Effect of lipid composition upon fusion of liposomes with Sendai virus membranes

How the lipid composition of liposomes determines their ability to fuse with Sendai virus membranes was tested. Liposomes were made of compositions designed to test postulated mechanisms of membrane fusion that require specific lipids. Fusion does not require the presence of lipids that can form micelles such as gangliosides or lipids that can undergo lamellar to hexagonal phase transitions such as phosphatidylethanolamine (PE), nor is a phosphatidylinositol (PI) to phosphatidic acid (PA) conversion required, since fusion occurs with liposomes containing phosphatidylcholine (PC) and any one of many different negatively charged lipids such as gangliosides, phosphatidylserine (PS), phosphatidylglycerol, dicetyl phosphate, PI, or PA. A negatively charged lipid is required since fusion does not occur with neutral liposomes containing PC and a neutral lipid such as globoside, sphingomyelin, or PE. Fusion of Sendai virus membranes with liposomes that contain PC and PS does not require Ca2+, so an anhydrous complex with Ca2+ or a Ca2+-induced lateral phase separation is not required although the possibility remains that viral binding causes a lateral phase separation. Sendai virus membranes can fuse with liposomes containing only PS, so a packing defect between domains of two different lipids is not required. The concentration of PS required for fusion to occur is approximately 10-fold higher than that required for ganglioside GD1a, which has been shown to act as a Sendai virus receptor. When cholesterol is added as a third lipid to liposomes containing PC and GD1a, the amount of fusion decreases if the GD1a concentration is low.(ABSTRACT TRUNCATED AT 250 WORDS)     

Modulation of vitronectin receptor binding by membrane lipid composition.

The vitronectin (Vn) receptor belongs to the integrin family of proteins and although its biochemical structure is fully characterized little is known about its binding affinity and specificity. We report here that Vn receptor binding to different matrix proteins is influenced by the surrounding lipid composition of the membrane. Human placenta affinity purified Vn receptor was inserted into liposomes of different composition: (i) phosphatidylcholine (PC); (ii) PC+phosphatidylethanolamine (PE); (iii) PC+PE+phosphatidylserine (PS) + phosphatidylinositol (PI) + cholesterol (chol). The amount of purified material that could be incorporated into the three lipid vesicle preparations was proportional to the efficiency of the vesicle formation that increased from PC (38%) to PC+PE and PC+PE+PS+PI+chol (about 50%) vesicles. Electron microscopy analysis showed that the homogeneity and size of the three liposome preparations were comparable (20-nm diameter) but their binding capacity to a series of substrates differed widely. Vn receptor inserted in PC liposomes bound only Vn, but when it was inserted in PC+PE and PC+PE+PS+PI+chol liposomes it also attached to von Willebrand factor (vWF) and fibronectin (Fn). Vn receptor had higher binding capacity for substrates when it was inserted in PC+PE+PS+PI+chol than PC+PE liposomes. Antibodies to Vn receptor blocked Vn receptor liposome binding to Vn, vWF, and Fn. The intrinsic emission fluorescence spectrum of the Vn receptor reconstituted in PC+PE+PS+PI+chol liposomes was blue-shifted in relation to PC liposomes, suggesting a conformational change of the receptor in the membranes. These data provide direct evidence that the Vn receptor is "promiscuous" and can associate with Vn, vWF and Fn. The nature of the membrane lipid composition surrounding the receptor could thus influence its binding affinity, possibly by changing its conformation or exposure or both.     

Lateral segregation of anionic phospholipids in model membranes induced by cytochrome P450 2B1: bi-directional coupling between CYP2B1 and anionic phospholipid

The lateral segregation of anionic phospholipids phosphatidic acid (PA), phosphatidylinositol (PI), and phosphatidylserine (PS) was detected after addition of cytochrome P450 2B1 (CYP2B1). The tendency of lipid clustering was highly dependent on the type of anionic phospholipids examined. PA was the most highly clustered while PI and PS clustered to a lesser degree. Moreover, liposomes containing anionic phospholipids form anionic phospholipid-rich microdomains in the presence of CYP2B1. Anionic phospholipids (mostly notably PA) also increased the ability of CYP2B1 to bind to lipid monolayers. In addition to the ability of CYP2B1 to modulate the physical properties of the membrane, the membrane itself can have reciprocal effects on the activity and conformation of CYP2B1. The catalytic activity of CYP2B1 increased as a function of anionic phospholipid concentration and in the presence of 10 mol% PA, the activity increased by 85%. These results suggest a bi-directional coupling between the CYP2B1 and anionic phospholipids.     

Self-adaptive modification of red-cell membrane lipids in lecithin: Cholesterol acyltransferase deficiency. Lipid analysis and spin labeling

In a patient with lecithin: cholesterol acyltransferase deficiency, free cholesterol was markedly increased, and esterified cholesterol was diminished. In the patient's plasma, an increase in phosphatidylcholine (PC) and a decrease in sphingomyelin were observed. Concomitantly, an increase in a shorter acyl chain 16:0 was noted in PC, sphingomyelin and phosphatidylethanolamine (PE). In contrast to these results, longer chains such as 22:0 and 24:0 were decreased, especially in sphingomyelin. Unsaturated double bonds such as 18:1 was also increased in PC and PE. In the red-cell membrane lipids, the increase in free cholesterol was counteracted by an increase in PC and by a decrease in sphingomyelin and PE, reflecting changes in the patient's plasma lipids. Increased 16:0 (in PC) and decreased 18:0 and 24:0 were observed. The increased plasma free cholesterol due to metabolic defect (lecithin:cholesterol acyltransferase deficiency) led to decreased red-cell membrane fluidity. This effect appeared to be counteracted by changing phospholipid composition (increased PC and decreased sphingomyelin and PE), by increasing shorter chains (16:0), by decreasing longer chains (18:0 and 24:0) and by increasing unsaturated double bonds (18:2). These results can be interpreted as a self-adaptive modification of lecithin:cholesterol acyltransferase deficiency-induced red-cell membrane abnormalities, to maintain normal membrane fluidity. This speculation was supported by the ESR spin-label studies on the patient's membrane lipids. The normal order parameters in intact red cells and in total lipid liposomes were decreased if cholesterol-depleted membrane liposomes were prepared. Thus, the hardening effect of cholesterol appeared to be counteracted by the softening effects described above. Overall membrane fluidity in intact red cells of the lecithin:cholesterol acyltransferase-deficient patient was maintained normally, judged by order parameters in ESR spin-label studies.     

Effects of lipids on the transport activity of the reconstituted glucose transport system from rat adipocyte

The glucose transport system, isolated from rat adipocyte membrane fractions, was reconstituted into phospholipid vesicles. Vesicles composed of crude egg yolk phospholipids, containing primarily phosphatidylcholine (PC) and phosphatidylethanolamine (PE), demonstrated specific d-glucose uptake. Purified vesicles made of PC and PE also supported such activity but PC or PE by themselves did not. The modulation of this uptake activity has been studied by systematically altering the lipid composition of the reconstituted system with respect to: (1) polar headgroups; (2) acyl chains, and (3) charge. Addition of small amounts (20 mol%) of PS, phosphatidylinositol (PI), cholesterol, or sphingomyelin significantly reduced glucose transport activity. A similar effect was seen with the charged lipid, phosphatidic acid. In the case of PS, this effect was independent of the acyl chain composition. Polar headgroup modification of PE, however, did not appreciably affect transport activity. Free fatty acids, on the other hand, increased or decreased activity based on the degree of saturation and charge. These results indicate that glucose transport activity is sensitive to specific alterations in both the polar headgroup and acyl chain composition of the surrounding membrane lipids.     

Phospholipid effects on SGLT1-mediated glucose transport in rabbit ileum brush border membrane vesicles

In small intestine, sodium-glucose cotransporter SGLT1 provides the main mechanism for sugar uptake. We investigated the effect of membrane phospholipids (PL) on this transport in rabbit ileal brush border membrane vesicles (BBMV). For this, PL of different charge, length, and saturation were incorporated into BBMV. Transport was measured related to (i) membrane surface charge (membrane-bound MC540 fluorescence), (ii) membrane thickness (PL incorporation of different acyl chain length), and (iii) membrane fluidity (r_12AS, fluorescence anisotropy of 12-AS).Compared to phosphatidylcholine (PC) carrying a neutral head group, inhibition of SGLT1 increased considerably with the acidic phosphatidic acid (PA) and phosphatidylinositol (PI) that increase membrane negative surface charge. The order of PL potency was PI>PA > PE = PS > PC. Inhibition by acidic PA-oleate was 5-times more effective than with neutral PE (phosphatidylethanolamine)-oleate. Lineweaver-Burk plot indicated uncompetitive inhibition of SGLT1 by PA.When membrane thickness was increased by neutral PC of varying acyl chain length, transport was increasingly inhibited by 16:1 PC to 22:1 PC. Even more pronounced inhibition was observed with mono-unsaturated instead of saturated acyl chains which increased membrane fluidity (indicated by decreased r_12AS).In conclusion, sodium-dependent glucose transport of rabbit ileal BBMV is modulated by (i) altered membrane surface charge, (ii) length of acyl chains via membrane thickness, and (iii) saturation of PL acyl chains altering membrane fluidity. Transport was attenuated by charged PL with longer and unsaturated acyl residues. Alterations of PL may provide a principle for attenuating dietary glucose uptake.     

Fusion of liposomes containing a novel cationic lipid, N-[2,3-(dioleyloxy)propyl]-N,N,N-trimethylammonium: induction by multivalent anions and asymmetric fusion with acidic phospholipid vesicles

The fusion behavior of large unilamellar liposomes composed of N-[2,3-(dioleyloxy)propyl]-N,N,N-trimethylammonium (DOTMA) and either phosphatidylcholine (PC) or phosphatidylethanolamine (PE) has been investigated by a fluorescence resonance energy transfer assay for lipid mixing, dynamic light scattering, and electron microscopy. Polyvalent anions induced the fusion of DOTMA/PE (1:1) liposomes with the following sequence of effectiveness: citrate greater than EDTA greater than phosphate, in the presence 100 mM NaCl, pH 7.4. Sulfate, dipicolinate, and acetate were ineffective. DOTMA/PC (1:1) vesicles were completely refractory to fusion in the presence of multivalent anions in the concentration range studied, consistent with the inhibitory effect of PC in divalent cation induced fusion of negatively charged vesicles. DOTMA/PE vesicles could fuse with DOTMA/PC vesicles in the presence of high concentrations of citrate, but not of phosphate. Mixing of DOTMA/PE liposomes with negatively charged phosphatidylserine (PS)/PE or PS/PC (1:1) vesicles resulted in membrane fusion in the absence of multivalent anions. DOTMA/PC liposomes also fused with PS/PE liposomes and, to a limited extent, with PS/PC liposomes. These observations suggest that the interaction of the negatively charged PS polar group with the positively charged trimethylammonium of DOTMA is sufficient to mediate fusion between the two membranes containing these lipids and that the nature of the zwitterionic phospholipid component of these vesicles is an additional determinant of membrane fusion.     

Effects of thallium(I) and thallium(III) on liposome membrane physical properties

The hypothesis that thallium (Tl) interaction with membrane phospholipids could result in the alteration of membrane physical properties was investigated. Working with liposomes composed of brain phosphatidylcholine and phosphatidylserine, we found that Tl(+), Tl(3+), and Tl(OH)(3) (0.5-25 microM): (a) increased membrane surface potential, (b) decreased the fluidity of the anionic regions of the membrane, in association with an increased fluidity in the cationic regions, and (c) promoted the rearrangement of lipids through lateral phase separation. The magnitude of these effects followed the order Tl(3+), Tl(OH)(3)>Tl(+). In addition, Tl(3+) also decreased the hydration of phospholipid polar headgroups and induced membrane permeabilization. The present results show that Tl interacts with membranes inducing major alterations in the rheology of the bilayer, which could be partially responsible for the neurotoxic effects of this metal.     

The response of plasma membrane lipid composition in callus of the halophyte Spartina patens (Poaceae) to salinity stress

Callus cultures of the salt marsh grass Spartina patens were examined to determine changes and consistencies in membrane lipid composition in response to salt. Major membrane lipid classes remained stable at all salinity levels (0, 170, 340 mmol/L). However, the membrane protein to lipid ratio decreased significantly in response to elevated NaCl. Callus plasma membrane (PM) consisted predominantly of sterols, about 60% (mol%) of the total lipids. Glycolipid was the second largest lipid class, making up about 20% (mol%) of the total. With increasing salinity, the relative percentage of sitosterol decreased, while that of campesterol increased. The phospholipid species detected were phosphatidylethanolamine (PE), phosphatidylcholine (PC), phosphatidylserine (PS), and phosphatidylinositol (PI). When callus was grown at 340 mmol/L NaCl, PC increased significantly. PI and PS were also significantly elevated in salinity treatments. Only 24-32% of the PM fatty acids were common plant membrane fatty acids, C16, C18, C20, and C22, while over 60% were the less common fatty acids, C11 and C14. Membrane fluidity remained stable in response to growth medium salinity. The findings on membrane responses to salinity will facilitate a better understanding of this halophyte's tactics for salt tolerance.     

Cardiolipin is essential for higher proton translocation activity of reconstituted Fo

The Fo membrane domain of FoF1-ATPase complex had been purifiedfrom porcine heart mitochondria. SDS-PAGE with silver staining indicated that the purity of Fo was about 85% and the sample contained no subunits of F1-ATPase. The purified Fo was reconstituted into liposomes with different phospholipid composition, and the effect of CL (cardiolipin), PA (phosphatidic acid), PI (phosphatidylinositol) and PS (phosphatidylserine) on the H+ translocation activity of Fo was investigated. The results demonstrated that CL, PA and PI could promote the proton translocation of Fo with the order of CL＞PA＞＞PI, while PS inhibited it. Meanwhile ADM (adriamycin) severely impaired the proton translocation activity of Fo vesicles containing CL, which suggested that CL's stimulation of the activity of reconstituted Fo might correlate with its non-bilayer propensity. After Fo was incorporated into the liposomes containing PE (phosphatidylethanolamine), DOPE (dioleoylphosphatidylethanolamine) as well as DEPE (dielaidoylphosphatidylethanolamine), it was found that the proton translocation activity of Fo vesicles increased with the increasing content of PE or DOPE, which has high propensity of forming non-bilayer structure, but was independent of DEPE. The dynamic quenching of the intrinsic fluorescence of tryptophan by HB (hypocrellin B) as well as fluorescent spectrum of acrylodan labeling Fo at cysteine indicated that CL could induce Fo to a suitable conformation resulting in higher proton translocation activity.     

Effects of lipid composition on membrane permeabilization by sticholysin I and II, two cytolysins of the sea anemone Stichodactyla helianthus

Sticholysin I and II (St I and St II), two basic cytolysins purified from the Caribbean sea anemone Stichodactyla helianthus, efficiently permeabilize lipid vesicles by forming pores in their membranes. A general characteristic of these toxins is their preference for membranes containing sphingomyelin (SM). As a consequence, vesicles formed by equimolar mixtures of SM with phosphatidylcholine (PC) are very good targets for St I and II. To better characterize the lipid dependence of the cytolysin-membrane interaction, we have now evaluated the effect of including different lipids in the composition of the vesicles. We observed that at low doses of either St I or St II vesicles composed of SM and phosphatidic acid (PA) were permeabilized faster and to a higher extent than vesicles of PC and SM. As in the case of PC/SM mixtures, permeabilization was optimal when the molar ratio of PA/SM was ~1. The preference for membranes containing PA was confirmed by inhibition experiments in which the hemolytic activity of St I was diminished by pre-incubation with vesicles of different composition. The inclusion of even small proportions of PA into PC/SM LUVs led to a marked increase in calcein release caused by both St I and St II, reaching maximal effect at ~5 mol % of PA. Inclusion of other negatively charged lipids (phosphatidylserine (PS), phosphatidylglycerol (PG), phosphatidylinositol (PI), or cardiolipin (CL)), all at 5 mol %, also elicited an increase in calcein release, the potency being in the order CL approximately PA > PG approximately PI approximately PS. However, some boosting effect was also obtained, including the zwitterionic lipid phosphatidylethanolamine (PE) or even, albeit to a lesser extent, the positively charged lipid stearylamine (SA). This indicated that the effect was not mediated by electrostatic interactions between the cytolysin and the negative surface of the vesicles. In fact, increasing the ionic strength of the medium had only a small inhibitory effect on the interaction, but this was actually larger with uncharged vesicles than with negatively charged vesicles. A study of the fluidity of the different vesicles, probed by the environment-sensitive fluorescent dye diphenylhexatriene (DPH), showed that toxin activity was also not correlated to the average membrane fluidity. It is suggested that the insertion of the toxin channel could imply the formation in the bilayer of a nonlamellar structure, a toroidal lipid pore. In this case, the presence of lipids favoring a nonlamellar phase, in particular PA and CL, strong inducers of negative curvature in the bilayer, could help in the formation of the pore. This possibility is confirmed by the fact that the formation of toxin pores strongly promotes the rate of transbilayer movement of lipid molecules, which indicates local disruption of the lamellar structure.     

Phospholipid dependence of calcium ion effects on electrophoretic mobilities of liposomes

Electrophoretic light scattering (ELS) and depolarization of fluorescence have been used to determine the effect of membrane fluidity on the binding of Ca2+ to liposomes. ELS was used to measure the electrophoretic mobilities of the liposomes. Fluorescence depolarization was used to determine membrane fluidity. Zero to 30 mol% phosphatidylserine (PS) was incorporated into liposomes containing, as bulk phospholipids, one of the following: dimyristoyl-phosphatidylcholine (DMPC), dipalmitoylphosphatidylcholine (DPPC), egg phosphatidylcholine (PC), or hydrogenated egg phosphatidylcholine (H egg PC). The binding of Ca2+ to the liposomes appears to be influenced by membrane fluidity. Liposomes containing bulk phospholipids whose phase transition temperature is higher than the experimental temperature exhibit enhanced binding of CA2+.     

Cardiolipin is essential for higher proton translocation activity of reconstituted F_o

The Fo membrane domain of FoF1-ATPase complex had been purified from porcine heart mitochondria. SDS-PAGE with silver staining indicated that the purity of Fo was about 85% and the sample contained no subunits of F1-ATPase. The purified Fo was reconstituted into liposomes with different phospholipid composition, and the effect of CL (cardiolipin), PA (phosphatidic acid), PI (phosphatidylinositol) and PS (phosphatidylserine) on the H+ translocation activity of Fo was investigated. The results demonstrated that CL, PA and PI could promote the proton translocation of Fo with the order of CL>PA>>PI, while PS inhibited it. Meanwhile ADM (adriamycin) severely impaired the proton translocation activity of Fo vesicles containing CL, which suggested that CL's stimulation of the activity of reconstituted Fo might correlate with its non-bilayer propensity. After Fo was incorporated into the liposomes containing PE (phosphatidylethanolamine), DOPE (dioleoylphosphatidylethanolamine) as well as DEPE (dielaidoylphospha     

Decreased lipid order induced by microsomal cytochrome P-450 and NADPH-cytochrome P-450 reductase in model membranes: fluorescence and electron spin resonance studies

Cytochrome P-450 and NADPH-cytochrome P-450 reductase were reconstituted in unilamellar lipid vesicles prepared by the cholate dialysis technique from pure dimyristoylphosphatidylcholine (DMPC), pure dipalmitoylphosphatidylcholine (DPPC), pure dioleoylphosphatidylcholine (DOPC), and phosphatidylcholine/phosphatidylethanolamine/phosphatidylserine (PC/PE/PS) (10:5:1). As probes for the vesicles' hydrocarbon region, 1,6-diphenyl-1,3,5-hexatriene (DPH) and spin-labeled PC were used. The steady-state and time-resolved fluorescence parameters of DPH were determined as a function of temperature and composition of liposomes. Incorporation of either protein alone or together increased the steady-state fluorescence anisotropy (rs) of DPH in DOPC and PC/PE/PS (10:5:1) liposomes. In DMPC and DPPC vesicles, the proteins decreased rs significantly below the transition temperature (Tc) of the gel to liquid-crystalline phase transition. Time-resolved fluorescence measurements of DPH performed in reconstituted PC/PE/PS and DMPC proteoliposomes showed that the proteins disorder the bilayer both in the gel and in the liquid-crystalline phase. Little disordering by the proteins was observed by a spin-label located near the mid-zone of the bilayer 1-palmitoyl-2-(5-doxylstearoyl)-3-sn-phosphatidylcholine (8-doxyl-PC), whereas pronounced disordering was detected by 1-palmitoyl-2-(8-doxylpalmitoyl)-3-sn-phosphatidylcholine (5-doxyl-PC), which probes the lipid zone closer to the polar part of the membrane. Fluorescence lifetime measurements of DPH indicate an average distance of greater than or equal to 60 A between the heme of cytochrome P-450 and DPH.     

肺叶切除术对红细胞及淋巴细胞膜流动性的影响

目的：研究肺叶切除术对红细胞及淋巴细胞膜流坳性的影响。方法：选择２０例择期开胸手术病人,均作肺叶切除术。分别用微量滴定法、高效液权色谱法和ＤＰＨ荧光探剂法测定血浆和红细胞膜ＰＬＡ２活性,红细胞膜磷脂ＰＳ、ＰＥ、ＰＣ和红细胞、淋巴膜脂流动性。结果：手术１０ｍｉｎ、手术６０ｍｉｎ和手术结束后３０ｍｉｎ血浆和红细胞膜ＰＬＡ２活性均显著高于麻醉诱导前;手术６０ｍｉｎ和手术结束后３０ｍｉｎ 红细胞膜ＰＳ、Ｐ     

Al(3+)-mediated changes in membrane physical properties participate in the inhibition of polyphosphoinositide hydrolysis

We investigated the possible involvement of Al(3+)-induced alterations in membrane physical properties in Al(3+)-mediated inhibition of polyphosphoinositide (PPI) hydrolysis by the enzyme phosphatidylinositol-specific phospholipase C (PI-PLC). Liposomes composed of brain phosphatidylcholine (PC) or of PC and a mixture of brain PPI (PC:PPI) were incubated in the presence of Al(3+) (1-100 microM). We evaluated: (1) the amount of membrane-bound Al(3+), (2) the effects of Al(3+) on key membrane physical properties (surface potential, lipid fluidity, and lipid arrangement), and (3) the hydrolysis of PPI. Al(3+) binding to PC:PPI (60:40 mol/mol) liposomes was 1.3 times higher than to PC:PPI (90:10 mol/mol) liposomes and did not change after treatment with Triton X-100. Al(3+) increased membrane surface potential, promoted the loss of membrane fluidity, and caused lateral phase separation in PC:PPI liposomes. Phosphatidylinositol and phosphatidylinositol monophosphate hydrolysis in the presence of PI-PLC was not affected by Al(3+), but a significant and concentration-dependent inhibition of PIP(2) hydrolysis was observed, an effect that was prevented by previous bilayer disruption with Triton X-100. The obtained results support the hypothesis that Al(3+) binding to liposomes promotes the formation of rigid clusters enriched in PPI, restricting the accessibility of the enzyme to the substrate and subsequently inhibiting PIP(2) hydrolysis by PI-PLC.     

Interaction of rat brain cytidylate cyclase with phospholipids

The interaction of rat brain cytidylate cyclase with some phospholipids such as L-alpha-phosphatidylcholine (PC), L-alpha-phosphatidylserine (PS), L-alpha-phosphatidylethanolamine (PE) and L-alpha-phosphatidic acid (PA) was studied. Cytidylate cyclase activity of Triton X-100 - solubilized fraction was inhibited by PS, PE and PA, but not with PC. The addition of PC to the incubation mixture containing PS, PE or PA dose - dependently reversed the inhibition of enzyme activity by these phospholipids. Phospholipids showed similar effect on the intact membrane - bound enzyme. PC could reactivate the enzyme which was inactivated by deoxycholate treatment, suggesting that PC may be an important factor to reconstitute an active conformation of the enzyme. These findings indicate that cytidylate cyclase could be regulated by phospholipids constituting its microenvironment of the membrane.     

Effect of hormones on phospholipid metabolism in human cultured fibroblasts

The effect of hormones on phospholipid metabolism, pool size, 32P labeling and changes in fatty acid of human adult fibroblasts was determined. Simultaneously the change in membrane fluidity of single cells was recorded via fluorescence recovery after photobleaching under the influence of hormones. From all substances tested (isoproterenol, phenylephrine, adrenalin, histamine, angiotensin II, dansylcadaverine, propranolol) only isoproterenol and adrenalin slightly decreased total amount of phosphatidylcholine (PC). The amount of the other phospholipids analyzed remained unchanged. The 32P incorporation rate into phospholipids (PC, phosphatidylinositol (PI), phosphatidylethanolamine (PE)) was affected basicly different analyzing either PC, PI or PE. Histamine and propranolol provoked the highest incorporation of 32P (240% increase in PI labeling). Isoproterenol and adrenalin decreased PC labeling (45% and 18%) whereas isoproterenol decreased 32P incorporation into PI (18%), and adrenalin led to an increase (37%). PE labeling showed no or a slight increase in 32P incorporation applying the other agonists or antagonists. The fatty acid pattern of the respective phospholipids changed only to a minor extend. A decrease in hexadecanoic acid content of PI was found after administration of either isoproterenol, adrenalin or histamine. Parallel determination of membrane fluidity of single cells by fluorescence recovery after photobleaching showed an increase in the diffusion coefficient of a fluorescent lipid probe sticking in the membrane, following administration of isoproterenol and adrenalin, other substances tested exerted no effect. A relationship to changes in phospholipid metabolism became obvious. These results are discussed considering known mechanisms of receptor coupling and change in phospholipid metabolism and fluidity.     

Lipid composition of subcellular particles from sheep platelets. Location of phosphatidylethanolamine and phosphatidylserine in plasma membranes and platelet liposomes

The lipid composition of whole sheep platelets and their subcellular fractions was determined. The basic lipids show similar distributions in granules, microsomes, plasma membranes and whole platelets. Phospholipid (about 70% of total lipids) and cholesterol (25% of total lipids) are the principal lipid components. Free cholesterol represents about 98% of the total, whereas cholesteryl ester is a minor component. The phospholipid composition found in intact platelets and their subcellular particles is about: 35% phosphatidylethanolamine (PE), 30% phosphatidylcholine (PC), 20% sphingomyelin and 15% phosphatidylserine (PS). We also investigated aminophospholipid topology in intact platelet plasma membranes and platelet liposomes by using the nonpenetrating chemical probe trinitrobenzenesulfonic acid (TNBS), because they are the major components of total lipids. In intact platelets, PS is not accessible to TNBS during the initial 15 min of incubation, whereas 18% PE is labelled after 15 min. In contrast, in phospholipid extracted from platelets 80% PE and 67% PS react with TNBS within 5 min, while 27 and 25% PE and 15 and 19% PS from liposomes and isolated plasma membranes, respectively, were modified after 15 min of incubation. In view of this chemical modification, it is concluded that 22% of PE and less than 1% of PS are located on the external surface of intact platelet plasma membranes. The asymmetric orientation of aminophospholipids is similar between liposomes and isolated plasma membrane. PS (23 and 28%) and PE (34 and 31%) are scarcely represented outside the bilayer. The data found are consistent with the nonrandom phospholipid distribution of blood cell surface membranes.     

Al(3+)-mediated changes on membrane fluidity affects the activity of PI-PLC but not of PLC

We investigated whether Al(3+)-mediated changes in membrane fluidity can affect the activity of prokaryotic enzymes phospholipase C (PLC) and phospholipase C-phosphatidyl inositol specific (PI-PLC) in liposomes of phosphatidyl choline (PC), PC:phosphatidyl inositol (PI), or PC and polyphosphoinositides (PPI). Al(3+) (10-100 microM) promoted membrane rigidification, evaluated with the probes 1,6-diphenyl-1,3,5-hexatriene and Laurdan, and followed the order: PC:PPI>PC:PI>PC. Al(3+) (25 and 50 microM) did not affect PLC-mediated hydrolysis of PC, PI and PIP(2), but stimulated PIP hydrolysis (48.6%). PI-PLC did not affect PC, PI, and PIP concentrations, but caused a 67% decrease in PIP(2). Al(3+) significantly inhibited PIP(2) hydrolysis in a concentration-dependent (25-50 microM) manner. Results suggest that the inhibition of PIP(2) hydrolysis by Al(3+) could be partially due to a higher lipid packing induced by Al(3+) which could affect the interaction between the enzyme and its substrate.