylthiouracil-treated rats. In summary, hormones such as triiodothyronine, insulin and glucagon may at least in part exert their biological effects on metabolism by altering the structure of the liver plasma membranes.     

Activatory effect of regucalcin on GTPase activity in rat liver plasma membranes

The effect of regucalcin, a regulatory protein of Ca²⁺ signaling, on guanosine-5-triphosphatase (GTPase) activity in isolated rat liver plasma membranes was investigated. GTPase activity was significantly increased by the addition of Ca²⁺ (25–100 M) in the enzyme reaction mixture. Such an increase was not seen by other metals (Mg, Co, Zn, Cu, Ni, and Mn) with 50 M. The activatory effect of calcium (50 M) was significantly decreased by calmodulin (2.5 and 5 g/ml), indicating that it does not depend on calmodulin. The presence of regucalcin (0.1–0.5 M) in the enzyme reaction mixture caused a significant increase in GTPase activity. This increase was not significantly enhanced by calcium (50 M). GTPase activity was significantly increased by dithiothreitol (DTT; 5 mM), a protecting reagent of thiol (SH)-groups, while it was decreased by N-ethylmaleimide (NEM; 5 mM), a modifying reagent of SH-groups. The effect of calcium or regucalcin in increasing GTPase activity was not seen in the presence of NEM. Also, the activatory effect of calcium or regucalcin on GTPase was not seen in the presence of vanadate, an inhibitor of protein phosphorylation, which could inhibit GTPase activity. Moreover, the effect of regucalcin was not seen in the presence of digitonin (0.01%), a solubilizing reagent of membranous lipids, while the effect of calcium was not inhibited by digitonin. The present study demonstrates that regucalcin has an activatory effect on GTPase activity independently of Ca²⁺ in rat liver plasma membranes.     

过氧化氢诱导酿酒酵母细胞膜透性和组成的变化

以下简述了过氧化氢(H2O2)作为一种信号分子诱导并调节酿酒酵母(Saccharomyces cerevisiae)细胞膜的变化。H2O2是一种强氧化剂,可以跨膜扩散进入细胞中,形成跨膜梯度;当外源H2O2达到亚致死剂量时,酿酒酵母的细胞膜透性和流动性降低,产生跨膜梯度,从而限制H2O2向细胞内的扩散速率,保护细胞免受氧化胁迫的伤害。研究表明,由H2O2引起的膜透性和流动性的变化与膜的组成有关:当酵母细胞对H2O2产生适应时,与膜组成和微区域变化有关的几个基因的表达发生了改变。膜组成的变化和微区域的调整还可能与H2O2依赖的信号途径有关,即以H2O2为信号分子,调节膜的变化并赋予细胞对氧化压力更高的适应性,但这种信号分子的具体传递途径及机制还需要进一步研究。     

Effects of semen preservation on boar spermatozoa head membranes

Head plasma membranes were isolated from the sperm-rich fraction of boar semen and from sperm-rich semen that had been subjected to three commercial preservation processes: Ex tended for fresh insemination (extended), prepared for freezing but not frozen (cooled), and stored frozen for 3-5 weeks (frozen-thawed). Fluorescence polarization was used to determine fluidity of the membranes of all samples for 160 min at 25°C and also for membranes from the sperm-rich and extended semen during cooling and reheating (25 to 5 to 40°C, 0.4°C/min). Head plasma membranes from extended semen were initially more fluid than from other sources (P < 0.05). Fluidity of head membranes from all sources decreased at 25°C, but the rate of decrease was significantly lower for membranes from cooled and lower again for membranes from frozen-thawed semen. Cooling to 5°C reduced the rate of fluidity change for plasma membranes from the spernvrich fraction, while heating over 30°C caused a signifi cantly greater decrease. The presence of Ca⁺⁺ (10 mM) lowered the fluidity of the head plasma membranes from sperm-rich and extended semen over time at 25°C but did not affect the membranes from the cooled or frozen-thawed semen. The change in head plasma membrane fluidity at 25°C may reflect the dynamic nature of spermatozoa membranes prior to fertilization. Extenders, preservation processes and temperature changes have a strong influence on head plasma membrane fluidity and therefore the molecular organization of this membrane.     

Effects of cold shock and phospholipase A2 on intact boar spermatozoa and sperm head plasma membranes

Head plasma membranes (HPM) isolated from cryopreserved boar spermatozoa show an excessive fluidization, which might be involved in the loss of fertility. The current study assessed the ability of cold shock (5 degrees C) and phospholipase A2 (PA2) to duplicate these effects on membrane structure and to affect 45Ca2+ uptake and gross morphological characteristics of whole, fresh boar-sperm. The HPM from cold-shocked sperm showed a significantly greater rate of fluidization over time than did HPM from control sperm. Addition of PA2 (bee or snake venom, 0.1 or 10.0 ng/ml) to HPM from control sperm caused fluidization similar to cold shocking, but to a lesser degree (P less than 0.05). Cold-shocked intact sperm exhibited severe acrosomal disruption, loss of motility, and increased 45Ca2+ uptake relative to control sperm. Addition of PA2 (bee or snake venom, 0.1, 1.0., 10.0, and 1,000 ng/ml) to control sperm had no effect on gross morphology or motility while maintaining or increasing sperm extrusion of 45Ca2+. Therefore, although PA2 can, to some extent, duplicate the effects of cold shock on HPM molecular organization, its lipid hydrolytic action is insufficient to cause all the gross disruptions of severe thermal shock. Both PA2 and cold shock disrupted HPM structure, but only cold shock increased 45Ca2+ uptake, suggesting that cold shock may be increasing 45Ca2+ uptake in areas other than the head. Cold shock disrupts sperm on three levels; membrane molecular organization, intracellular Ca2+ regulation, and gross morphology/motility.     

Temperature-induced modifications of glycosphingolipids in plasma membranes of Neurospora crassa

Plasma membranes isolated from a cell-wall-less mutant of Neurospora crassa grown at 37 and 15°C display large differences in lipid compositions. A free sterol-to-phospholipid ratio of 0.8 was found in 37°C membranes, while 15°C plasma membranes exhibited a ratio of nearly 2.0. Membranes formed under both growth conditions were found to contain glycosphingolipids. Cultures grown at the low temperature, however, were found to contain 6-fold higher levels of glycosphingolipids and a corresponding 2-fold reduction of phospholipid levels. The high glycosphingolipid content at 15°C compensates for the reduced levels of phospholipids in such a way that sterol/polar lipid ratios are almost the same in plasma membranes under the two growth conditions. Temperature-dependent changes in plasma-membrane phospholipid and glycosphingolipid species were also observed. Phosphatidylethanolamine levels were sharply reduced at 15°C, in addition to a moderate increase in levels of unsaturated phospholipid fatty acids. Glycosphingolipids contained high levels of long-chain hydroxy fatty acids, which constituted 75% of the total fraction at 37°C, but only 50% at 15°C. Compositional changes were also observed in the long-chain base component of glycosphingolipids with respect to growth temperature. Fluorescence polarization studies indicate that the observed lipid modifications in 15°C plasma membranes act to modulate bulk fluidity of the plasma-membrane lipids with respect to growth temperature. These studies suggest that coordinate modulation of glycosphingolipid, phospholipid and sterol content may be involved in regulation of plasma-membrane fluid properties during temperature acclimation.     

Enzymatic,biophysical and ultrastructural changes of plasma membranes in chemical-induced rat hepatoma

Plasma membranes from liver of control rats or from chemical-induced hepatoma were prepared. The basal activity of adenylate cyclase was increased significantly in the rat plasma membranes of DEN-induced hepatoma compared to normal tissue. The glucagon-induced response on the cellular effector systems via guanine nucleotide-binding regulatory proteins (G proteins) was inhibited in hepatoma plasma membranes. These findings suggest that in hepatoma membranes, unlike normal hepatic membranes, the response to hormonal stimuli through regulatory G proteins results in a loss of response to glucagon, as well as to GTP plus glucagon or to GTPγS. However, the activating effects of forskolin, which catalyses the formation of cyclic AMP from ATP acting on the catalytic subunit, were to some extent retained. The methyltransferase-I behaved in the opposite direction to the adenylate cyclase, showing a decreased activity in hepatoma plasma membranes compared to control membranes. In contrast, the activity of the ecto-5′-nucleotidase was significantly increased in hepatoma. These enzymatic changes have been found to influence the membrane fluidity and to be responsible for the ultrastructural modifications of hepatoma plasma membranes which are induced by chemical carcinogens.     

Thiol reactivity and the molecular individuality of α- and β-adrenoreceptors in rat liver plasma membranes

Whether or not α- and β-adrenoreceptors are non-identical binding sites on the same protein is still an open question. We investigated the effects of sulfhydryl reagents and dithiothreitol on the binding of [³H]dihydroalprenolol and [³H]dihydroergocryptine to β- and α-adrenoreceptors of rat liver plasma membranes. Dithiothreitol inhibited the binding of [³H]dihydroalprenolol to the β-adrenoreceptor, whereas it had no effect on the specific binding of [³H]dihydroergocryptine to the α-adrenoreceptor. In contrast, mersalyl, a mercurial SH reagent, readily blocked the α-adrenoreceptor and, although to a lesser extent, the β-adrenoreceptor. The interaction of mersalyl with the α-adrenoreceptors was almost instantaneous. In contrast, under the same experimental conditions, the inactivation of the β-adrenoreceptors was much slower ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{:7}\text{min}$). Finally, a marked difference in the accessibility of the SH groups to mersalyl was observed between the α- and β-adrenoceptors. The presence of 15 μM (?)-epinephrine or 1.5 μM phentolamine was sufficient to prevent the blockade of the α-adrenoreceptor by mersalyl, but inactivation of the β-adrenoreceptor by mersalyl was not modified by 500 μM (?)-epinephrine and was only slightly decreased by 50 μM (?)-propranolol. Thus, the α- and β-adrenoreceptors from rat liver plasma membranes exhibited biochemical differences which may be interpreted in favor of their molecular individuality.     

Differential effects of conjugated linoleic acid isomers on the biophysical and biochemical properties of model membranes

Conjugated linoleic acids (CLA) are known to exert several isomer-specific biological effects, but their mechanisms of action are unclear. In order to determine whether the physicochemical effects of CLA on membranes play a role in their isomer-specific effects, we synthesized phosphatidylcholines (PCs) with 16:0 at sn-1 position and one of four CLA isomers (trans 10 cis 12 (A), trans 9 trans 11 (B), cis 9 trans 11 (C), and cis 9 cis 11 (D)) at sn-2, and determined their biophysical properties in monolayers and bilayers. The surface areas of the PCs with the two natural CLA (A and C) were similar at all pressures, but they differed significantly in the presence of cholesterol, with PC-A condensing more than PC-C. Liposomes of PC-A similarly showed increased binding of cholesterol compared to PC-C liposomes. PC-A liposomes were less permeable to carboxyfluorescein compared to PC-C liposomes. The PC with two trans double bonds (B) showed the highest affinity to cholesterol and lowest permeability. The two natural CLA-PCs (A and C) stimulated lecithin-cholesterol acyltransferase activity by 2-fold, whereas the unnatural CLA-PCs (B and D) were inhibitory. These results suggest that the differences in the biophysical properties of CLA isomers A and C may partly contribute to the known differences in their biological effects.     

Lipid peroxidation and fluidity of plasma membranes from rat liver and Morris hepatoma 3924A

Plasma membranes isolated from the fast-growing, maximal-deviation, Morris hepatoma 3924A exhibit remarkable changes in lipid composition, lipid peroxidation and to some extent in the physical state with respect to rat liver plasmalemmas. A correlation appears to exit between the lower phospholipid: protein ratio, higher cholesterol: phospholipid ratio, lower rate of lipid peroxidation and decrease in fluidity in tumor plasma membranes.     

Calmodulin selectively modulates the guanylate cyclase activity by repressing the lipid phase separation temperature in the inner half of the bilayer of rat brain synaptosomal plasma membranes

The association of [¹²⁵I-]calmodulin with rat brain synaptosomal plasma membranes, when incubated for 1 h at 25° in the presence or in absence of 20 M Ca²⁺, follows a sigmoid path with a Hill coefficient h=1.79±0.12 and h=1.72±0.11, respectively. The total association of calmodulin with the membrane increased approx. 60%–80% at all the range of calmodulin concentrations used in the presence of 20 M Ca²⁺. A three fold increase of guanylate cyclase activity was shown in the presence of low concentrations of calmodulin (up to 10 mM); higher concentrations (up to 40 mM) however, led to a progressive inhibition of the enzyme activity with respect to maximal stimulation. Calmodulin increased the lipid fluidity of synaptosomal plasma membranes labeled with 1,6-diphenyl-1,3,5-hexatriene (DPH), as indicated by the steady-state fluorescence anisotropy [(r_o/r)-1]^–1. Arrhenius-type plots of [(r_o/r)-1]^–1 indicated that the lipid separation of the membrane at 22.7±1.2° was perturbed by calmodulin such that the temperature was reduced to 16.3±0.9° and 15.5±0.8° in the absence or in the presence of 20 M Ca²⁺. Arrhenius plots of guanylate cyclase and acetylcholinesterase activities exhibited brak points at 25.7±1.4° and 22.3±1.0° in control synaptosomal plasma membranes, respectively. The break point for the guanylate cyclase was reduced to 16.3±0.9° in calmodulin treated synaptosomal plasma membranes whereas that of acetylcholinesterase remained unaffected (21.1±0.9°). The allosteric properties of guanylate cyclase by Mn-GTP (as reflected by changes in the Hill coefficient) were modulated by calmodulin while those of acetylcholinesterase by fluoride (F^–) were not altered. We propose that calmodulin achieves these effects through asymmetric perturbations of the membrane lipid structure and that increase in membrane fluidity of the inner leaflet of the membrane induced by calmodulin may be an early key event to the process of neurotransmitter release.     

Generation of hydrogen peroxide on oxidation of NADH by hepatic plasma membranes

The oxidation of NADH by mouse liver plasma membranes was shown to be accompanied by the formation of H₂O₂. The rate of H₂O₂ formation was less than one-tenth the rate of oxygen uptake and much slower than the rate of reduction of artificial electron acceptors. The optimum pH for this reaction was 7.0 and theK _m value for NADH was found to be 3×10^–6 M. The H₂O₂-generating system of plasma membranes was inhibited by quinacrine and azide, thus distinguishing it from similar activities in endoplasmic reticulum and mitochondria. Both NADH and NADPH served as substrates for plasma membrane H₂O₂ generation. Superoxide dismutase and adriamycin inhibited the reaction. Vanadate, known to stimulate the oxidation of NADH by plasma membranes, did not increase the formation of H₂O₂. In view of the growing evidence that H₂O₂ can be involved in metabolic control, the formation of H₂O₂ by a plasma membrane NAD(P)H oxidase system may be pertinent to control sites at the plasma membrane.     

Epigallocatechin-3-gallate location and interaction with late endosomal and plasma membrane model membranes by molecular dynamics

Epigallocatechin-3-gallate (EGCG) is the most abundant polyphenol in green tea and it has been reported to have many beneficial properties against many different types of illnesses and infections. However, the exact mechanism/s underlying its biological effects are unknown. It has been previously shown that EGCG is capable of binding to and disrupting the membrane, so that some of its effects on biological systems could be ascribed to its capacity to incorporate into the biological membrane and modulate its structure. In this work, we have used atomistic molecular dynamics (MD) to discern the location and orientation of EGCG in model membranes and the possible existence of specific interactions with membrane lipids. For that goal, we have used in our simulation two complex model membranes, one resembling the plasma membrane (PM) and the other one the late endosome (LE) membrane. Our results support that EGCG tends to associate with the membrane and exists inside it in a relatively stable and steady location with a low propensity to be associated with other EGCG molecules. Interestingly, EGCG forms hydrogen bonds with POPC and POPE in the PM system but POPC and BMP and no POPE in the LE. These data suggest that the broad beneficial effects of EGCG could be mediated, at least in part, through its membranotropic effects and therefore membrane functioning.

Communicated by Ramaswamy H. Sarma     

Molecular mechanism of action of chlorogenic acid on erythrocyte and lipid membranes

Abstract

The high antioxidant capacity of chlorogenic acid (CGA) in respect to biological systems is commonly known, though the molecular mechanism underlying that activity is not known. The aim of the study was to determine that mechanism at the molecular and cell level, in particular with regard to the erythrocyte and the lipid phase of its membrane. The effect of CGA on erythrocytes and lipid membranes was studied using microscopic, spectrophotometric and electric methods. The biological activity of the acid was determined on the basis of changes in the physical parameters of the membrane, in particular its osmotic resistance and shapes of erythrocytes, polar head packing order and fluidity of erythrocyte membrane as well as capacity and resistivity of black lipid membrane (BLM). The study showed that CGA becomes localized mainly in the outer part of membrane, does not induce hemolysis or change the osmotic resistance of erythrocytes, and induces formation of echinocytes. The values of generalized polarization and fluorescence anisotropy indicate that CGA alters the hydrophilic region of the membrane, practically without changing the fluidity in the hydrophobic region. The assay of electric parameters showed that CGA causes decreased capacity and resistivity of black lipid membranes. The overall result is that CGA takes position mainly in the hydrophilic region of the membrane, modifying its properties. Such localization allows the acid to reduce free radicals in the immediate vicinity of the cell and hinders their diffusion into the membrane interior.     

Anion effects on the structural organization of spinach thylakoid membranes

Changes in the conformation of spinach thylakoid membranes were monitored in 5-doxyl stearic acid (SAL)-treated thylakoid membranes in the presence of various anions (Cl⁻, Br⁻, I⁻, NO₂ ⁻, SO₄ ²⁻, PO₄ ³⁻). The presence of anions made the thylakoid membrane more fluid. The extent of change in membrane fluidity differed with different anion and was reversible.