Investigation of the interaction of pig muscle lactate dehydrogenase with acidic phospholipids at low pH

Interaction of pig muscle lactate dehydrogenase (LDH) with acidic phospholipids is strongly dependent on pH and is most efficient at pH values<6.5. The interaction is ionic strength sensitive and is not observed when bilayer structures are disrupted by detergents. Bilayers made of phosphatidylcholine (PC) do not bind the enzyme. The LDH interaction with mixed composition bilayers phosphatidylserine/phosphatidylcholine (PS/PC) and cardiolipin/phosphatidylcholine (CL/PC) leads to dramatic changes in the specific activity of the enzyme above a threshold of acidic phospholipid concentration likely when a necessary surface charge density is achieved. The threshold is dependent on the kind of phospholipid. Cardiolipin (CL) is much more effective compared to phosphatidylserine, which is explained as an effect of availability of both phosphate groups in a CL molecule for interaction with the enzyme. A requirement of more than one binding point on the enzyme molecule for the modification of the specific activity is postulated and discussed. Changes in CD spectra induced by the presence of CL and PS vesicles evidence modification of the conformational state of the protein molecules. In vivo qualitative as well as quantitative phospholipid composition of membrane binding sites for LDH molecules would be crucial for the yield of the binding and its consequences for the enzyme activity in the conditions of lowered pH.     

Action of α-l-fucoside fromOctopus vulgaris hepatopancreas on phospholipid vesicles containing the fucosylated ganglioside FucGM1

The behaviour of a highly purified -l-fucosidase (E.C. 3.2.1.51) extracted from octopus hepatopancreas was studied with phospholipid vesicles composed of phosphatidylcholine (PC) and phosphatidylserine (PS) containing the fucosylated ganglioside FucGM1, a potential natural substrate of the enzyme. The substrate recognition and hydrolysis take place only with PS/FucGM1 mixtures via an association process of the enzyme with the vesicles at acidic pH; the enzyme rapidly and stably binds to PS vesicles but not to PC vesicles. The data suggest that only the PS-associated enzyme is able to hydrolyse FucGM1 embedded in the same bilayer. The enzyme association with FucGM1/PS vesicles is a prerequisite for ganglioside hydrolysis but is followed by irreversible enzyme inactivation.     

Efficacy of soluble phospholipids in the prothrombinase reaction

The prothrombinase complex is comprised of an enzyme, factor Xa, and a cofactor, factor Va, that each bind peripherally to membranes containing phosphatidylserine (PS) and activate the substrate, prothrombin. The mechanism by which the membrane contributes to enhanced catalytic efficacy of prothrombinase is not precisely known but is generally attributed to some aspect of enzyme and substrate assembly on the multisite surface of the membrane. A recent proposal has suggested a radically different role in which individual phospholipid molecules, either in the membrane or as single soluble molecules, act by an entirely allosteric mechanism that does not involve the multisite feature of the membrane [Zhai, X., Srivastava, A., Drummond, D. C., Daleke, D., and Lentz, B. R. (2002) Biochemistry 41, 5675-5684]. Our study measured prothrombinse activity in the presence of phospholipids such as short-chain phosphatidylserine and lysophosphatidylserine (lyso-PS). Both enhanced prothrombinase activity, and the increase was consistent with the requirement for extended bilayer structure. Even then, prothrombinase activity was low when compared with activity on bilayer membranes of mixed PS and phosphatidylcholine (PC). Lyso-PS approached the activity of PS/PC membranes only when it was mixed with PC bilayers. The results suggest that the two-dimensional membrane bilayer surface is necessary for the support of full prothrombinase activity.     

Human factor VIII procoagulant activity and phospholipid interaction

The interaction between purified human factor VIII and phospholipid vesicles was investigated. The binding of factor VIII to an equimolecular mixture of phosphatidylserine (PS) and phosphatidylcholine (PC) was studied by sucrose gradient ultracentrifugation (10–40% w/v saccharose in 0.01 M Tris-HCl/0.15 M NaCl buffer (pH 7). In the absence of phospholipids all factor VIII activities (VIII : C, VIII R : WF and VIII R : AG) were found in the zone of highest sucrose density including the factor VIII related protein subunit (200 000 molecular weight). In the presence of an equimolecular mixture of PS/PC VIII R : WF activity, VIII R : AG and a factor VIII related protein still migrated to the bottom of the tube, while VIII : C activity remained at the top where phospholipids were found. Thus a dissociation phenomenon between VIII : C and the other factor VIII relateda activities was apparent in the presence of phospholipids. These results also demonstrate the binding of factor VIII : C to certain active phospholipids.     

Membrane properties induced by anionic phospholipids and phosphatidylethanolamine are critical for the membrane binding and catalytic activity of human cytochrome P450 3A4

Human cytochrome P450 (CYP) 3A4, a membrane anchoring protein, is the major CYP enzyme present in both liver and small intestine. The enzyme plays a major role in the metabolism of many drugs and procarcinogens. The roles of individual phospholipids and membrane properties in the catalytic activity, membrane binding, and insertion into the membrane of CYP3A4 are poorly understood. Here we report that the catalytic activity of testosterone 6beta-hydroxylation, membrane binding, and membrane insertion of CYP3A4 increase as a function of anionic phospholipid concentration in the order phosphatidic acid (PA) > phosphatidylserine (PS) in a binary system of phosphatidylcholine (PC)/anionic phospholipid and as a function of phosphatidylethanolamine (PE) content in ternary systems of PC/PE/PA or PC/PE/PS having a fixed concentration of anionic phospholipids. These results suggest that PA and PE might help the binding of CYP3A4 to the membrane and the interaction with NPR. Cytochrome b(5) (b(5)) and apolipoprotein b(5) further enhanced the testosterone 6beta-hydroxylation activities of CYP3A4 in all tested phospholipids vesicles with various compositions. Phospholipid-dependent changes of the CYP3A4 conformation were also revealed by altered Trp fluorescence and CD spectra. We also found that PE induced the formation of anionic phospholipid-enriched domains in ternary systems using extrinsic fluorescent probes incorporated into lipid bilayers. Taken together, it can be suggested that the chemical and physical properties of membranes induced by anionic phospholipids and PE are critical for the membrane binding and catalytic activity of CYP3A4.     

New reagents for phosphatidylserine recognition and detection of apoptosis

The phospholipid bilayer surrounding animal cells is made up of four principle phospholipid components, phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS), and sphingomyelin (SM). These four phospholipids are distributed between the two monolayers of the membrane in an asymmetrical fashion, with PC and SM largely populating the extracellular leaflet and PE and PS restricted primarily to the inner leaflet. Breakdown in this transmembrane phospholipid asymmetry is a hallmark of the early to middle stages of apoptosis. The consequent appearance of PS on the extracellular membrane leaflet is commonly monitored using dye-labeled Annexin V, a 36 kDa, Ca2+-dependent PS binding protein. Substitutes for Annexin V are described, including small molecules, nanoparticles, cationic liposomes, and other proteins that can recognize PS in a membrane surface. Particular attention is given to the use of these reagents for detecting apoptosis.     

Ovalbumin-induced fusion of acidic phospholipid vesicles at low pH

The interactions of ovalbumin (OA) with large unilamellar vesicles (LUV) of phosphatidylserine (PS) and PS/phosphatidylethanolamine (PE) were studied. It was observed that OA induces aggregation, destabilization, and fusion of these LUV composed of acidic phospholipids at low pH levels. The fusion of LUV by OA was monitored by measuring the intermixing of internal aqueous contents of vesicles, by resonance energy transfer assay which follows the mixing of the membrane components, and by thin-sectioning electron microscopy. The pH profile of fusion was found to be similar to the pH-dependent binding of OA to the same phospholipid vesicles. Proteolytic digestion and hydrophobic labeling with dansyl chloride and photoreactive phosphatidylcholine (PC) of the OA-vesicle complex showed that a segment of OA with a molecular weight of approximately 2,500 penetrates the bilayer. The amino acid composition of this segment indicated that it is the 291-322 fragment and not the putative signal sequence.     

Interfacial binding of bee venom secreted phospholipase A2 to membranes occurs predominantly by a nonelectrostatic mechanism

The secreted phospholipase A(2) from bee venom (bvPLA(2)) contains a membrane binding surface composed mainly of hydrophobic residues and two basic residues that come in close contact with the membrane. Previous studies have shown that the mutant in which these two basic residues (K14 and R23) as well as three other nearby basic residues were collectively changed to glutamate (charge reversal), like wild-type enzyme, binds with high affinity to anionic phospholipid vesicles. In the present study, we have measured the equilibrium constants for the interaction of wild-type bvPLA(2), the charge-reversal mutant (bvPLA(2)-E5), and the mutant in which the five basic residues were changed to neutral glutamine (bvPLA(2)-Q5) with phosphatidylcholine (PC) vesicles containing various amounts of the anionic phosphatidylserine (PS). Remarkably, bvPLA(2)-E5 with an anionic membrane binding surface binds more tightly to vesicles as the mole percent of PS is increased. Computational studies predict that this is due to a significant upward shift in the pK(a) of E14 (and to some extent E23) when the enzyme binds to PC/PS vesicles such that the carboxylate of the glutamate side chain near the membrane surface undergoes protonation. The experimental pH dependence of vesicle binding supports this prediction. bvPLA(2)-E5 binds more weakly to PS/PC vesicles than does wild-type enzyme due to electrostatic protein-vesicle repulsion coupled with the similar energetics of desolvation of basic residues and glutamates that accompanies enzyme-vesicle contact. Studies with bvPLA(2)-Q5 show that only a small fraction of the total bvPLA(2) interfacial binding energy ( approximately 10%) is due to electrostatics.     

Effect of exogenous N-stearoylethanolamine on fatty acid composition of individual phospholipids in the isolated rat heart under postischemic reperfusion

Changes of the individual phospholipid fatty acid composition under the normothermal short-time ischemia with following reperfusion were investigated. Modification of the phospholipid fatty acid (FA) composition under ischemia-reperfusion didn't bear total character and was more manifested in cardiolipin (CL) and phosphatidylethanolamine (PE). The decrease of short chain FA in these phospholipids (more than by 50%) was observed. The amount of unsaturated FA included in CL increased and whole the saturated ones decreased. This caused the rise of the unsaturation index. The selective type of the changes suggested that they had an adaptive character. The addition of the N-stearoilethanolamine (NSE) into the perfusion solution caused a normalization of saturated and unsaturated FA relative amount, as well as of omega-3 and omega-9 FA level in CL. The modification of the FA composition of phosphatidylcholine (PC), phosphatidylserine (PS) and phosphatidylinositol (PI) was also found. The quantity of arachidonic acid in PC increased by 26% and the amount of stearinic acid enhanced in PS. The labeled N-([1-(14)C]-palmitoil)-ethanolamine was found in different lipid classes of the rat organs immediately 5 min following intraperitoneal injection. Approximately 1/3 of all incorporated label accumulated in the phospholipid fraction, and more than 50% of the labels were found in CL.     

The tricalbin C2 domains: lipid-binding properties of a novel, synaptotagmin-like yeast protein family

The tricalbins are a recently discovered family of Saccharomyces cerevisae proteins containing a predicted N-terminal transmembrane domain and at least three C2 domains. They are thought to be yeast homologues of synaptotagmin, a hypothesis supported by structural similarities and past studies that implicated tricalbins in processes of membrane trafficking and sorting. We expressed and purified constructs consisting of single tricalbin C2 domains, and assayed their ability to bind lipids in response to calcium. Protein-lipid overlay assays indicated that the C-terminal C2 domains (C2C) of tricalbins 1 and 3 bind numerous species of acidic phospholipid, including phosphatidylserine and several phosphoinositides, and the amount of protein bound was greatly enhanced in the presence of 1 mM calcium. Sedimentation assays using mixed phosphatidylserine/phosphatidylcholine (PS/PC) vesicles confirmed that the C2C domains of tricalbin 1 and 3 bind membranes in a calcium-responsive manner and showed that they are more sensitive to calcium than the C2A domain of synaptotagmin I. Both assays revealed that all of the C2 domains of tricalbin 2 are insensitive to calcium. Fluorimetric assays exploiting the position of naturally occurring tryptophans in tricalbin 1 C2C and tricalbin 3 C2C confirmed that these domains are capable of binding calcium and that this is coupled to the binding of acidic phospholipid. Combining this with past protein-protein interaction data, we theorize that the calcium-insensitive tricalbin 2 mediates the creation of hetero-oligomeric tricalbin complexes in which tricalbin 1 or 3 or both supply a calcium-dependent membrane binding activity.     

Binding of phosphoinositide-specific phospholipase C-zeta (PLC-zeta) to phospholipid membranes: potential role of an unstructured cluster of basic residues

Phospholipase C-zeta (PLC-zeta) is a sperm-specific enzyme that initiates the Ca2+ oscillations in mammalian eggs that activate embryo development. It shares considerable sequence homology with PLC-delta1, but lacks the PH domain that anchors PLC-delta1 to phosphatidylinositol 4,5-bisphosphate, PIP2. Thus it is unclear how PLC-zeta interacts with membranes. The linker region between the X and Y catalytic domains of PLC-zeta, however, contains a cluster of basic residues not present in PLC-delta1. Application of electrostatic theory to a homology model of PLC-zeta suggests this basic cluster could interact with acidic lipids. We measured the binding of catalytically competent mouse PLC-zeta to phospholipid vesicles: for 2:1 phosphatidylcholine/phosphatidylserine (PC/PS) vesicles, the molar partition coefficient, K, is too weak to be of physiological significance. Incorporating 1% PIP2 into the 2:1 PC/PS vesicles increases K about 10-fold, to 5x10(3) M-1, a biologically relevant value. Expressed fragments corresponding to the PLC-zeta X-Y linker region also bind with higher affinity to polyvalent than monovalent phosphoinositides on nitrocellulose filters. A peptide corresponding to the basic cluster (charge=+7) within the linker region, PLC-zeta-(374-385), binds to PC/PS vesicles with higher affinity than PLC-zeta, but its binding is less sensitive to incorporating PIP2. The acidic residues flanking this basic cluster in PLC-zeta may account for both these phenomena. FRET experiments suggest the basic cluster could not only anchor the protein to the membrane, but also enhance the local concentration of PIP2 adjacent to the catalytic domain.     

Association of protein kinase C with phospholipid vesicles

The Ca2+- and phospholipid-dependent protein kinase, protein kinase C (PKC), was purified from bovine brain by a modified procedure that provided sufficient quantities of stable protein for analysis of physical properties of protein-membrane binding. The binding of PKC to phospholipid vesicles of various compositions was investigated by light-scattering and fluorescence energy transfer measurements. The binding properties for membranes of low phosphatidylserine (PS) content were consistent with a peripheral membrane association; PKC showed Ca2+ -dependent binding to phospholipid vesicles containing phosphatidylserine, phosphatidylinositol, or phosphatidylglycerol. Membranes containing 0-20% PS (the remainder of the phospholipid was phosphatidylcholine) bound less protein than membranes containing greater than 20% PS; the factor limiting protein binding to membranes containing low PS appeared to be the availability of acidic phospholipids. Increasing the PS content above 20% did not increase the amount of membrane-bound protein at saturation, and the limiting factor was probably steric packing of protein on the membrane surface. The membranes bound about 1 g of protein/g of phospholipid at steric saturation. Binding was of relatively high affinity (Kd less than 5 nM), and the association rate was rapid on the time scale of the experiments. Addition of ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid to phospholipid-bound PKC caused dissociation of the complex, and the properties of this dissociation indicated an equilibrium binding of protein to membrane. However, only partial dissociation of PKC was achieved when the PS content of the vesicles exceeded 20%. A number of comparisons revealed that binding of protein to the membrane, even in the presence of phorbol esters, was insufficient for development of enzyme activity.(ABSTRACT TRUNCATED AT 250 WORDS)     

Mechanism of protein-induced membrane fusion: fusion of phospholipid vesicles by clathrin associated with its membrane binding and conformational change

The clathrin-induced fusion of liposome membranes, the membrane binding of clathrin, and the conformational states of clathrin were investigated over a wide pH range using large unilamellar and multilamellar vesicles composed of phosphatidylserine (PS), phosphatidylcholine (PC), PS/PC (2:1), PS/PC (1:1), or PS/PC (1:2). The pH profiles of clathrin-induced fusion of all types of liposomes containing PS showed biphasic patterns. Their pH thresholds were found in the pH range of 5-6 and shifted to lower pH values with decrease in the PS content. Similar shifts were observed in the pH range of 5-6 and shifted to lower pH values with decrease in the PS content. Similar shifts were observed in the pH profiles of clathrin binding to these vesicles, but the pH profiles of binding were different from the biphasic fusion patterns. With PC vesicles, only small degrees of fusion and clathrin binding were observed at pH 2-4. The pH dependences of the conformation and hydrophobicity of clathrin were determined by measuring the extent of the blue shift of the fluorescence maximum of 1-anilinonaphthalene-8-sulfonate in the presence of the protein, the fluorescence intensity of N-(1-anilinonaphthyl-4)maleimide bound to the clathrin molecule, the resonance energy transfer from its tryptophan to anilinonaphthyl residues, the partitioning of the protein in Triton X-114 solution, and the hydrophobicity index of clathrin using cis-parinaric acid. These measurements indicated that conformational change and exposure of hydrophobic regions occur below pH 6 and suggested that clathrin may adopt different conformational states in the pH region where it induced membrane fusion.(ABSTRACT TRUNCATED AT 250 WORDS)     

High cooperativity, specificity, and multiplicity in the protein kinase C-lipid interaction

The number of phosphatidylserine molecules involved in activating protein kinase C was determined in a mixed micelle system where one monomer of protein kinase C binds to one detergent:lipid micelle of fixed composition. Unusually high cooperativity, specificity, and multiplicity in the protein kinase C-phospholipid interaction are demonstrated by examining the lipid dependence of enzymatic activity. The rates of autophosphorylation and substrate (histone) phosphorylation are specifically regulated by the phosphatidylserine content of the micelles. Hill coefficients of 8-11 were calculated for phosphatidylserine-dependent stimulation of enzyme activity, with a maximum occurring in micelles containing greater than or equal to 12 phosphatidylserine molecules. The high specificity that exists is illustrated by the fact that phosphatidylethanolamine and phosphatidylglycerol, but not phosphatidylcholine or phosphatidic acid, can replace only some of the phosphatidylserine molecules. We propose that Ca2+ and acidic phospholipids cause the protein to undergo a conformation change revealing multiple phosphatidylserine binding sites and resulting in the highly cooperative and specific interaction of protein kinase C with phosphatidylserine. Consistent with this, the proteolytic sensitivity of protein kinase C increases approximately 10-fold in the presence of phosphatidylserine and Ca2+ compared to Ca2+ alone. The high degree of cooperativity and specificity may provide a sensitive method for the physiological regulation of protein kinase C by phospholipid.     

Quantitative fluorescence analysis of the adsorption of lysozyme to phospholipid vesicles

Experiments directed to measure the interaction of lysozyme with liposomes consisting of phosphatidylcholine (PC) and phosphatidylserine (PS) have been conducted by monitoring both protein and lipid fluorescence and fluorescence anisotropy of the protein. The binding of lysozyme to the unilamellar vesicles was quantified using a novel method of analysis in which the fractional contribution at moderate binding conditions is determined from either total fluorescence decay or anisotropy decay curves of tryptophan at limiting binding conditions. In the energy transfer experiments PC and PS lipids labelled with two pyrene acyl chains served as energy acceptors of the excited tryptophan residues in lysozyme. The binding was strongly dependent on the molar fraction of negatively charged PS in neutral PC membranes and on the ionic strength. Changes in the tryptophan fluorescence decay characteristics were found to be connected with long correlation times, indicating conformational rearrangements induced by binding of the protein to these lipid membranes. The dynamics of membrane bound protein appeared to be dependent on the physical state of the membrane. Independent of protein fluorescence studies, formation of a protein-membrane complex can also be observed from the lipid properties of the system. The interaction of lysozyme with di-pyrenyl-labelled phosphatidylserine in anionic PS/PC membranes resulted in a substantial decrease of the intramolecular excimer formation, while the excimer formation of dipyrenyl-labelled phosphatidylcholine in neutral PC membranes barely changed in the presence of lysozyme.Abbreviations dipyr₄ sn-1,2-(pyrenylbutyl) - dipyr₁₀ sn-1,2-(pyrenyldecanoyl). - DMPC dimyristoyl-phosphatidylcholine - DOPC dioleoyl-phosphatidylcholine - DPPC dipalmitoyl-phosphatidylcholine - DPPC dipalmitoylphosphatidylcholine - PC phosphatidylcholine - PS phosphatidylserine Correspondence to: A. J. W. G. Visser     

31P MRS analysis of the phospholipid composition of normal human peripheral blood mononuclear cells (PBMC)

The aim of this investigation was to characterize the phospholipid composition of normal human blood mononuclear cells using 31P NMR spectroscopy. Mononuclear cells of peripheral blood were obtained from 10 volunteers. Phospholipid extracts were prepared from 60x10(6) cells according to modified Folch's method. An AMX 300 Bruker spectrometer 7.05 T was used. The 31P spectrum of phospholipid extracts from normal human PBMC consisted of 9 peaks, with one each for phosphatidylcholine (PC), plasmalogen of phosphatidylcholine (CPLAS), lysophosphatidylcholine (LPC), sphingomyelin (SM), phosphatidylethanolamine (PE), phosphatidylinositol (PI), phosphatidylserine (PS) and cardiolipin (CL), and another one due to the external reference substance, methylenediphosphonic acid (MDPA). The concentrations of these phospholipids (PL), based on the integral intensities, were as follows: 0.398 +/- 0.078 mmole/l for PC; 0.033 +/- 0.019 mmole/l for CPLAS; 0.155 +/- 0.043 mmole/l for SM; 0.266 +/- 0.104 mmole/l for PI+PE; 0.101 +/- 0.040 mmole/l for PS, and 0.026 +/- 0.033 mmole/l for CL. The results of this study confirmed that 31P MRS is a convenient tool for measuring the phospholipid concentrations of biological samples.     

Membrane fusion activity of influenza virus. Effects of gangliosides and negatively charged phospholipids in target liposomes

Fusion of influenza virus with liposomes composed of negatively charged phospholipids differs from fusion with biological membranes or zwitterionic liposomes with ganglioside receptors [Stegmann, T., Hoekstra, D., Scherphof, G., & Wilschut, J. (1986) J. Biol. Chem. 261, 10966-10969]. In this study, we investigated how the kinetics and extent of fusion of influenza virus, monitored with a fluorescence resonance energy-transfer assay, are influenced by the surface charge and the presence of receptors on liposomal membranes. The results were analyzed in terms of mass action kinetic model, providing separate rate constants for the initial virus-liposome adhesion, or aggregation, and for the actual fusion reaction. Incorporation of increasing amounts of cardiolipin (CL) or phosphatidylserine (PS) into otherwise zwitterionic phosphatidylcholine (PC)/phosphatidylethanolamine (PE) vesicles results in a gradual shift of the pH threshold of fusion to neutral, relative to the pH threshold obtained with PC/PE vesicles containing the ganglioside GD1a, while also the rate of fusion increases. This indicates the emergence of a fusion mechanism not involving the well-documented conformational change in the viral hemagglutinin (HA). However, only with pure CL liposomes this nonphysiological fusion reaction dominates the overall fusion process; with pure PS or with zwitterionic vesicles containing CL or PS, the contribution of the nonphysiological fusion reaction is small. Accordingly, preincubation of the virus alone at low pH results in a rapid inactivation of the viral fusion capacity toward all liposome compositions studied, except pure CL liposomes. The results of the kinetic analyses show that with pure CL liposomes the rates of both virus-liposome adhesion and fusion are considerably higher than with all other liposome compositions studied.(ABSTRACT TRUNCATED AT 250 WORDS)     

Low pH induced membrane fusion of lipid vesicles containing proton-sensitive polymer

For the purpose of cytoplasmic delivery of aqueous content in liposomes through endosomes, we synthesized a pH-sensitive polymer, cetylacetyl(imidazol-4-ylmethyl)polyethylenimine (CAIPEI), which generates polycations at acidic pH. CAIPEI in its aqueous phase caused aggregation of sonicated vesicles composed of phosphatidylserine (PS) and phosphatidylcholine (PC) (molar ratio 1:4) when the pH of the solution was lowered. The polymer also induced membrane intermixing as measured by resonance energy transfer between vesicles containing N-(7-nitro-2,1,3-benz[d]oxadiazol-4-yl)phosphatidylethanolamine and those containing N-Rhodamine phosphatidylethanolamine at pH 4-5, while the addition of CAIPEI caused neither aggregation of PC vesicles nor the intermixing of liposomal membranes between PC and PC/PS vesicles at any pH. The CAIPEI-induced membrane intermixing was dependent on the polymer/vesicle ratio rather than on the polymer concentration. Then the polymer was incorporated into the bilayers of PC vesicles. These CAIPEI vesicles also caused membrane intermixing with liposomes containing PS under acidic conditions. The reconstituted CAIPEI did not reduce the trapping efficiency of vesicles or increase their permeability to glucose even at low pH. The vesicles caused the low pH induced aggregation and membrane intermixing with other negatively charged liposomes containing phosphatidic acid or phosphatidylglycerol. These results suggest that the protonation of the polymer at acidic pH endows the CAIPEI vesicles with the activity to fuse with negatively charged liposomes.     

Phospholipid and phospholipase changes by jasmonic acid during stolon to tuber transition of potato

Potato tuber formation starts with the stolon swelling and is regulated by jasmonates. The cascade of events leading to tuber formation is not completely understood. The aim of this study was to evaluate phospholipid composition and phospholipase activities during four stages of stolon-to-tuber transition of Solanum tuberosum L., cv. Spunta, and involvement of phosphatidic acid (PA) in stolon cell expansion during early stages. Effects of jasmonic acid (JA) treatment on phospholipid content and activation of phospholipase D (PLD) (EC 3.1.4.4) and phosphatidylinositol-4,5-bisphosphate-specific phospholipase C (PIP₂-PLC) (EC 3.1.4.3) were studied in the early stages (first stage, hooked apex stolon; second stage, initial swelling stolon) of tuberization. All the phospholipid species identified, phosphatidylinositol (PI), phosphatidylserine (PS), phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylglycerol (PG), PA, and cardiolipin (CL), decreased as tuber formation progressed. PLD and PLC were activated in control tissues at an early stage. JA treatment caused a decrease of PC and PS in first stage stolons, accumulation of PA in second stage stolons, and modification of PLD and PLC activities. PA increased stolon cell area in the first and second stages. These findings indicate that phospholipid catabolism is activated from the early stages of tuber formation, and that JA treatment modifies the pattern of phospholipid (PC, PS, and PA) composition and phospholipase (PLD and PLC) activity. These phospholipids therefore may play a role in activation of an intracellular mechanism that switches the developmental fate of stolon meristem cells, causing differentiation into a tuber.     

Investigation of interaction of human platelet membrane components with anticoagulant drugs Abciximab and Eptifibatide

Abciximab (Abci) and eptifibatide (Epti) are antiaggregate drugs which may reduce thrombotic complications in acute coronary syndromes. The aim of this work was the investigation of the interaction between the phospholipid-GPIIb/IIIa glycoprotein complex and Abci or Epti, and the influence of these drugs on the phospholipid ratio in the platelet membrane. The interaction between the phospholipid-GPIIb/IIIa glycoprotein complex and antiaggregate drugs were investigated using the Surface Plasmon Resonance Imaging technique (SPRI). Phospholipids phosphatidylinositol (PI), phosphatidylserine (PS), phosphatidylethanolamine (PE), phosphatidylcholine (PC) and sphingomyelin (SM) were first immobilized onto the gold chip surface. The phospholipid ratio in the platelet membrane was determined by the HPLC. Only PI, PS, PE and PC were determined. Human platelets treated 'in vitro' with Abci or Epti exhibit changes in the phospholipid ratio in the platelet membrane. The ratio of PS decreases and PC rises. The SPRI distinctly shows interactions between phospholipids and glycoprotein GPIIb/IIIa, and between the phospholipid-glycoprotein GPIIb/IIIa complex and Abci or Epti. The interaction between phospholipids and glycoprotein GPIIb/IIIa is growing in the sequence: PI相似文献