Modification Of Membrane Structure Research Articles

Surfactant as modulating agent of enzyme-loaded liposome activity

Large phosphatydilcholine unilamellar vesicles appear to be suitable controlled and protective delivery systems of beta-galactosidase. Kinetic measurements carried out on intact loaded liposomes show that most of the enzyme is entrapped inside the liposomes and its activity is latent. Nevertheless, intact liposomes also show significant activity, which can be controlled by addition of detergent. At sublytic detergent concentrations, liposome enzymatic activity reaches values two or three times greater than those of intact liposomes. This increase seems to be due to membrane structure modification that also enhances the substrate permeability across the bilayer. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 55: 261-266, 1997.

Effect of a low-osmolality nonionic contrast medium (iopromide) on blood platelet membrane structure and aggregation

Nonionic contrast media (NICM) used in radiology exhibit weaker anticoagulant properties than ionic media. This study was intended to evaluate the effect of iopromide NICM upon the platelet aggregation and their membrane structure (investigated by electron paramagnetic resonance method with a spin label). Blood was collected directly from the left ventricle during coronary angiography prior to and following the application of the contrast medium. These results were compared with in vitro examinations. Based on the spectral curves parameters that reflect conformational changes in membrane proteins were determined. The iopromide-induced decrease in platelet aggregation examined in vitro with adenosine diphosphate (ADP), collagen and ristocetine was statistically significant. In vitro platelet membrane structure was modified significantly with regard to both protein conformation and the accessibility of sulfhydryl (- SH) and amino (- NH2) groups. In ex vivo studies the changes in platelet aggregation was only observed with ADP, whereas in the membrane-structure modifications were only noted regarding accessibility of - SH and - NH2 groups. We conclude that iopromide-induced platelet aggregation changes are most probably due to their membrane-structure modifications, whereas the less-pronounced contrast effect ex vivo is due to its rapid dilution in the inflowing blood. The results confirm weak antiplatelet and anticoagulant influence of NICM in vivo.

Effect of anthracyclines on phospholipase A 2 activity and prostaglandin E 2 production in rat gastric mucosa

The purpose of this study was to investigate in rats the effects of three anthracyclines, pirarubicin, doxorubicin and epirubicin on gastric prostaglandin E 2 (PGE 2) metabolism and phospholipase A 2 (PLA 2, EC 3.1.1.4) activity. The level of the membrane precursor, arachidonic acid, and the stability of the membrane were investigated by analysis of the composition of fatty acids. Enzymatic activities involved in the turnover of membrane phospholipids such as lysophospholipase (LPase, EC 3.1.1.5) and acyl-CoA lysophosphatidylcholine: acyltransferase (ACLAT, EC 2.3.1.23), and in the detoxification of lipid hydroperoxides, selenium-dependent glutathione-peroxidase (GSH-PX, EC 1.11.1.9) were measured after injection of the drugs for 4 consecutive days. Pirarubicin does not give rise to any changes in these activities but doxorubicin and epirubicin decreased PGE 2 production and the activities of PLA 2, LPase and ACLAT. GSH-PX activity was not changed by any of the drugs. The decrease in PLA 2 activity does not seem to be related to variations in membrane lipid composition because the total phospholipids content was unchanged. The P/S (polyunsaturated/ saturated) ratio increased in the doxorubicin group and decreased in the epirubicin group, and the unsaturation index was moderately modified. Arachidonic acid was increased only in the doxorubicin group. In vitro, PLA 2 activity was not inhibited by the three drugs in the micromolar range. A marked inhibition was observed at 2.5 mM for pirarubicin and at 1.0 mM for doxorubicin and epirubicin. The Lineweaver-Burk representation showed that these inhibitions were of an uncompetitive type. Pirarubicin may therefore be considered to be an anthracycline without marked side-effects on gastric mucosa. However, the in vitro inhibition of PLA 2 activity by anthracyclines does not fully explain the in vitro decrease in PLA 2 specific activity observed after doxorubicin and epirubicin treatment, and in this context membrane structure modifications unconnected with the lipid composition can not be excluded. In vivo these phenomena may affect PGE 2 synthesis, whose level was lower in the doxorubicin and epirubicin groups than in control group.

Omega-3 fatty acid modification of membrane structure and function. I. Dietary manipulation of tumor cell susceptibility to cell- and complement-mediated lysis.

Omega-3 fatty acids, abundant in fish oil, are reported to alter membrane properties when incorporated into membrane phospholipids. We report that dietary omega-3 fatty acids, incorporated into tumor cell membranes, alter tumor recognition and cytolysis by the immune system. Mice were fed diets rich in corn oil, hydrogenated coconut oil, or menhaden (fish) oil. T27A leukemia cells were grown as an ascites tumor in these mice and harvested for biochemical and immunologic assays. The incorporation of the long-chain omega-3 fatty acid docosahexaenoic acid (22:6) into tumor plasma membranes correlated with an increased susceptibility to tumor cytolysis by alloreactive cytotoxic T lymphocytes and decreased expression of a class I major histocompatibility complex epitope, monitored by complement-mediated lysis and radioimmunoassay. Thus the immunologic phenotype of this ascites tumor reflected the source of oil present in the diet during tumor growth.

Omega‐3 fatty acid modification of membrane structure and function. II. Alteration by docosahexaenoic acid of tumor cell sensitivity to immune cytolysis

Docosahexaenoic acid (DHA, 22:6) is a long-chain omega-3 fatty acid abundant in cold water fish; it is the most unsaturated fatty acid found in biologic systems and is reported to alter membrane structure. To explore DHA's effect on membrane function, we have fused tumor cells with synthetic phosphatidylcholine (PC) containing stearic acid in the sn-1 position and DHA in the sn-2 position (18:0, 22:6 PC) and have found the lipid-modified tumor cells to be more sensitive to cytolysis by alloreactive cytotoxic T lymphocytes. Cold target competition experiments suggested that fusion of tumor plasma membranes with 18:0, 22:6 PC produced a qualitative change in expression of surface antigens recognized by cytotoxic T lymphocytes. We monitored the expression of various epitopes on tumor cells by complement-mediated lysis and radioimmunoassay with monoclonal antibodies against H-2 class I antigens. Our results suggest that membrane-bound DHA increases the expression of some epitopes while decreasing the expression of others and that different tumor lines vary in the magnitude of DHA's effect. Our findings are consistent with a model in which DHA-containing phospholipids segregate into membrane domains, in turn altering the expression of membrane proteins.

Effects of severe dehydration on leaf photosynthesis in Quercus petraea (Matt.) Liebl.: photosystem II efficiency, photochemical and nonphotochemical fluorescence quenching and electrolyte leakage.

Leaf disks of oak (Quercus petraea (Matt.) Liebl.) trees were subjected to rapid dehydration in air in the dark. Optimal photochemical efficiency of PS II (F(v)/F(M)), photochemical (q(P)) and nonphotochemical (q(NP)) quenchings of chlorophyll a fluorescence, and relative conductivity (C(r)) of leaf disk diffusate were measured in leaf disks with different water deficits (D). No effect of dehydration was detected before D reached values above 0.30. When D increased from 0.30 to 0.50, q(NP) increased without any change in q(P), which may indicate that thermal deexcitation of PS II increased, allowing reduced photochemical activity and maintenance of a large pool of oxidized primary acceptors (QA), although carbon reduction was impaired. Large changes in electron transport chain activity, leading to decreases in both q(P) and q(NP), appeared only in leaf disks subjected to severe water deficits (D > 0.60) and were correlated with a modification of membrane structure. However, stability of F(v)/F(M) indicated that the functional integrity of PS II was not altered until D reached values above 0.75. We conclude that the photosynthetic apparatus of Q. petraea is rather insensitive to leaf dehydration per se during drought under natural conditions.

Changes in fatty acid and lipid composition in normal and habituated sugar beet calli

Analysis of fatty acids and other classes of lipids in normal and habituated calli of Beta vulgaris shows that their lipid composition is identical during early stages of culture. However, after 14 days this composition turns out to be very different, as a result of membrane structure modifications during the growth of both types of calli. The fatty acid composition of normal callus remains very stable. The loss of phospholipids during culture is compensated for by increased sterol contents. The normal callus, therefore, seems to maintain its membrane integrity throughout growth. On the contrary, the abundance of free fatty acids and malondialdehyde in habituated callus, even at the juvenile stage, together with a decrease of the unsaturation level of fatty acids during culture are indicative of high lipid peroxidation. Peroxidation of polyunsaturated fatty acids and increases in neutral lipids could generate a loss of fluidity in membranes of habituated callus; the more permeable calli would be unable to maintain their homeostasis. This disturbed cellular homeostasis could be correlated with the vitrified aspect of the habituated callus and its relation with the acquisition of hormonal autonomy could be questioned.

Fatty acid composition of phospholipids in mitochondria and microsomes during diethylnitrosamine carcinogenesis in rat liver.

Changes in lipid composition and function of subcellular organelles have been described in transplanted and primary tumours. We examine here the fatty acid composition of individual phospholipids (PL) in hyperplastic nodules and primary hepatoma induced by diethylnitrosamine (DEN), compared to that of normal liver and of transplantable Yoshida AH-130 hepatoma. Phosphatidylcholine and phosphatidylethanolamine fatty acid composition in mitochondria and microsomes from primary hepatoma were markedly different from normal liver; C18:0/C18:1 ratio was lower and the ratio between monosaturated and polyunsaturated fatty acids was higher. Linoleic acid content of mitochondrial cardiolipin, usually very high in normal rat liver, was notably lower in primary hepatoma. Cholesterol/phospholipid ratio in both microsomes and mitochondria from DEN-induced hepatoma was higher than in normal liver. Hyperplastic nodules showed no changes in cholesterol content whereas modifications in fatty acid composition were already observable. These modifications of membrane structure may be related to the functional changes found in nodular cells. Changes in fatty acid composition of membrane phospholipids, occurring in both primary hepatoma and preneoplastic nodules, might be one of the causes for decreased rate of lipid peroxidation peculiar to these tissues.

Novel tools for determining mechanically induced modifications of membrane structure: Electrochemical and fluorescence methods

Two methods for investigating the effects of mechanical strain on the structure of biological membranes are proposed and illustrated in the case of the erythrocytic membrane. Firstly, we show that the diffusion limiting current measured at a rotating disc electrode for a biological suspension allows one to follow the variation of the adhesion of the cells to the wall with the hydrodynamic conditions, provided that the results be interpreted in the framework of partially blocked electrodes. Secondly, we propose an entirely novel method, which consists in following in a Couette flow, the variation of the light intensity of a fluorescent probe (for instance a membrane potential probe) with the shear rate. Original results concerning the erythrocytic membrane are provided both under stationary and non-stationary conditions.

Differential effects of ethanol on two synaptic membrane Ca 2+ transport systems

The Na +Ca 2+ exchange activity in synaptic plasma membranes is inhibited by very low concentrations of ethanol (<25 mM). The high affinity Mg 2+- and ATP-dependent Ca 2+ transport in highly purified synaptic membranes is much less sensitive to inhibition by ethanol, with no statistically significant inhibition observed until an ethanol concentration of nearly 800 mM was used. Manipulations of the lipid environment designed to increase membrane fluidity enhanced the activity of the Na +Ca 2+ exchange system but inhibited the ATP-dependent Ca 2+ pump. The differential responses of the two synaptic plasma membrane Ca 2+ transporting systems to such modifications of membrane structure suggest that these two ion transport processes differ in the extent to which their activity is dependent on the lipid microenvironment in which they reside. Thus, the effects of ethanol on the Na +Ca 2+ antiporter may represent a fairly selective inhibitory process.

Genotoxicity of N-methyl- N-nitrosourea in the presence of amphiphilic membrane-active compounds

With V79 Chinese hamster lung cells the dose-response relationship of sister chromatid exchange (SCE) induction by NMU shows a sigmoidal-shaped curve. In vitro, the time schedule of the short-lived (<60 min) SCE inducer (NMU) and the sister chromatid marker 5-bromodeoxyuridine (BrdU) addition to the cells has a significant effect on the SCE response suggesting a direct chemical and/or indirect biochemical interaction between both compounds. Certain amphiphilic molecules of a definite length, consisting of a hydrophobic chain and a polar end, have been shown to alter functions of various membrane integrated proteins by modifying membrane lipid structures. In vitro, three chemically distinct membrane-active compounds inhibit induction of SCEs following N-methyl- N-nitrosourea (NMU) treatment to different extents in a concentration-dependent manner. These findings with Chinese hamster lung cells confirm the results of in vivo experiments with bone marrow cells from Chinese hamsters. The anti-SCEs inducer effects are interpreted in terms of a decrease of nuclear bioavailability of NMU. Experimental results are not in favor of a competitive inhibition of SCE induction as a consequence of eventual alkylations of the amphiphilic molecules at their polar end. It is hypothesized that cell membrane structure modifications enhance the probability of chemical reactions of the strongly reactive SN 1 alkylating compound NMU with nucleophilic membrane sites. Membrane-active compounds with anti-SCE inducer capacities should be useful probes for the study of possible relationships between SCE induction and the carcinogenic as well as antitumor efficiency of NMU.

Changes in cell surface glycoproteins on variant hamster cell lines

1. 1. Resistance to concanavalin A was accompanied by modifications to the fundamental growth and membrane-associated properties of two concanavalin A-resistant cell lines which were independently selected by either single step or cycling procedures. 2. 2. Using the galactose-oxidase [ 3H]borohydride surface labelling procedure, several modifications in surface glycoproteins were detected on variant cells when compared to parental wild type cells; a prominent difference was the presence of an additional surface component, with an apparent molecular weight of 155,000 on variant cells which was missing from both wild type populations. 3. 3. These observations provide strong support for the view that modified cell surface glycoproteins are directly involved in the complex concanavalin A-resistant phenotype which includes changes to some fundamental biological properties; concanavalin A-resistant variants provide an opportunity to correlate changes in membrane function with modifications in membrane structure.

Membrane structure and function with different lipid-supplemented diets.

In 1966 we made the observation that the (Na+, K+)-ATPase and (Mg2+)-ATPase from rat erythrocyte of animals fed with a commercial diet showed a lesser degree of cooperativity as compared with animals fed with the same diet supplemented either with lard or cod liver oil (Farias, 1967). The work performed by our group in the last ten years confirmed our early hypothesis that changes in the lipid composition of the cell membranes modify the regulatory activity of the membrane-associated enzymes (Farias et al., 1975). Conclusive evidence has been accumulated indicating that allosteric transitions and modification of other kinetic parameters of a given enzyme could be correlated with changes in the “conformation” or “state” of the protein involved. The determination of these kinetic parameters in membrane-associated enzymes may give some clues as to how these enzymes are regulated by the membrane. On the other side, changes in the behavior of enzymes may indicate if some modifications in membrane structure take place under special situations such as changes in lipid composition or action of hormones (Massa et al., 1975; Moreno & Farias, 1976; De Mendoza et al., 1977) or other chemical agents (Domenech et al., 1976).

Hydrogen-ion titration studies on erythrocyte membranes.

1. H+ titration was used to detect the presence of ionizable groups on human erythrocyte plasma membranes. Between pH2.9 and 11.3, two significant peaks of H+ association/dissociation occur in the differential from of the titration curve, one at pH3. 1. And the other at pH10.3. 2. After disruption of membrane structure by exposure to high pH or by the addition of sodium dodecyl sulphate, maxima of H+ association/dissociation were seen at pH3.1,4.3,6.5,10.3 and 10.7. 3. Spectrophotometric assay and selective chemical treatments were used to identify several of the titratable residues. 4. The degree of eleectrostatic interaction between titratable charged groups was investigated by comparing the titration characteristics of the membranes before and after modification of membrane structure.

Structural and Functional Responses of Wheat Mitochondrial Membranes to Growth at Low Temperatures

The responses in membrane lipid composition, structure, and function of four cultivars of wheat (Triticum aestivum L.) to growth at low temperature have been investigated. Marked growth temperature-dependent alterations in the fatty acid composition and unsaturation of the mitochondrial phospholipids correlate with changes in respiratory activity in all the varieties. Parameters such as the respiratory control ratio and the phosphorylative efficiency decrease in cold-adapted seedlings. Three temperature-dependent structural transitions were identified in the mitochondria by the spin-labeling method. The structural transitions occur at lower temperatures in the cold-grown material. The shift in one transition appears to be quantitatively greater in the winter hardy varieties. Cold-induced changes in all of the other measured parameters were indistinguishable in hardy and nonhardy varieties. The results indicate major involvement of the phospholipid matrix in cold acclimation. A link between cold acclimation and winter survival may exist involving the structural and functional modifications in membrane structure which occur during acclimation.

Control of excitation transfer in photosynthesis V. Correlation of membrane structure to regulation of excitation transfer between two pigment systems in isolated spinach chloroplasts

1. Changes in fluorescence yield of chlorophyll a in isolated chloroplasts have been interpreted by means of regulation of excitation transfer between two pigment systems of photosynthesis 5–7. In order to investigate the relationship between the membrane structure of chloroplasts and the regulation of excitation transfer, changes of light scattering and chlorophyll a fluorescence of isolated spinach chloroplasts were measured upon addition of cations, Mg 2+ and Na +. The cations increased the intensities of both light scattering and fluorescence yield. The changes showed similar time courses and concentration dependences. These facts suggest that modification of membrane structure produced by the cations suppresses the excitation transfer between the two pigment systems. 2. In another case of structural change which is induced by light in the presence of N-methylphenazonium methosulfate, there was little correlation between light-scattering and fluorescence changes. 3. Changes in fluorescence yield induced by the addition of Mg 2+ were measured in disintegrated chloroplasts and fractionated particles. The effects of Mg 2+ on fluorescence were observed only in preparations of grana stacks, but not in preparations of stroma lamellae. These findings suggest that the excitation transfer is regulated between the two pigment systems located in the grana thylacoid membranes.