Phospholipid Polar Head Research Articles

Dehydration: A new alcohol theory

A central belief about ethanol is that it acts mainly by partitioning into the lipid bilayer of membranes. Newer ideas focus on the neuronal synapse and suggest that ethanol can allosterically change protein conformation, as is suggested by studies on GABA-receptor-mediated chloride uptake and on (Na +-K +)-ATPase. Several studies from my laboratory suggest that ethanol enhances enzymatic cleavage of sialic acid (SA) from gangliosides, and perhaps also glycoproteins, but does so without stimulating enzyme activity, suggesting conformational changes that affect accessibility. I propose a new model for the cell membrane in the synaptic region, which features gangliosides surrounding membrane proteins, with an interspersed film of water creating hydrogen bonds that anchor SA moieties to membrane protein. I believe that we should consider the possibility that an important action of ethanol, and polar anesthetics, is due to hydrophilic, not hydrophobic, properties and the ability to dehydrate the cell-surface microdomain. Our laboratory has recently advanced the theory that ethanol dehydrates a “solvent regulatory site” of membrane (Na +-K +)-ATPase. This principle might be extended to other enzymes and receptor proteins, as well as to the accessibility of sialoglycoconjugates to sialidase (neuraminidase). Hydrogen bonding between SA and polar regions of receptor protein, and the conformation on both imposed by it, would surely be changed by minor degrees of dehydration and substitution of alcohol molecules for water. Ethanol, unlike water, can only hydrogen bond “at one end”. Displacement of water by ethanol would not only “free” the SA groups and make them more vulnerable to enzymatic cleavage but also could simultaneously change the conformation of receptor protein. Similarly, ethanol may displace water that links the polar heads of phospholipids to polar portions of receptor proteins. Ethanol may have an even more important and direct effect of substituting for hydrogen-bonded water within protein itself.

Brain asymmetry in phospholipid polar head group metabolism: Parallel in vivo and in vitro studies

Phospholipid content and 32P-incorporation have been studied in individual rat cerebral hemispheres. The total phospholipid content was 44.9 +/- 0.9 and 47.9 +/- 1.3 mumol lipid P/100 mg protein for the right and left hemispheres respectively. Individually, only sphingomyelin was significantly (about 30%) higher in the left hemisphere. Metabolic experiments have been conducted in vivo using i.p. injection of 32P and following its incorporation into total and individual phospholipids in each cerebral hemisphere. Higher incorporations were attained by phosphatidate and phosphatidylinositol-4,5-bisphosphate (PIP2) in the left cerebral hemisphere than in the right. In an attempt to determine whether phospholipid metabolism is also lateralized in specific subcellular compartments related with the neurotransmission process, we have studied in vitro the [32P] incorporation into phosphoglycerides of synaptosomal fractions obtained from each cerebral cortex. The precursor was taken up differently by the two cerebral cortex preparations, resulting in different profiles of distribution among lipids. In addition, the kinetics of lipid labeling showed higher rates of 32P-incorporation in fractions derived from the left cerebral cortex, mainly in PIP and PIP2. These results are interpreted to indicate that several enzymes involved in lipid metabolism are modulated to a different extent in the two hemispheres.

Interactions among phospholipids of guinea pig rough microsomes, effect of fat deficiency.

The fatty acid composition and the steady-state fluorescence anisotropy (rs) of 1,6-diphenyl-1,3,5-hexatriene (DPH) were determined for each of the five major phospholipid (PL) classes present in the liver rough microsomes (RM) of guinea pigs fed with control and fat-deprived diets. In order to obtain information about PL-PL interactions and their contribution to the overall membrane fluidity the experimental rs of phospholipid mixtures were compared to the molar weighed average values of the individual phospholipid rs values. The PL ratios in the mixtures were the same to those found in the RM membranes. Binary mixtures of phosphatidylcholine (PC) with phosphatidylethanolamine (PE), phosphatidylserine (PS), phosphatidylinositol (PI) and sphingomyelin (SM) show higher values of rs than those estimated from the individual component parameters indicating a 'rigidizing' interaction. The rigidizing effect of PE was also observed when this phospholipid was sonicated with mixtures of PC with PS and PI. However, no rigidizing effect of PE was observed in the PC bilayers when SM was included in the composition suggesting that PE-SM interactions prevent 'rigidizing' effects of PE. Besides, in spite that PC-PI and PC-PS mixtures have 'rigidizing' interactions, the incorporation of PI and PS to PC-PS and PC-PI mixtures, respectively, have a 'fluidizing' effect. In consequence, phospholipid polar head groups interact in RM membranes modifying the molecular packing and/or the rotational diffusion of acyl chains. The complexity and variety of constituent phospholipids could prevent major changes in the fluidity. The comparison of results obtained with PL mixtures of control and fat-deficient animals indicate that a change in the number of double bonds does not evoke a significant difference between either the 'rigidizing' of 'fluidizing' effects. However, there is a general tendency indicating that phospholipids with higher double bond index evoke lower 'rigidizing' and 'fluidizing' interactions. Moreover, PL of animals fed a fat-deficient diet have less fluidity than those of control animals.

Effect of membrane phospholipids on activation of the alternative complement pathway.

Although some of the membrane glycoproteins that serve as activators or regulators of C activation have been identified, the influence of membrane lipids has not been studied extensively. A model of alternative C pathway activation was established using liposomes composed of cholesterol and synthetic phospholipids. Liposomes containing phosphatidylcholine (PC) as the sole phospholipid did not activate C as measured by C3 binding after incubation in normal human serum containing 2.5 mM MgCl2 and 10 mM EGTA. When phosphatidylethanolamine (PE) was included as 20% or more of the phospholipid, C3 binding was observed. C3 binding to liposomes was inhibited by salicylhydroxamic acid indicating binding through the C3 thioester bond. The phospholipid composition did not influence C3 binding to liposomes in an unregulated system of C3, B, D, and P indicating equivalent C3b binding sites on activating and nonactivating liposomes. When the regulatory proteins H and I were added to the other components, liposomes containing PE bound three times more C3 than PC liposomes suggesting that the phospholipid affects C3 regulation. This was tested directly in a radiolabeled H binding assay. In the presence of equal amounts of C3b, PC liposomes showed a greater number of high affinity H binding sites than PE liposomes. Using different PE derivatives, C activation could be directly related to the phospholipid polar head group. Liposomes containing PE, trinitrophenyl-PE or monomethyl-PE did activate the alternative C pathway, whereas those containing dimethyl-PE, PC, or phosphatidylserine did not. These studies provide evidence that primary and secondary amino groups on lipid membranes can decrease the interaction between H and C3b and provide sites for alternative pathway activation.

Phospholipid polymers & new haemocompatible materials

AbstractWhen blood comes into contact with an artificial surface, a number of events occur which include protein adsorption, platelet activation and the activation of the intrinsic pathway of blood coagulation. With the increased application of blood containing artificial devices, there is a great demand to develop new biomaterials which retard thrombus formation. Our new approach to solving this problem is to mimic the non‐thrombogenic surface of natural biological membranes at least in a simple form. We have developed a polymerisable phospholipid and polyesters based on the major phospholipid polar head group present on the erythrocyte outer membrane surface. The coagulation of blood exposed to these polymers was examined by the technique of Material Thrombelastography, a relatively simple test for the in vitro screening of polymer thrombogenicity. We present results which indicate that the polymerised phospholipid and polyesters show reduced thrombogenicity, and may therefore have potential for future biomaterials.

Lateral proton conduction along a lipid-water interface layer: a molecular mechanism for the role of hydration water molecules

Using a fluorescence method applied to phospholipids spread at the air-water interface, we showed the occurence of a localized proton pathway along the phospholipid polar heads. From the systematic investigation of this phenomenon, we conclude that the protons diffuse through a hydrogen bond network which is a matrix of hydrated polar head groups.

Characterization of shell gland lipids from chicken ( gallus domesticus) producing strong or weak egg shells

1. 1. The reduction in egg shell strength in hens at the end of the first reproductive cycle was not associated with abnormal uterine or body fat accumulation. 2. 2. The phosphatidylcholine concentration was greater in the shell glands of hens producing strong egg shells compared to that of hens producing weak egg shells. 3. 3. Differences were not detected in the total lipid or cholesterol concentrations or the fatty acid profiles of phosphatidylcholine or phosphatidylethanolamine fractions. 4. 4. Following an induced molt, the fatty acid profile of the total lipid fraction of the shell gland was altered with a decrease in stearic acid and an increase in oleic acid. 5. 5. The results demonstrate that alterations in egg shell strength are mainly associated with changes in shell gland phospholipid polar head group composition and not with changes in phospholipid fatty acid profile or cholesterol concentration.

The role of car☐yl groups of Na +/K +-ATPase in the interaction with divalent cations

Mg 2+-induced subconformational changes of the E 1 conformation of partly purified pig kidney Na +/K +-ATPase were studied by fluorescence techniques. In the enzyme with car☐yl groups modified by carbodiimide in the presence of an exogenous nucleophile the efficiency to pass through conformational substates was substantially lower than in the unmodified enzyme. Magnesium could form bridges between car☐yl groups near the membrane/water interface and negatively charged phospholipid polar heads.

Membrane phospholipid polar heads influence the coupling of M2 muscarinic receptors to G proteins

Treating membranes from rat heart with phospholipase C (phosphatidylcholine choline phosphohydrolase) from Clostridium perfringens increased the affinity of muscarinic acetylcholine receptors (M2) for the agonists carbachol and oxotremorine. The affinity for antagonists was not affected. Phospholipase C activity, i.e., the cleavage of polar heads of membrane phospholipids, led to the disappearance of the guanine nucleotide-dependent rightward shift of the isotherm for agonist binding. The treatment of tracheal smooth muscle with phospholipase C led to a decrease in the maximum contractile effect of muscarinic (M2) stimulation with no modification of the agonist EC50, i.e., to the uncoupling of the stimulation-contraction process. These results demonstrate that when phospholipid polar heads are hydrolysed by phospholipase C, M2 receptors are uncoupled from G proteins, which enhances their affinity for agonists but prevents information transfer.

Development of the Gastrointestinal Mucosal Barrier: Changes in Phospholipid Head Groups and Fatty Add Composition of Intestinal Microvillus Membranes from Newborn and Adult Rats

Phospholipids in microvillus membrane (MVM) prepared from the proximal small intestine of newborn (less than 24 h of age) and adult rats were characterized to determine if differences in composition existed. Our hypothesis is that developmental differences in composition of the MVM may account for mucosal barrier differences to microorganisms and antigens during the perinatal period. Lipid analysis showed that both cholesterol and total phospholipids, per mg of membrane protein, were much higher in the newborn MVM than the adult, but the molar ratio of these two lipid classes remained unchanged. Increased phosphatidylcholine and decreased phosphatidylinositol levels were found in the newborn MVM compared to the adult. In addition, the major difference in fatty acid composition of total lipid extracts and three major membrane phospholipids, phosphatidylcholine, phosphatidylethanolamine, and phosphatidylinositol appeared to be an increased ratio of palmitate (16:0) to stearate (18:0) and of oleate (18:1) to linoleate (18:2) in the newborn. These data suggest that changes in the proportion of phospholipid polar head groups and fatty acyl groups as well as changes in the amount of total phospholipids and cholesterol exist in the newborn MVM. These observations are consistent with differences in MVM fluidity previously noted and may help explain the increased uptake of antigens and other mucosal barrier defects in the newborn intestine.

Phospholipid profile and production of prostanoids by murine colonic epithelium: effect of dietary fat.

Dietary fat and abnormal production of various prostanoids have been linked to various disease states of the large bowel, including cancer of the colon. Studies were conducted to determine the effect of dietary fat (beef tallow or corn oil) on the lipid composition and prostanoid production of the murine colon. Female C57BL/6J mice were fed high-fat (HF) diets (47% of calories as fat) or low-fat (LF) diets (10% of calories as fat). After four wk of dietary treatment, the mucosa was scraped, and lipids were extracted from the mucosal and muscle layers. The fat content of the diets did not significantly alter the amount of phospholipid (PL) or neutral lipid in the colonic tissue. However, the HF affected the PL profile of the colonic mucosa. For example, the ratio of phosphatidylcholine (PC) to phosphatidylethanolamine (PE) was significantly higher for both the HF groups compared with that of the two LF groups (0.76 +/- 0.15 and 0.80 +/- 0.13 vs 0.31 +/- 0.20 and 0.34 +/- 0.18). Production of 13,14-dihydro-15-keto-PGE2 (measured as bicyclic PGE2) and TXB2 (a stable metabolite of TXA2) and PGF1 alpha (a stable metabolite of PGI2) was unaffected by the dietary treatments. The muscle had a different PL profile (PC:PE is 2.6 +/- 0.1) than the mucosa and contributed a larger proportion of the prostanoids formed. This study demonstrates that the phospholipid polar head group composition of normal colonic mucosa is altered by dietary fat, but the ability of the mucosa to synthesize metabolites of PGE2, TXA2 and PGI2 is not affected.

Polyamine inhibition of lipoperoxidation. The influence of polyamines on iron oxidation in the presence of compounds mimicking phospholipid polar heads.

Polyamines appear to inhibit peroxidation of vesicles containing acidic phospholipids. A correlation exists between polyamine binding to phospholipid vesicles and its protective effect. However, phosphatidylinositol-containing vesicles which bind spermine are not protected by the polyamine [Tadolini, Cabrini, Landi, Varani & Pasquali (1985) Biogenic Amines 3, 97-106]. In the present paper I tested the hypothesis that polyamines, in particular spermine, by forming a ternary complex with iron and the phospholipid polar head may change the susceptibility of Fe2+ to autoxidation and thus its ability to generate free oxygen radicals. Different compounds mimicking phospholipid polar heads were studied, namely AMP, mimicking phosphatidic acid, CDP-choline, mimicking phosphatidylcholine, and glycerophosphoinositol, mimicking phosphatidylinositol. The results support the proposed hypothesis. In the presence of CDP-choline or of glycerophosphoinositol, spermine poorly affects Fe2+ autoxidation, whereas a considerable inhibition is observed in the presence of AMP. The ability of other phosphorus-containing compounds (ATP, ADP, cyclic AMP, sodium phosphate) to affect Fe2+ autoxidation in the presence of polyamines was also evaluated to understand the molecular mechanism of this phenomenon. It is proposed that polyamines may be part of the passive cellular defence mechanism against the oxidative damage caused by Fe2+.

Effect of choline, ethanolamine and serine supplementation on the membrane properties of Microsporum gypseum.

Phospholipid bases, choline and ethanolamine, when supplemented in the growth medium of Microsporum gypseum resulted in an increase in the corresponding phospholipid and total phospholipid content. However, when serine was supplemented, marginal changes were observed. The fatty acid profile of phospholipids remained almost unchanged. The uptake of lysine, leucine and aspartic acid in the spheroplasts of choline- and ethanolamine-grown cells was higher as compared to the control. 1-Anilinonaphathalene-8-sulfonate (ANS) binding to the spheroplast membrane, as calculated from Scatchard plots, demonstrated an increase in the number of binding sites in choline- and ethanolamine-grown cells, while a decrease was observed in the serine-supplemented cells. The results are discussed in terms of the effect of phospholipid polar head group composition on the membrane structure and function of this fungus.

Role of the physical state of the hepatic microsomal membrane in the formation of bilirubin diglucuronide

In vitro formation of bilirubin diglucuronide by rat hepatic microsomes proceeds efficiently only under specific conditions; i.e., a low level of bilirubin, temperature greater than 23-24 degrees C and treatment of the microsomes with a very specific level of perturbant (J. Biol. Chem., 258: 15028-15036). In the present study, the effect of temperature and detergents on the anisotropy of fluorescent probes in the rat hepatic microsomal membrane was used to determine the role of the physical state of the membrane in controlling the formation of bilirubin diglucuronide. A lipid phase separation that occurred at 23 +/- 2 degrees C was identified in these membranes indicating that bilirubin diglucuronide is formed efficiently only above the lipid phase separation when the phospholipids are in the liquid crystalline state. In addition, use of fluorescent probes for the surface and core of the membrane indicates that alterations in the physical state of the hydrophobic core rather than the phospholipid polar head group region of the membrane controls the in vitro formation of bilirubin diglucuronide.

Role of phospholipid fatty acids on the kinetics of high and low affinity sites of cytochrome c oxidase.

The nature of the interactions between cytochrome c oxidase and the phospholipids in mitochondrial membranes has been investigated by varying the nature of the fatty acyl components of Saccharomyces cerevisiae. A double fatty acid yeast mutant, FAI-4C, grown in combinations of unsaturated (oleic, linoleic, linolenic, and eicosenoic) and saturated (lauric and palmitic) fatty acids, was employed to modify mitochondrial membranes. The supplemented fatty acids constituted a unique combination of different acyl chain lengths with varying degrees of unsaturation which were subsequently incorporated into mitochondrial phospholipids. Phosphatidylethanolamine and cardiolipin, the predominant phospholipids of the inner mitochondrial membrane, were characterized by their high levels of supplemented unsaturated fatty acids. Increasing the chain length or the degree of unsaturation of mitochondrial membrane phospholipids had no effect on altering the nature of the phospholipid polar head group but did result in a profound change on the specific activity of cytochrome c oxidase. When studied under conditions of different ionic strengths and pHs the enzyme's activity, as documented by Eadie-Hofstee plots, showed biphasic kinetics. The kinetic parameters for the low affinity reaction were greatly influenced by the changes in the membrane fatty acids and only marginal effects were noted at the high affinity reaction site. The discontinuities in the steady-state fluorescence anisotropy of 1,6-diphenyl-1,3,5-hexatriene, monitored at increasing temperatures, suggested that changes in membrane fluidity were conditioned by alterations in mitochondrial membrane fatty acid constituents. These results indicate that the lipid changes affecting the low affinity binding site of cytochrome c oxidase may be the result of lipid-protein interactions which lead to enzyme conformational changes or may be due to gross changes in membrane fluidity. It may, therefore, follow that this enzyme site may be embedded in or be juxtaposed to the outer surface of the inner mitochondrial membrane bilayer in contrast to the high affinity site which has been shown to be significantly above the membrane plane.

Effect of manipulation of phospholipid polar head group on low density lipoprotein metabolism in human cultured fibroblasts

Modification of phospholipid polar head group was achieved by growing human cultured fibroblasts in medium devoid of serum and supplemented with N-methyl ethanolamine or N, N-dimethylethanolamine during 48 h. The corresponding phospholipids accounted for approximately 45 % of total phospholipids. Whereas low density lipoprotein (LDL) binding was unaffected, LDL internalization and degradation appeared to be markedly reduced in the presence of N-methylethanolamine. N,N-dimethylethanolamine had no effect on the three studied parameters. These results emphasize the importance of phospholipid polar head group in LDL processing by receptor-mediated endocytosis.

Lipid-protein interactions in biomembranes studied through the phospholipid specificity of d-β-hydroxybutyrate dehydrogenase

Since the biological membranes are fundamental units in the living cells, the studies of lipid-protein interactions are crucial for the understanding of their structure, functions and properties. Beside hydrophobic interactions between fatty acids chain of phospholipids and intrinsic membrane proteins, the interactions between charged groups of the protein with the polar heads of phospholipids generally confer the specificity which may be absolute or preferential. This paper reports essential results obtained these last few years with D-beta-hydroxybutyrate dehydrogenase (BDH) from inner mitochondrial membrane, one of the most interesting and best documented examples of a lipid-requiring enzyme. This is a review of the molecular basis--knowledge and strategy of study--of the lipid specificity for membrane protein functions.

Regulation of transbilayer distribution of a fluorescent sterol in tumor cell plasma membranes

The lipid composition and transbilayer distribution of plasma membrane isolated from primary tumor (L-929, LM, A-9 and C 3H) and nine metastatic cell lines cultured under identical conditions was examined. Cultured primary tumor and metastatic cells differed two-fold in sterol/phospholipid molar ratios. There was a direct correlation between plasma membrane anionic phospholipid (phosphatidylinositol and phosphatidylserine) content and plasma membrane sterol/phospholipid ratio. This finding may bear on the possible link between oncogenes and inositol lipids. The fluorescent sterol, dehydroergosterol, was incorporated into primary tumor and metastatic cell lines. Selective quenching of outer monolayer fluorescence by covalently linked trinitrophenyl groups demonstrated an asymmetric transbilayer distribution of sterol in the plasma membranes. The inner monolayer of the plasma membranes from both cultured primary and metastatic tumor cells was enriched in sterol as compared with the outer monolayer Consistent with this, the inner monolayer was distinctly more rigid as determined by the limiting anisotropy of 1,6-diphenyl-l,3,5-hexatriene. Dehydroergosterol fluorescence was temperature dependent and sensitive to lateral phase separations in phosphatidylcholine vesicles and in LM cell plasma membranes. Dehydroergosterol detected phase separations near 24°C in the outer monolayer and at 21°C and 37°C in the inner monolayer of LM plasma membranes. Yet, no change in transbilayer sterol distribution was detected in ascending or descending temperature scans between 4 and 45°C. Alterations in plasma membrane phospholipid polar head group composition by choline analogues ( N,N- dimethylethanolamine, N- methylethanolamine , and ethanolamine) also did not perturb transbilayer sterol asymmetry. Treatment with phenobarbital or prilocaine, drugs that selectively fluidize the outer and inner monolayer of LM plasma membranes, respectively, did not change dehydroergosterol transbilayer distribution.

Interfacial properties of model membranes and plasma lipoproteins containing ether lipids.

The interfacial properties of synthetic ester and ether phosphatidylcholines (PCs) were investigated by using the polarity-sensitive fluorescent probes 6-propionyl-2-(dimethylamino)naphthalene (Prodan) and pyrene. The physical state of the phospholipid matrix was determined by fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene (DPH). Single-bilayer phospholipid vesicles formed by sonication and model high-density lipoproteins were studied. On the basis of a number of spectroscopic and thermodynamic criteria, the interfacial regions of PCs and their ether analogues are similar. The fluorescence properties of Prodan in model lipoproteins or single-bilayer vesicles were independent of the phospholipid fatty acyl chain length and polar head group, as well as the substitution of ether linkage for ester bonds in the phospholipid. The spectral shifts correlated mainly with the physical state of the phospholipid. The emission spectrum of Prodan appeared at shorter wavelengths upon transfer from water to liquid-crystalline phospholipid and blue shifted further when the lipid was cooled to its gel phase. The effect of cholesterol in model high-density lipoproteins on the emission spectrum of Prodan was dose dependent and, at 18 mol % cholesterol, the spectrum was similar to that observed in a pure gel-phase lipid and was independent of temperature. The quantum yield of Prodan fluorescence in an ether-PC matrix was similar to that observed in water, whereas in an ester-PC matrix it was enhanced by a factor of about 5. Phospholipid-water partition coefficients of Prodan were independent of the physical state of 1,2-dimyristoyl-sn-glycero-3-phosphocholine or 1,2-tetradecyl-sn-glycero-3-phosphocholine.(ABSTRACT TRUNCATED AT 250 WORDS)

Differences in amino acid transport and phospholipid contents during the cell cycle of Candida albicans.

Drugs like L-ethionine, 1,10-phenanthroline and 3-(2-thienyl)-DL-alanine which arrest Saccharomyces cerevisiae cells in the G1 phase, were unable to arrest Candida albicans cells. However, C. albicans could be arrested in G1 after a prolonged stationary phase. As compared to normal cells, there was a selective reduction in the level of accumulation of valine and glutamate in G1-arrested cells, while the phospholipid polar head group ratio was not significantly altered. When G1-arrested C. albicans cells were again allowed to grow, the level of different phospholipids started increasing at about the time of bud emergence (2.5 h) whereas reduced levels of accumulated valine and glutamate recovered within 1 h. The recovery of phospholipids and amino acid transport are two distinct events during the progression of C. albicans cells from G1 to S phase.