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PubMed1996

The effects of dietary phospholipids enriched with phosphatidylethanolamine on bile and red cell membrane lipids in humans.

The role of phospholipids in biliary cholesterol solubilization and crystallization has only recently begun to be appreciated. Phospholipid vesicles are believed to be the metastable carrier from which cholesterol nucleates. Cholesterol crystallization is influenced by the phospholipid species in bile. Feeding rats and hamsters with diets enriched in phospholipids or their precursors, especially ethanolamine, resulted in reduced cholesterol saturation of bile. Although whole phospholipids are normal dietary constituents, the effects and safety of phospholipid components have not been tested in humans. In the present study, we have evaluated the effects of a dietary phospholipid mixture, enriched with phosphatidylethanolamine, on human bile and red blood cell membrane lipid composition. Five ambulatory volunteers having a chronic indwelling T-tube, with an intact enterohepatic circulation, were investigated. Thirty-six grams of phospholipids (54% phosphatidylethanolamine, 54% linoleyl acyl chains) were added to their daily diet for fourteen days. Biliary nucleation time, cholesterol carriers, as well as plasma, red blood cell membrane, and bile lipid compositions, were monitored. Following phospholipid supplementation, the proportion of linoleyl chains (18:2) in biliary phospholipids increased significantly from 31.1 +/- 1.2 to 37.7 +/- 5.3%, while that of oleyl chains (18:1) decreased from 11.4 +/- 1.6 to 9.6 +/- 1.1%. These changes were accompanied by an increase of linoleate and its metabolite, arachidonate, in red cell membranes. Phospholipid feeding did not cause any side effects, and no significant changes in biliary nucleation time, cholesterol, phospholipid, or bile salt concentrations, or in the distribution of cholesterol within micelles or vesicles. We conclude that phospholipid feeding is safe, and can be effective as a vehicle for lecithin fatty acyl chain modulation of bile and lipid membranes. These findings may provide a basis for a controlled modulation of biliary phospholipids to increase cholesterol solubility in bile.

Adult↗
PubMed1991

Extensive segregation of acidic phospholipids in membranes induced by protein kinase C and related proteins.

Protein kinase C and two other proteins with molecular masses of 64 and 32 kDa, purified from bovine brain, constitute a type of protein that binds a large number of calcium ions in a phospholipid-dependent manner. This study suggested that these proteins also induced extensive clustering of acidic phospholipids in the membranes. Clustering of acidic phospholipids was detected by the self-quenching of a fluorescence probe that was attached to acidic phospholipids (phosphatidic acid or phosphatidylglycerol). Addition of these proteins to phospholipid vesicles containing 15% fluorescently labeled phosphatidic acid dispersed in neutral phosphatidylcholine resulted in extensive, rapid, and calcium-dependent quenching of the fluorescence signal. Fluorescence-quenching requirements coincided with protein-membrane binding characteristics. As expected, the addition of these proteins to phospholipid vesicles containing fluorescent phospholipids dispersed with large excess of acidic phospholipids produced only small fluorescence changes. In addition, association of these proteins with vesicles composed of 100% fluorescent phospholipids resulted in no fluorescence quenching. Protein binding to vesicles containing 5-50% fluorescent phospholipid showed different levels of fluorescence quenching that closely resemble the behavior expected for extensive segregation of the acidic phospholipids in the outer layer of the vesicles. Thus, the fluorescence quenching appeared to result from self-quenching of the fluorophores that become clustered upon protein-membrane binding. These results were consistent with protein-membrane binding that was maintained by calcium bridges between the proteins and acidic phospholipids in the membrane. Since each protein bound eight or more calcium ions in the presence of phospholipid, they may each induce clustering of a related number of acidic phospholipids.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1998

Membrane insertion of cytochrome P450 1A2 promoted by anionic phospholipids.

The role of phospholipids in the membrane binding and subsequent insertion of the microsomal protein rabbit cytochrome P450 (P450) 1A2 into phospholipid bilayers was investigated. The insertion of P450 1A2 into phospholipid bilayers was measured by the quenching of Trp fluorescence of P450 1A2 by pyrene and brominated and doxyl-labeled phospholipids. When the phosphatidylcholine (PC) matrix was replaced with acidic phospholipids [phosphatidic acid (PA), phosphatidylserine, and phosphatidylinositol] and phosphatidylethanolamine (PE), the extent of insertion into lipid bilayers was strictly dependent on the type of acidic phospholipids. All anionic phospholipids caused the penetration of P450 1A2 into lipid bilayers, but PA was the most efficient in facilitating deep penetration of P450 1A2 into bilayers. On the other hand, binding of P450 1A2 to liposomes was increased by acidic phospholipids to the same degree regardless of the type of acidic phospholipids. PE was found to act as an inert matrix phospholipid, similar to PC, as it exerted very little effect on the insertion of P450 1A2 into lipid bilayers and the binding of P450 1A2 to membranes. It was also found that the phospholipid-dependent membrane insertion of P450 1A2 was associated with altered enzyme activity, increased alpha-helix content, and increased Trp fluorescence of P450 1A2. These results indicate that negative charges on the acidic phospholipids are important for the initial binding of P450 1A2 to membranes, but the penetration of P450 1A2 into lipid bilayers is regulated by the type of acidic phospholipids, and that phospholipid-dependent insertion of P450 1A2 is accompanied by a structural change of P450 1A2.

Animals↗
PubMed2002

The fatty acid profile of buccal cheek cell phospholipids is a noninvasive marker of long-chain polyunsaturated Fatty Acid status in piglets.

The fatty acid pattern of cheek cell phospholipids has been proposed as a noninvasive marker of long-chain polyunsaturated fatty acid (PUFA) status. However, the cheek cell phospholipid fatty acid pattern has been compared only with that of plasma and erythrocytes. The objective of this study was to assess the extent to which the fatty acid profile of cheek cell phospholipids reflects that of tissue phospholipids. Piglets (n = 31; 6 d old) were fed five formula diets differing in total fat and fatty acid composition. After 14 d of consuming the assigned diets, cheek cell plasma, erythrocyte, liver, muscle, adipose tissue, retina and brain samples were collected for determination of the phospholipid fatty acid patterns. There were significant correlations between the cheek cell phospholipid content of most PUFA and the content of these fatty acids in tissue phospholipids (r = 0.509-0.951, P < 0.01). The cheek cell phospholipid content of most of the PUFA, except 20:4(n-6), reflected that of other tissue phospholipids as well as, or nearly as well as the contents of plasma and/or erythrocyte phospholipids. The correlations between the 22:6(n-3) contents of cheek cell, plasma, or erythrocyte phospholipids and those of brain and retina phospholipids were relatively poor (r = 0.596-0.737, P < 0.001). We conclude that the fatty acid pattern of cheek cell phospholipid can be used as a noninvasive marker of PUFA status, but it is not a better index than the pattern of plasma or erythrocyte phospholipids, particularly for assessing the fatty acid pattern of organs with slow fatty acid incorporation and/or turnover rates.

Adipose Tissue↗
PubMed1992

The effect of phospholipids, calcium ions and protein S on rate constants of human factor Va inactivation by activated human protein C.

Rate constants for human factor Va inactivation by activated human protein C (APC) were determined in the absence and presence of Ca2+ ions, protein S and varying concentrations of phospholipid vesicles of different lipid composition. APC-catalyzed factor Va inactivation in free solution (in the presence of 2 mM Ca2+) was studied under first-order reaction conditions with respect to both APC and factor Va and was characterized by an apparent second-order rate constant of 6.1 x 10(5) M-1 s-1. Stimulation of APC-catalyzed factor Va inactivation by phospholipids was dependent on the concentration and composition of the phospholipid vesicles. Optimal acceleration (230-fold) of factor Va inactivation was observed with 10 microM phospholipid vesicles composed of 20 mol% dioleoylglycerophosphoserine (Ole2GroPSer) and 80 mol% dioleoylglycerophosphocholine (Ole2GroPCho). At higher vesicle concentrations and at higher molar fractions of Ole2GroPSer some inhibition of APC-catalyzed factor Va inactivation was observed. Membranes that contained anionic phospholipids other than phosphatidylserine also promoted factor Va inactivation. The ability of different anionic lipids to enhance factor Va inactivation increased in the order phosphatidylethanolamine less than oleic acid less than phosphatidic acid less than phosphatidylglycerol less than phosphatidylmethanol less than phosphatidylserine. APC-catalyzed factor Va inactivation in the presence of phospholipid vesicles could be saturated with respect to factor Va and the reaction obeyed Michaelis-Menten kinetics. Both the Km for factor Va and the Vmax of factor Va inactivation were a function of the phospholipid concentration. The Km increased from 1 nM at 2.5 microM phospholipid (Ole2GroPSer/Ole2GroPCho 20:80, mol/mol) to 65 nM at 250 microM phospholipid. The Vmax increased from 20 mol factor Va inactivated.min-1.mol APC-1 at 2.5 microM phospholipid to 62 mol factor Va inactivated.min-1.mol APC-1 at 10 microM phospholipid and remained constant at higher phospholipid concentrations. Protein S appeared to be a rather poor stimulator of APC-catalyzed factor Va inactivation. Protein-S-dependent rate enhancements were only observed in reaction mixtures that contained negatively charged phospholipid vesicles. Independent of the concentration and the lipid composition of the vesicles, protein S caused a twofold stimulation of APC-catalyzed factor Va inactivation. This suggests that, in the human system, enhancement of APC binding to phospholipid vesicles by protein S is of minor importance. Considering that protein S is a physiologically essential antithrombotic agent, it is likely that other factors or phenomena contribute to the in vivo antithrombotic action of protein S.

Calcium↗
PubMed2002

Plasma and muscle phospholipids are involved in the metabolic response to long-distance migration in a shorebird.

We studied: (1) concentrations and fatty acid compositions of plasma non-esterified fatty acids, neutral lipids, and phospholipids, and (2) fatty acid composition of flight muscle phospholipids in wintering, premigratory, and spring and fall migrating western sandpipers ( Calidris mauri). Plasma neutral lipid and phospholipid levels were elevated in migrants, reflecting high rates of fat deposition. An important role of phospholipids in fattening is suggested by the fact that the amount of fatty acids in plasma phospholipids was similar to, or in spring as much as twice, that of neutral lipids. Changes in the ratio of plasma neutral lipids to phospholipids may indicate seasonal changes in triacylglycerol stores of invertebrate prey. Monounsaturation and total unsaturation of plasma neutral lipids and phospholipids increased during migration. Muscle phospholipids were more monounsaturated in spring and fall, but total unsaturation was reduced in fall. Arachidonic acid [20:4(n-6)] was especially abundant in muscle phospholipids in winter (29%) and declined during migration (19-22%), contributing to a decline in the ratio of n-6 to n-3 fatty acids. The abundance of plasma phospholipids and variability of neutral lipid to phospholipid ratio indicates that measurement of plasma phospholipids will improve methods for assessment of fattening rates of birds. The functional significance of changes in muscle phospholipids is unclear, but may relate to depletion of essential n-6 fatty acids during exercise.

Adaptation, Physiological↗
PubMed1986

Phospholipid transfer activity in synchronous populations of Rhodobacter sphaeroides.

Studies of intracytoplasmic membrane biogenesis employing steady-state synchronously dividing populations of Rhodobacter sphaeroides reveal that the translocation of pre-existing phospholipid into the growing membrane is concurrent with cell division (Cain, B.D., Deal, C.D., Fraley, R.T. and Kaplan, S. (1981) J. Bacteriol. 145, 1154-1166), yet the mechanism of phospholipid movement is unknown. However, the discovery of phospholipid transfer protein activity in R. sphaeroides (Cohen, L.K., Lueking, D.R. and Kaplan, S. (1979) J. Biol. Chem. 254, 721-728) provides one possible mechanism for phospholipid movement. Therefore the level of phospholipid transfer activity in cell lysates of synchronized cultures was measured and was shown to increase stepwise coinciding precisely with the increase in cell number of the culture. Although the amount of transfer activity per cell remained constant throughout the cell cycle, the specific activity of the phospholipid transfer activity showed a cyclical oscillation with its highest value coincident with the completion of cell division. Purified intracytoplasmic membrane can be used as phospholipid acceptor in the developed phospholipid transfer assay by employing either cytoplasmic membrane or liposomes as the phospholipid donor. Intracytoplasmic membrane isolated from the cells prior to division (high protein to phospholipid ratio) served as a better phospholipid acceptor in the phospholipid transfer system when compared with membranes derived from the cells following cell division (low protein to phospholipid ratio).

Chromatophores↗
PubMed1992

Resistance to ethanol disordering of membranes from ethanol-fed rats is conferred by all phospholipid classes.

Phospholipids extracted from liver microsomes and mitochondria of ethanol-fed rats retained the resistance to membrane disordered by ethanol which is observed in the intact isolated membranes. The lipid extracts were separated into the major phospholipid classes (phosphatidylcholine, phosphatidylethanolamine and phosphatidylinositol from microsomes and phosphatidylcholine, phosphatidylethanolamine and cardiolipin from mitochondria) by preparative TLC. The extent of membrane disordering by ethanol of phospholipid vesicles composed of a mixture of phospholipids from ethanol-fed rats and controls was determined from the reduction of the order parameter of the spin-probe 12-doxyl-stearate. In contrast to previous reports, we found that all phospholipid classes from ethanol-fed rats confer resistance to disordering by ethanol. To a first approximation the extent of resistance was proportional to the fraction of lipids from ethanol-fed rats, regardless of the phospholipid head-group. Subtle differences between phospholipid classes may exist but were too small to measure accurately. Except for phosphatidylethanol, incorporation of anionic phospholipids did not have a significant effect on the sensitivity of phospholipid vesicles to the disordering effect of ethanol. Vesicles prepared from mixtures of various dioleoyl phospholipids and natural phospholipids did not indicate a clear effect of fatty acid saturation on the sensitivity to disordering by ethanol. Although the precise molecular changes that occur in phospholipids from ethanol-fed rats have not been fully characterized it appears that subtle changes in all phospholipid classes contribute to the resistance to ethanol disordering of these membranes.

Administration, Oral↗
PubMed2006

Plasma metabolite profiles: effects of dietary phospholipids in a migratory passerine (Zonotrichia leucophrys gambelii).

Plasma metabolites, including triglycerides, beta -hydroxybutyrate, and glycerol, can be used to estimate mass change in birds. Although dietary fatty acids can be ingested and absorbed as phospholipids, they have been largely overlooked as a potential indicator of mass change. The plasma ratio of triglyceride to phospholipid could also provide insight into diet quality because a high ratio in food items indicates high relative energy content. Variability in dietary phospholipid content and triglyceride : phospholipid may also affect the relationships between metabolites and mass change. We fed Gambel's white-crowned sparrows (Zonotrichia leucophrys gambelii) diets differing in phospholipid content and triglyceride : phospholipid and measured metabolites during mass loss and mass gain. Plasma phospholipids were higher and triglyceride : phospholipid was lower in birds fed a diet higher in phospholipid content and lower in triglyceride : phospholipid. Contrary to our expectations, plasma phospholipids were negatively related to mass change. Plasma triglyceride levels were positively related to mass change and unaffected by diet. The relationships between mass change and both plasma beta -hydroxybutyrate and glycerol were affected by diet. Plasma triglyceride appears to be the most reliable metabolite predicting body mass changes, but inclusion of plasma phospholipids and triglyceride : phospholipid into metabolite profiles may provide additional information on diet quality.

Animal Migration↗
PubMed1999

Modulation of rat atrial G protein-coupled K+ channel function by phospholipids.

1. G protein-gated K+ channels (KACh channels) in the heart and brain are activated by the betagamma subunit of inhibitory G protein. Phosphatidylinositol-4,5-bisphosphate (PIP2) has recently been reported to directly activate KACh channels (GIRK) expressed in oocytes, as well as to support activation by the betagamma subunit in the presence of Na+. We examined the effect of Na+, PIP2 and other phospholipids on the KACh channel to understand better their role in KACh channel activation and modulation. 2. In atrial membrane patches, none of the phospholipids tested including PIP2 caused activation of the KACh channel in either the presence or the absence of 30 mM Na+. PIP2 (3 microM) and other phospholipids (30 microM) blocked acetylcholine-induced activation of the KACh channel. 3. When KACh channels were first activated with GTPgammaS, however, all phospholipids (100 microM) tested augmented the KACh channel activity 1.5- to 2-fold. Phosphatidylinositol-4-phosphate (PIP) and PIP2 were an order of magnitude more potent than other phospholipids. The increase in KACh channel activity was the result of a shift in the gating mode of the channel from a short-lived to a longer-lived open state. Such a modulatory effect was qualitatively similar to that produced by intracellular ATP. Trypsin blocked the ATP effect but not the phospholipid effect on the KACh channel kinetics. 4. The phosphate group linked to the glycerol backbone was important for KACh channel modulation by phospholipids. The higher potency of PIP and PIP2 was due to the presence of inositol phosphates. 5. Intracellular Na+ (30 mM) increased the frequency of KACh channel opening approximately 2-fold if the channels were already active, but did not affect modulation by phospholipids. The effects of Na+ and phospholipids on KACh channel activity were additive. 6. A low concentration of ATP (20 microM), which had no effect on the KACh channel by itself, potentiated the stimulatory action of phospholipids, indicating that ATP and phospholipids interacted to modulate KACh channel function. 7. We conclude that exogenously applied PIP2 and other phospholipids block agonist-mediated KACh channel activation. However, if the KACh channel is already activated with GTPgammaS, phospholipids augment the existing activity by increasing the number of longer-lived channel openings. The evidence for and against the role of PIP and PIP2 in the stimulatory effect of ATP on the KACh channel is presented and discussed.

Acetylcholine↗
PubMed2006

Effect of amiodarone on phospholipid content and composition in heart, lung, kidney and skeletal muscle: relationship to alteration of thyroid function.

To investigate the effect of chronic amiodarone treatment on tissue phospholipids, a marker of amiodarone-induced toxicity, and to test the hypothesis that tissue phospholipids changes are related to amiodarone-induced effects on thyroid function, male Wistar rats were treated with amiodarone and tissue phospholipid content and fractions were assessed. Twenty-six animals were allocated to 4 groups: (i) group 1 received amiodarone, 20 mg/kg per day, for 3 weeks (n = 6); (ii) group 2 received amiodarone for 5 weeks (n = 6); (iii) group 3 received drug for 6 weeks (n = 6), and (iv) group 4 (control group) received the diluent for 6 weeks (n = 8). Total phospholipid content of lung, kidney and skeletal muscle but not heart was increased after 3 weeks of amiodarone treatment. With longer durations of treatment, the phospholipid content was significantly (p < 0.05) reduced in all four organs. The proportion of phospholipids in different classes was modified by amiodarone treatment with the most consistent changes across different tissues being reductions in phosphatidylethanolamine and increases in phosphatidylserine. Serum thyroxine concentration was significantly (p < 0.05) reduced at 5 weeks of treatment and thereafter. There was a significant correlation between serum thyroxine and total phospholipid concentration in heart (r = 0.555; p < 0.05) and lung (r = 0.502; p < 0.05). For heart, there was a significant correlation between serum thyroxine and the distribution of phospholipid classes, mainly for phosphatidylserine even after considering amiodarone dose. The same was found in the lung. In the kidney and skeletal muscle, there was a significant (p < 0.05) correlation between serum thyroxine and the proportion of phospholipids in phosphatidylcholine and sphingomyelin. In conclusion, this study presents the novel finding of a biphasic tissue phospholipid response to amiodarone characterized by a short term increase in phospholipids in lung, kidney and skeletal muscle but not the heart followed by a long term decline in phospholipids in all four organs that is likely due to a direct action of amiodarone on phospholipid metabolism and potentially the result of amiodarone-induced reduction in thyroid function.

Amiodarone↗
PubMed1985

Evidence for glucose-responsive and -unresponsive pools of phospholipid in pancreatic islets.

The effect of glucose on the metabolism of phospholipids in pancreatic islets was studied with three radioactive phospholipid precursors, [32P]orthophosphate, [3H]myoinositol, and [3H]arachidonic acid, to determine the conditions necessary for studying the breakdown of prelabeled phospholipids. Islets were incubated in the presence of a radioactive precursor for 60 or 90 min and in the presence of either 3.3 or 16.7 mM glucose to prelabel phospholipids. To study the breakdown of prelabeled phospholipid, the unincorporated precursor was removed and the islets were reincubated for 15 or 20 min under conditions that either did or did not stimulate insulin release. Prelabeling in the presence of a noninsulinotropic concentration of glucose (3.3 mM) supported the incorporation of precursors into almost all islet phospholipids studied. Prelabeling in an insulinotropic concentration of glucose (16.7 mM) increased the incorporation of precursors into a number of phospholipids even more; and reincubation in 16.7 mM glucose caused a rapid loss of radioactivity from specific phospholipids (phosphatidylinositol and/or phosphatidylcholine, depending on the precursor). This breakdown was observed only when islets had been prelabeled in 16.7 mM glucose. The amount of radioactivity lost from phospholipid corresponded roughly to the additional amount incorporated during the prelabeling in the high concentration of glucose. Radioactivity in phospholipids in islets prelabeled in 3.3 mM glucose or in nonsecretagogue metabolic fuels, such as malate plus pyruvate, did not decrease when the islets were subsequently exposed to 16.7 mM glucose, nor did it decrease in 3.3 mM glucose when these islets had been prelabeled in 16.7 mM glucose. Glyceraldehyde, an insulin secretagogue, but not galactose or L-glucose which are not insulin secretagogues, stimulated phospholipid breakdown in islets that had been prelabeled in 16.7 mM glucose. Depriving islets of extracellular calcium, a condition that inhibits insulin release, inhibited phospholipid breakdown. The results suggest that pancreatic islets contain a glucose-responsive and a glucose-unresponsive phospholipid pool. The glucose-responsive pool becomes labeled and undergoes rapid turnover only under stimulatory conditions and may play a role in the stimulus-secretion coupling of insulin release.

Animals↗
PubMed1976

Role of gamma-carboxyglutamic acid. Cation specificity of prothrombin and factor X-phospholipid binding.

Divalent cations are required for two roles in prothrombin-phospholipid interaction. The first role, catalysis of a prothrombin protein transition has a reaction half-life of 100 min at 0 degrees and is a prerequisite to phospholipid binding. The binding sites required for the transition have a very low cation specificity. All di- and trivalent cations tested were effective in this role with the exception of beryllium. Barium catalyzed the transition but only at high concentrations (6.6 mM was required for half-reaction). Blood-clotting Factor X, another gamma-carboxyglutamic acid-containing protein, also undergoes a cation-catalyzed protein transition which is a prerequisite to Factor X-phospholipid binding. In both proteins, the transition can be monitored by a decrease in the protein's intrinsic fluorescence. Compared to prothrombin, the Factor X transition occurs much more rapidly, has a somewhat greater specificity for cations, and requires higher concentrations of cations. This indicates that the cation binding sites provided by gamma-carboxyglutamic acid are not completely uniform in all proteins. The second role of divalent cations in prothrombin-phospholipid interaction is in the actual protein-phospholipid binding. This interaction was studied by protein fluorescence quenching resulting from excitation energy transfer to a chromophore attached to the phospholipid membrane. Only strontium and barium satisfactorily replaced calcium in this role. A number of other cations form protein-phospholipid complexes but of the wrong structure. These cations inhibit the prothrombinase complex (Factor Xa, calcium, phospholipid, Factor V). The cation specificity for Factor X-phospholipid binding is the same as for prothrombin except that higher concentrations of cations are required. Factor Xa (generated by action of Russell's viper venom on Factor X) displayed the same calcium requirements for the protein transition and phospholipid interaction as Factor X. The cation requirements of the prothrombinase complex correlate with the cation requirements of prothrombin and Factor X-phospholipid binding. Strontium is the only cation that will singly replace calcium. Barium is ineffective alone because the concentrations required to catalyze the protein transitions cause precipitation of the phospholipid. Combination of certain other cations with barium will, however, substitute for calcium. The other cations (specifically magnesium or manganous ion) catalyze the protein transitions and barium forms the correct protein-phospholipid complexes.

Animals↗
PubMedDate not supplied

Loss of membrane phospholipid asymmetry during activation of blood platelets and sickled red cells; mechanisms and physiological significance.

Membrane phospholipid asymmetry is considered to be a general property of biological membranes. Detailed information is presently available on the non-random orientation of phospholipids in red cell- and platelet membranes. The outer leaflet of the lipid bilayer membrane is rich in choline-phospholipids, whereas amino-phospholipids are abundant in the inner leaflet. Studies with blood platelets have shown that these asymmetries are not maintained when the cells are activated in various ways. Undoing the normal asymmetry of membrane phospholipids in activated blood cells is presumably mediated by increased transbilayer movement of phospholipids. This process, which leads to increased exposure of negatively charged phosphatidylserine at the outer surface, plays an important physiological role in local blood clotting reactions. A similar phenomenon occurs in sickled red cells. Phospholipid vesicles breaking off from reversibly sickled cells contribute similarly to intravascular clotting in the crisis phase of sickle cell disease. The loss of membrane phospholipid asymmetry in activated platelets seem to be strictly correlated with degradation of cytoskeletal proteins by endogenous calpain. It is remarkable that membrane phospholipid asymmetry can be (partly) restored when activated platelets are treated with reducing agents. This leads to disappearance of phosphatidylserine from the outer leaflet where it was previously exposed during cell activation. These observations will be discussed in relation to two mechanisms which have been recognized to play a role in the regulation of membrane phospholipid asymmetry; i.e. the interaction of amino-phospholipids to cytoskeletal proteins, and the involvement of a phospholpid-translocase catalyzing outward-inward transbilayer movement of amino-phospholipids.

Anemia, Sickle Cell↗
PubMed1979

Levels and distributions of phospholipids and cholesterol in the plasma membrane of neuroblastoma cells.

Murine neuroblastoma cells (clone N-2A) grown in suspension (spinner cells) or attached on a plastic surface (monolayer cells) were used in studies of the phospholipid and cholesterol composition of whole cells, primary plasma membranes, plasma membranes internalized during phagocytosis of polystyrene latex beads, mitochondria and microsomes. Monolayer cells contained higher concentrations of total phospholipid, phosphatidylserine and phosphatidylcholine, and lower concentration of phosphatidylethanolamine than spinner cells. The cholesterol levels and the relative proportions of the various phospholipids were similar in both cell types except phosphatidylethanolamine and sphingomyelin whose proportions were lower in monolayer cells. The primary plasma membranes of the two cell types differed significantly in the relative proportions of all phospholipids, except sphingomyelin, and the phospholipid to protein and the cholesterol to protein ratios were all higher in the membranes of spinner cells. In contrast to these results, all the phospholipid to protein and the cholesterol to protein ratios of the internalized plasma membranes were higher in monolayer than in spinner cells, and the proportions of all phospholipids, except phosphatidylethanolamine, were similar in both cell types. The membrane distributions of individual phospholipids and cholesterol were inferred from comparison of the phospholipid and cholesterol compositions of primary plasma membranes and plasma membranes internalized during phagocytosis of polystyrene beads. The results are consistent with a non-random distribution of most phospholipids in both spinner and monolayer cells, but the patterns of these distributions were different in the two cell types. With regard to cholesterol the results are compatible with a random or a heterogeneous distribution. All the phospholipid to protein ratios of the mitochondrial fraction of both cell types were lower than those of the plasma membranes. However, these ratios of the microsomal fraction were higher than those of the plasma membranes of monolayer cells, whereas they were comparable, with a few exceptions, to those of spinner cell membranes. The cholesterol to phospholipid molar ratios of plasma membranes were 6.4 and 4.3 fold greater than those of the mitochondrial and microsomal fractions, respectively.

Animals↗
PubMed1995

Mycobacterium smegmatis malate dehydrogenase: activation of the lipid-depleted enzyme by anionic phospholipids and phosphatidylethanolamine.

Phospholipid-protein interactions have been investigated in a phospholipid-requiring enzyme, FAD-dependent malate dehydrogenase isolated from Mycobacterium smegmatis membranes, to correlate these interactions with enzyme function. The ability of several natural and synthetic phospholipids including CL and PE, which are major phospholipids in M. smegmatis membranes, to activate purified, lipid-depleted, enzymatically inactive malate dehydrogenase was examined. Anionic phospholipids and PE activated the enzyme, while zwitterionic phospholipids did not. A PE/PC mixture activated the enzyme in the form of both bilayer and non-bilayer structure. CL/PE mixtures activated malate dehydrogenase much more than each single phospholipid species. All anionic phospholipids used stabilized the enzyme, while PE and zwitterionic phospholipids did not. CL and a CL/PE mixture protected malate dehydrogenase from proteinase digestion, while PE did not. All phospholipids and phospholipid mixtures tested caused little secondary structural change in malate dehydrogenase. The results obtained in this study suggest that CL and CL/PE mixtures could form stable, enzymatically active complexes with malate dehydrogenase which might be similar to the native complex in M. smegmatis membranes. Although PE could activate malate dehydrogenase in both bilayer and non-bilayer form, it formed a complex with malate dehydrogenase which was inferior in terms of stability and susceptibility to proteinases, indicating that PE alone poorly reconstitutes the active enzyme-phospholipid complex.

Enzyme Activation↗
PubMed2005

Preparation of a chemically anchored phospholipid monolayer on an acrylated polymer substrate.

This paper describes a strategy for designing a chemically anchored phospholipid monolayer that could be used as coating materials for biomedical implants. To make a chemically anchored phospholipid monolayer on the polymer substrate, we prepared the mono-acrylated phospholipid (1-palmitoyl-2-[12-(acryloyloxy)-dodecanoyl]-sn-glycero-3-phosphocholine; acryloyl-PC) and the acrylated polymer (poly(octadecylacrylate-co-4-acryloyloxy butylacrylate)), which was synthesized by the acrylation of poly(octadecyl acrylate-co-hydroxybutyl acrylate, poly(OA-co-HA)) with acryloyl chloride. The chemically anchored phospholipid monolayer was prepared by using in situ photopolymerization of a pre-assembled phospholipid monolayer, produced by lipid vesicle fusion, onto the acrylated polymer coated silicon wafer. Optimal condition of vesicle fusion and irradiation time was determined from the degree of hydrophilicity rendered by the polymerized phospholipid surface. The physicochemical properties of polymerized phospholipid monolayer on the substrate were evaluated using water contact angle, field-emission scanning electron micrograph (FE-SEM), atomic force microscopy (AFM) and X-ray photoelectron spectroscopy (XPS). These results confirmed that the polymerized phospholipid monolayer was chemically anchored on the acrylated polymer substrate. The chemically anchored phospholipid monolayer was stable in aqueous condition for 2 weeks, but the physically adsorbed phospholipid monolayer got removed within 1 day. Moreover, the polymerized phospholipid monolayer also suppressed albumin absorption and platelet adhesion, in vitro. This polymerized phospholipid monolayer provides a new biomimetic system for coating medical devises.

Acrylates↗
PubMed1989

Phospholipid flip-out controls the cell cycle of Escherichia coli.

Phospholipids are the principal constituents of biological membranes. In Escherichia coli, phospholipids are involved in the metabolism of other envelope constituents such as lipoprotein, lipopolysaccharide, certain envelope proteins and peptidoglycan. They are also involved in the regulation of the cell cycle. DNAA, the key protein in the initiation of chromosome replication, is activated by acidic phospholipids only when these are in fluid bilayers, whilst interruptions of phospholipid synthesis inhibit both the initiation of chromosome replication and cell division. The transmembrane movement or flip-flop of phospholipids from one monolayer to the other requires the passage of the polar head group through the hydrophobic core of the bilayer. Hence, in many systems, flip-flop is a slow process with half-time of days. Flip-flop accompanies the formation of non-bilayer structure. Such structures form under certain conditions of packing density and composition and have been observed both in vitro and in vivo. In bacteria, flip-flop appears to be extremely rapid, with half-times as fast as 3 min being observed. However, such rapid flip-flop may not be characteristic of all phospholipids. The asymmetrical distribution of phosphatidylethanolamine in the plasma membrane of Bacillus megaterium has been attributed to the existence of two classes of this phospholipid. In E. coli, studies of the metabolic turnover of phosphatidylserine, phosphatidylglycerol and phosphatidic acid also reveal the existence of distinct classes of these phospholipids. In this article I propose that, in E. coli, a class of phospholipids does indeed escape the rapid flip-flop mechanism; this class probably includes a subpopulation of the acidic phospholipids. Therefore during the cell cycle these phospholipids accumulate in the inner monolayer of the cytoplasmic membrane and so cause an increase in its packing density; at a critical density, phospholipids "flip out" from the inner to the outer monolayer. This flip-out occurs once per cycle and initiates cell cycle events.

Cell Cycle↗