Influence of enzymatic phospholipid cleavage on the permeability of the erythrocyte membrane. I. Transport of non-electrolytes via the lipid domain

In order to further elucidate the influence of membrane lipids on transport via the lipid domain of the erythrocyte membrane, simple non-electrolyte diffusion was investigated by tracer flux measurements in whole cells after cleavage of up to 65% of phosphatidylcholine or sphingomyelin by phospholipase A₂ from Naja naja, or by sphingomyelinase.A new type of labelled model non-electrolyte was used in this study, readily available by reacting a non-labelled thiol with a labelled alkylating SH-reagent.In spite of the marked enzymatic alterations of the membrane, which lead to the occurrence of large quantities of lysophosphatidylcholine and long chain fatty acids, or of ceramide, the permeability of the lipid domain remained unaffected.This finding is very surprising, since the physical properties of the lipid phase (microviscosity, structure of the membrane interface) are likely to be perturbed in the enzyme-treated membranes.Sphingomyelinase-treated cells undergo stomatocytic shape changes followed by deep invaginations of the membrane and finally endocytosis, while phospholipase A₂-treated cells essentially maintain their normal shape.     

Evidence for a GTP-binding protein coupling thrombin receptor to PIP2-phospholipase C in membranes of hamster fibroblasts

Two different methods were used to study directly alpha-thrombin modulation of polyphosphoinositide breakdown in membranes prepared from Chinese hamster lung (CHL) fibroblasts. In the first one we labelled the lipid pool by incubating the intact cells with myo-[3H]inositol prior to membrane isolation; in the other we used exogenous [3H]PIP2 with phosphatidylethanolamine (1:10) added as liposomes to freshly isolated membranes. A Ca2+-dependent PIP2 and PIP phospholipase C activity was characterized by measuring the rate of formation of inositol tris- and bisphosphate. Basal phospholipase C activity was stimulated up to 3-fold by GTP or GTP-gamma-S. Of the two mitogens, alpha-thrombin and EGF, known to stimulate DNA synthesis in Chinese hamster fibroblasts, only alpha-thrombin is a potent activator of PIP2 breakdown in intact cells. Consistent with this observation, alpha-thrombin but not EGF potentiated GTP-gamma-S-dependent phospholipase C activity in membrane preparations. These results strongly support the hypothesis that a GTP-binding protein couples alpha-thrombin receptor to PIP2 hydrolysis. Because both methods used to assay phospholipase C gave identical results, we conclude that the coupling is at the level of PIP2-phosphodiesterase activity.     

Glucose transport into human erythrocytes treated with phospholipase A2 or C

Phospholipase A2 induced crenation of human erythrocytes and decreased glucose transport activity (influx rate) by 40% when 51% of phosphatidylcholine (PC) in the membrane was hydrolyzed. On the other hand, phospholipase C induced invagination of the cells and negligibly affected the glucose transport in the case of 21% hydrolysis of the PC. By altering the pH of the medium for suspending cells treated with phospholipase A2 from 7.4 to 6.0, cell shape was changed from clear crenation to slight invagination, but glucose transport activity was not affected. Cells that were treated with phospholipase A2 and then washed with albumin to remove free fatty acids produced in the cell membrane showed an almost normal cell shape and slightly higher glucose transport activity than did untreated cells. The ratios of beta-D-glucose transport rate to alpha-D-glucose transport rate in untreated cells, cells treated with phospholipase A2 and cells treated with phospholipase C were 1.13, 1.04, and 1.20, respectively. These results demonstrate that the drastic morphological change (invagination or crenation) induced by the treatment with phospholipases bears no clear relationship to the activity of glucose transport and suggest that the increase in the volume of the outer half of the lipid bilayer might reduce the rate of glucose transport across the human erythrocyte membrane and change the anomeric preference of glucose transport.     

Role of phosphatidylinositol 4,5-bisphosphate in the activation of cytosolic phospholipase A2-alpha

Cytosolic phospholipase A(2)-alpha (cPLA(2)) plays an important role in the release of arachidonic acid and in cell injury. Activation of cPLA(2) is dependent on a rise in cytosolic Ca(2+) concentration, membrane association via the Ca(2+)-dependent lipid binding (CaLB) domain, and phosphorylation. This study addresses the activation of cPLA(2) via potential association with membrane phosphatidylinositol 4,5-bisphosphate (PIP(2)), including the role of a "pleckstrin homology (PH)-like" region of cPLA(2) (amino acids 263-354). In cells incubated with complement, phorbol myristate acetate+the Ca(2+) ionophore, A23187, or epidermal growth factor+A23187, expression of the PH domain of phospholipase C-delta1 (which sequesters membrane PIP(2)) attenuated cPLA(2) activity. Stimulated cPLA(2) activity was also attenuated by the expression of cPLA(2) 135-366, or cPLA(2) 2-366, and expression of a PIP(2)-specific 5'-phosphatase. However, in a yeast-based assay that tests the ability of proteins to bind to membrane lipids, including PIP(2), with high affinity, only cPLA(2) 1-200 (CaLB domain) was able to interact with membrane lipids, whereas cPLA(2)s 135-366, 2-366, 201-648, and 1-648 were unable to do so. Therefore, cPLA(2) activity can be modulated by sequestration or depletion of cellular PIP(2), although the interaction of cPLA(2) with membrane PIP(2) appears to be indirect, or of weak affinity.     

Bacterial cytotoxins, amphotericin B and local anesthetics enhance transbilayer mobility of phospholipids in erythrocyte membranes. Consequences for phospholipid asymmetry

Incorporation of the channel-forming polyene antibiotic amphotericin B and of cytotoxins from Staphylococcus aureus (alpha-toxin) or Pseudomonas aeruginosa into erythrocyte membranes results in a concentration-dependent enhancement of the flip rates of exogenous lysophosphatidylcholine. The flip rate is also enhanced by incorporation of tetracaine and dibucaine. Removal of tetracaine and amphotericin B from the cells normalizes the flip rates. In parallel to the enhancement of flip rates, alpha-toxin produces a loss of transmembrane asymmetry of both phosphatidylethanolamine and phosphatidylserine. Pretreatment of cells with amphotericin or high concentrations (over 2.5 mmol . l-1) of tetracaine, followed by removal of the perturbing agent by washing, produces a selective loss of the asymmetric orientation of phosphatidylethanolamine to the inner membrane layer, as evaluated by the accessibility of the lipid towards cleavage by phospholipase A2. The extent to which asymmetry is lost depends on the time of pretreatment with amphotericin or tetracaine, indicating a limitation by the rate of reorientation of phosphatidylethanolamine to the outer membrane surface. Evaluation of the accessibility of phosphatidylethanolamine towards cleavage by phospholipase A2 in the presence of local anesthetics indicates accessible fractions much higher than those obtained after removal of the perturbant. In the presence of tetracaine, endofacial phosphatidylethanolamine seems somehow to become accessible to phospholipase A2. Phosphatidylserine does not exhibit this peculiarity. The results indicate that various types of perturbation of the lipid domain of the erythrocyte membrane may enhance the transbilayer mobility of phospholipids as well as destabilize the asymmetric distribution of aminophospholipids. However, as in other instances reported previously (Haest, C.W.M., Erusalimsky, J., Dressler, V., Kunze, I. and Deuticke B. (1983) Biomed. Biochim. Acta 42, 17-21), there is no tight coupling between transbilayer mobility and destabilization of asymmetry of the transbilayer distribution of phospholipids.     

The molecular basis of differential subcellular localization of C2 domains of protein kinase C-alpha and group IVa cytosolic phospholipase A2

The C2 domain is a Ca(2+)-dependent membrane-targeting module found in many cellular proteins involved in signal transduction or membrane trafficking. C2 domains are unique among membrane targeting domains in that they show a wide range of lipid selectivity for the major components of cell membranes, including phosphatidylserine and phosphatidylcholine. To understand how C2 domains show diverse lipid selectivity and how this functional diversity affects their subcellular targeting behaviors, we measured the binding of the C2 domains of group IVa cytosolic phospholipase A(2) (cPLA(2)) and protein kinase C-alpha (PKC-alpha) to vesicles that model cell membranes they are targeted to, and we monitored their subcellular targeting in living cells. The surface plasmon resonance analysis indicates that the PKC-alpha C2 domain strongly prefers the cytoplasmic plasma membrane mimic to the nuclear membrane mimic due to high phosphatidylserine content in the former and that Asn(189) plays a key role in this specificity. In contrast, the cPLA(2) C2 domain has specificity for the nuclear membrane mimic over the cytoplasmic plasma membrane mimic due to high phosphatidylcholine content in the former and aromatic and hydrophobic residues in the calcium binding loops of the cPLA(2) C2 domain are important for its lipid specificity. The subcellular localization of enhanced green fluorescent protein-tagged C2 domains and mutants transfected into HEK293 cells showed that the subcellular localization of the C2 domains is consistent with their lipid specificity and could be tailored by altering their in vitro lipid specificity. The relative cell membrane translocation rate of selected C2 domains was also consistent with their relative affinity for model membranes. Together, these results suggest that biophysical principles that govern the in vitro membrane binding of C2 domains can account for most of their subcellular targeting properties.     

Control of baculovirus gp64-induced syncytium formation by membrane lipid composition.

We have investigated the effects of membrane lipid composition on biological membrane fusion triggered by low pH and mediated by the baculovirus envelope glycoprotein gp64. Lysolipids, either added exogenously or produced in situ by phospholipase A2 treatment of cell membranes, reversibly inhibited syncytium formation. Lysolipids also decreased the baculovirus infection rate. In contrast, oleic and arachidonic acids and monoolein promoted cell-cell fusion. Membrane lipid composition affected pH-independent processes which followed the low-pH-induced change in fusion protein conformation. Inhibition and promotion of membrane fusion by a number of lipids could not be explained by mere binding or incorporation into membranes, but rather was correlated with the effective molecular shape of exogenous lipids. Our data are consistent with the hypothesis that membrane fusion proceeds through highly bent membrane intermediates (stalks) having a net negative curvature. Consequently, inverted cone-shaped lysolipids inhibit and cone-shaped cis-unsaturated fatty acids promote stalk formation and, ultimately, membrane fusion.     

Investigation into the role of phosphatidylserine in modifying the susceptibility of human lymphocytes to secretory phospholipase A(2) using cells deficient in the expression of scramblase

Normal human lymphocytes resisted the hydrolytic action of secretory phospholipase A(2) but became susceptible to the enzyme following treatment with a calcium ionophore, ionomycin. To test the hypothesis that this susceptibility requires exposure of the anionic lipid phosphatidylserine on the external face of the cell membrane, experiments were repeated with a human Burkitt's lymphoma cell line (Raji cells). In contrast to normal lymphocytes or S49 mouse lymphoma cells, most of the Raji cells (83%) did not translocate phosphatidylserine to the cell surface upon treatment with ionomycin. Those few that did display exposed phosphatidylserine were hydrolyzed immediately upon addition of phospholipase A(2). Interestingly, the remaining cells were also completely susceptible to the enzyme but were hydrolyzed at a slower rate and after a latency of about 100s. In contradistinction to the defect in phosphatidylserine translocation, Raji cells did display other physical membrane changes upon ionomycin treatment that may be relevant to hydrolysis by phospholipase A(2). These changes were detected by merocyanine 540 and trimethylammonium diphenylhexatriene fluorescence and were common among normal lymphocytes, S49 cells, and Raji cells. The levels of these latter effects corresponded well with the relative rates of hydrolysis among the three cell lines. These results suggested that while phosphatidylserine enhances the rate of cell membrane hydrolysis by secretory phospholipase A(2), it is not an absolute requirement. Other physical properties such as membrane order contribute to the level of membrane susceptibility to the enzyme independent of phosphatidylserine.     

Membrane orientation and position of the C2 domain from cPLA2 by site-directed spin labeling

The C2 domain is a ubiquitous Ca(2+)-binding motif that triggers the membrane docking of many key signaling proteins during intracellular Ca(2+) signals. Site-directed spin labeling was carried out on the C2 domain of cytosolic phospholipase A(2) in order to determine the depth of penetration and orientation of the domain at the membrane interface. Membrane depth parameters, Phi, were obtained by EPR spectroscopy for a series of selectively spin-labeled C2 domain cysteine mutants, and for spin-labeled lipids and spin-labeled bacteriorhodopsin cysteine mutants. Values of Phi were combined with several other constraints, including the solution NMR structure, to generate a model for the position of the C2 domain at the membrane interface. This modeling yielded an empirical expression for Phi, which for the first time defines its behavior from the bulk aqueous phase to the center of the lipid bilayer. In this model, the backbones of both the first and third Ca(2+)-binding loops are inserted approximately 10 A into the bilayer, with residues inserted as deep as 15 A. The backbone of the second Ca(2+)-binding loop is positioned near the lipid phosphate, and the two beta-sheets of the C2 domain are oriented so that the individual strands make angles of 30-45 degrees with respect to the bilayer surface. Upon membrane docking, spin labels in the Ca(2+)-binding loops exhibit decreases in local motion, suggesting either changes in tertiary contacts due to protein conformational changes and/or interactions with lipid.     

Membrane docking of the C2 domain from protein kinase Cα as seen by polarized ATR-IR. The role of PIP₂

We have used attenuated total internal reflection infrared spectroscopy (ATR-IR) spectroscopy to study the association of the C2 domain from protein kinase Cα (PKCα) with different phospholipid membranes, so as to characterise the mode of membrane docking and its modulation by the second-messenger lipid PIP?. In parallel, we have also examined the membrane interaction of the C2 domain from cytosolic phospholipase A?. PIP? did not induce significant changes in secondary structure of the membrane-bound PKCα-C2 domain, nor did binding of the PKCα-C2 domain change the dichroic ratios of the lipid chains, whereas the C2 domain from phospholipase A? did perturb the lipid chain orientation. Measurements of the dichroic ratios for the amide I and amide II protein bands were combined so as to distinguish the tilt of the β-sheets from that of the β-strands within the sheet. When associated with POPC/POPS membranes, the β-sandwich of the PKCα-C2 domain is inclined at an angle α=35° to the membrane normal, i.e., is oriented more nearly perpendicular than parallel to the membrane. In the process of membrane docking, the tilt angle increases to α=44° in the presence of PIP?, indicating that the β-sandwich comes closer to the membrane surface, so confirming the importance of this lipid in determining docking of the C2 domain and consequent activation of PKCα.     

Chronic ethanol ingestion modifies liver microsomal phosphatidylserine inducing resistance to hydrolysis by exogenous phospholipase A2.

Chronic ethanol ingestion leads to the acquisition of a tolerance to membrane lipid disordering, a lowered partition coefficient to hydrophobic compounds and a resistance to the hydrolysis of the phospholipids by exogenous phospholipase A2. Anionic phospholipids have been implicated as being responsible for the resistance to lipid disordering and a number of modifications to these phospholipids are known to occur as a result of chronic ethanol-ingestion. In this study the basis of the resistance to phospholipase A2 in hepatic microsomes was investigated. It was found that chronic ethanol-induced modifications to each of the major phospholipid classes was responsible to some extent for the resistance to phospholipase A2, however, PS was particularly potent considering it is a compositionally minor constituent. The effect was interpreted as a reduced ability to activate the phospholipase A2 since PS acts as an essential activator of phospholipase A2 (along with PI). Fatty acid analysis revealed that the chronic ethanol-treatment resulted in a elevated level of docosahexaenoate with a parallel reduction in arachidonate in phosphatidylserine. Lipid packing and organization is important in the regulating the level of exogenous phospholipase A2 activity but the activity was not found to correlate with lipid order of different phosphatidylserine species. It is concluded that subtle differences in the molecular species arrangement or disposition around the enzyme may be responsible for the altered phospholipase A2 interaction with the membrane induced by chronic ethanol-treatment. One implication of this study is that other anionic phospholipid dependent membrane proteins, of which there are many known examples, may also be modified as a result of chronic ethanol-ingestion.     

Rhnull human erythrocytes have an abnormal membrane phospholipid organization.

Rhnull human erythrocytes lack the antigens of the Rhesus blood group system, have an abnormal shape and an increased osmotic fragility, and are associated with mild chronic haemolytic anaemia. Studies with phospholipase A2 and sphingomyelinase C show that the asymmetric distribution of phosphatidylethanolamine (PtdEtn) in the membrane of these cells differs from that found in control cells. The amount of PtdEtn which can be hydrolysed by phospholipase A2 in the presence of sphingomyelinase C in intact Rhnull cells is twice as high as that in normal erythrocytes. In intact Rhnull cells all of the phosphatidylcholine (PtdCho) present in the membrane can be readily exchanged with a PtdCho-specific exchange protein, whereas in control cells 75% is readily exchanged and 25% at a much lower rate. This indicates that PtdCho experiences a relatively fast transbilayer movement in the Rhnull cells. The observation that the loss of two membrane polypeptides in the Rhnull cells leads to abnormal shape, increased osmotic fragility, abnormal PtdEtn distribution and enhanced transbilayer mobility of PtdCho strongly suggests that one or both polypeptides are essential for the maintenance of a proper membrane-membrane skeleton interaction.     

Selective removal of lipids from the outer membrane layer of human erythrocytes without hemolysis. Consequences for bilayer stability and cell shape

(1) Treatment of erythrocytes with phospholipase A₂ from bee venom cleaves about 55% of the phosphatidylcholine in the outer membrane lipid layer without changing the discoid shape of the cells. All of the fatty acids and 80% of the lysophosphatidylcholine produced under this conditions can be sequentially extracted by bovine serum albumin without hemolysis of the cells. (2) The cells remain discoid up to extraction of all of the fatty acids and 15% of the lysophosphatidylcholine. Removal of a higher fraction of lysophosphatidylcholine induces formation of stomatocytes and sphero-stomatocytes, probably going along with an internalization of membrane vesicles. Stomatocytosis can be explained on the basis of the ‘bilayer couple hypothesis’ (Sheetz, M.P. and Singer, S.J. (1974) Proc. Natl. Acad. Sci. 71, 4457–4461). The shape change will compensate for the differences in surface pressure between the two leaflets induced by selective removal of material from the outer leaf of the bilayer. (3) Increasing the shear modulus of the membrane by diamide prevents this compensatory shape change even after extraction of up to 80% of the lysophosphatidylcholine, which amounts to a loss of 34% of the phospholipids of the outer membrane layer or 22% of its area. This leads to the interesting situation of a membrane possibly having a strikingly diminished ratio of the numbers of phospholipid molecules in the outer to that in the inner lipid layer. A marked difference in surface pressures should arise in this situation, unless other compensatory mechanisms become operative. Evidence for a compensation for outer lipid loss by a constriction of the inner layer has been obtained. A compensation by transbilayer reorientation of phospholipids could not be demonstrated. This latter observation supports the concept of a stabilisation of the asymmetric phospholipid arrangement by proteins such as spectrin.     

Effect of membrane sterol content on the susceptibility of phospholipids to phospholipase A2

The effects of membrane sterol level on the susceptibility of LM cell plasma membranes to exogenous phospholipases A2 has been investigated. Isolated plasma membranes, containing normal or decreased sterol content, were prepared from mutant LM cell sterol auxotrophs. beta-Bungarotoxin-catalyzed hydrolysis of both endogenous phospholipids and phospholipids introduced into the membranes with beef liver phospholipid exchange proteins was monitored. In both cases, phosphatidylcholine (PC) and phosphatidylethanolamine (PE) were degraded at similar rates in normal membranes, while PC hydrolysis was specifically accelerated in sterol-depleted membranes. Additional data suggest that this preferential hydrolysis of PC is not a consequence of the phospholipid head group specificity of the phospholipase, nor of a difference in the accessibility of PC versus PE to the enzyme. Analysis of the reaction products formed during treatment of isolated membranes with phospholipase A2 showed almost no accumulation of lysophospholipids. This was found to be due to highly active lysophospholipase(s), present in LM cell plasma membranes, acting on the lysophospholipids formed by phospholipase A2 action. A soluble phospholipase A2 was partially purified from LM cells and found to behave as beta-bungarotoxin with regard to membrane sterol content. These results demonstrate that the nature of phospholipid hydrolysis, catalyzed by phospholipase A2, can be significantly affected by membrane lipid composition.     

Transformation by the k-ras oncogene correlates with increases in phospholipase A2 activity, glycerophosphoinositol production and phosphoinositide synthesis in thyroid cells.

Two cell lines transformed by the k-ras oncogene (KiKi and KiMol cells) and a temperature sensitive clone (Ts), all originated from a normal rat thyroid line (FRTL5 cells), have been employed to analyse the intracellular mechanisms affected by the ras p21. In k-ras transformed cells two phosphoinositide derivatives, glycerophosphoinositol and inositol monophosphate, were markedly increased, whereas inositol bisphosphate and trisphosphate maintained the same level as in normal cells. Cytosolic Ca2+ was also unaffected. This indicates that in epithelial cells the phospholipase C activity is not altered upon ras transformation. The formation of glycerophosphoinositol involved the activation of a phosphoinositide specific phospholipase A2. The higher phospholipase A2 activity in ras transformed cells could be further demonstrated by the increase in total arachidonic acid release. In the Ts clone the increase in glycerophosphoinositol and inositol monophosphate was evident only at the permissive temperature (33 degrees C), whereas it disappeared at 39 degrees C. At 33 degrees C the cells were also characterized by an enriched membrane pool of phosphoinositides. All these changes occurred in parallel with morphological transformation. We propose that cell transformation by the k-ras oncogene affects different steps of the membrane lipid metabolism, among which the most prominent one is the activation of a phosphoinositide specific phospholipase A2. These effects could originate mitogenic metabolites. Moreover, they correlate well with the induction of the malignant phenotype.     

Stimulation-dependent recruitment of cytosolic phospholipase A2-alpha to EA.hy.926 endothelial cell membranes leads to calcium-independent association.

Cytosolic phospholipase A2-alpha (cPLA2-alpha) is a calcium-activated enzyme involved in agonist-induced arachidonic acid release. In endothelial cells, free arachidonic acid is predominantly converted into prostacyclin, a potent vasodilator and inhibitor of platelet activation. As the rate-limiting step in prostacyclin production is the generation of free arachidonic acid by cPLA2-alpha, this enzyme has become an attractive pharmacological target and the focus of many studies. Following stimulation with calcium-mobilizing agonists, cPLA2-alpha translocates to intracellular phospholipid membranes via its C2 domain. In this study, the calcium-induced association of cPLA2-alpha with EA.hy.926 endothelial cell membranes was investigated. Subcellular fractionation and immunofluorescence studies showed that following stimulation with histamine, thrombin or the calcium ionophore A23187, cPLA2-alpha relocated to intracellular membranes. Treatment of A23187-stimulated cells with EGTA or BAPTA-AM demonstrated that a substantial pool of cPLA2-alpha remained associated with membrane fractions in a calcium-independent manner. Furthermore, immunofluorescence microscopy studies revealed that cells stimulated for periods of greater than 10 min showed a high proportion of calcium-independent membrane-associated cPLA2-alpha. Calcium-independent membrane association of cPLA2-alpha was not due to hydrophobic or cytoskeletal interactions. Finally, the recombinant C2 domain of cPLA2-alpha exhibited calcium-independent membrane binding to membranes isolated from A23187-stimulated cells but not those isolated from nonstimulated cells. These findings suggest that novel mechanisms involving accessory proteins at the target membrane play a role in the regulation of cPLA2-alpha. Such regulatory associations could enable the cell to discriminate between the varying levels of cytosolic calcium induced by different stimuli.     

A phospholipid substrate molecule residing in the membrane surface mediates opening of the lid region in group IVA cytosolic phospholipase A2

The Group IVA (GIVA) phospholipase A(2) associates with natural membranes in response to an increase in intracellular Ca(2+) along with increases in certain lipid mediators. This enzyme associates with the membrane surface as well as binding a single phospholipid molecule in the active site for catalysis. Employing deuterium exchange mass spectrometry, we have identified the regions of the protein binding the lipid surface and conformational changes upon a single phospholipid binding in the absence of a lipid surface. Experiments were carried out using natural palmitoyl arachidonyl phosphatidylcholine vesicles with the intact GIVA enzyme as well as the isolated C2 and catalytic domains. Lipid binding produced changes in deuterium exchange in eight different regions of the protein. The regions with decreased exchange included Ca(2+) binding loop one, which has been proposed to penetrate the membrane surface, and a charged patch of residues, which may be important in interacting with the polar head groups of phospholipids. The regions with an increase in exchange are all located either in the hydrophobic core underneath the lid region or near the lid and hinge regions from 403 to 457. Using the GIVA phospholipase A(2) irreversible inhibitor methyl-arachidonyl fluorophosphonate, we were able to isolate structural changes caused only by pseudo-substrate binding. This produced results that were very similar to natural lipid binding in the presence of a lipid interface with the exception of the C2 domain and region 466-470. This implies that most of the changes seen in the catalytic domain are due to a substrate-mediated, not interface-mediated, lid opening, which exposes the active site to water. Finally experiments carried out with inhibitor plus phospholipid vesicles showed decreases at the C2 domain as well as charged residues on the putative membrane binding surface of the catalytic domain revealing the binding sites of the enzyme to the lipid surface.     

Specific and non specific stimulation of prostaglandin release by human skin fibroblasts in culture.--Are changes of membrane fluidity involved?

In order to study a bidirectional relationship between changes of membrane fluidity and prostaglandin synthesis, the arachidonic acid cascade was stimulated in cultured human skin fibroblasts by unspecific stimuli (hypotonicity, low calcium concentrations) and by the specific stimulus, bradykinin. Fluorescence anisotropy of trimethylammoniumdiphenylhexatriene was used to measure membrane fluidity in cell monolayers. Hypotonicity or low calcium concentrations induce membrane fluidisation and prostaglandin synthesis. However, after specific stimulation of prostaglandins with bradykinin (at normocalcic and isotonic conditions) a rigidification of plasma membranes was observed in living cells. Fluidisation of membranes and bradykinin activate phospholipase A2 and induce prostaglandin synthesis. Although in cell membrane preparations increased phospholipase A2 activity leads to fluidisation, in our model a membrane fluidisation was not observed after stimulation of phospholipase with bradykinin. This suggests that in living cells a fluidizing effect of lysolecithin resulting from phospholipase A2 activation may be rapidly counteracted by its removal. A decrease of phosphatidylcholin content and consequently a rigidification of the membrane may ensue. Thus, the cell culture model using two different ways of stimulating phospholipase activity, helps to define the directional relationship between changes of membrane fluidity and activation of phospholipase and the arachidonic acid cascade in living human cells.     

Triggering of the activity of phospholipase A2 by an electric field

In this paper we show that the action of phospholipase A2 can be triggered by applying an electric field across a 1,2-didodecanoyl-sn-3-phosphoglycerol monolayer residing between an alkylated silicon surface and water. When the silicon wafer served as a cathode, rapid activation of porcine pancreatic phospholipase was observed and did depend on the magnitude of the applied potential. The degree of activation was different for the pancreatic phospholipase A2 and snake and bee venom enzymes. Maximally, a 7-fold activation of pancreatic phospholipase A2 was observed when the applied potential was 75 V. The effective field over the lipid film could be estimated to be approximately 25-175 mV, i.e., in the range of membrane potentials found in cells. On the basis of these results, we suggest that changes in membrane potential might be an important factor in the regulation of the action of intracellular phospholipases A2 in vivo.     

PLRP2 selectively localizes synaptic membrane proteins via acyl-chain remodeling of phospholipids

The plasma membrane of neurons consists of distinct domains, each of which carries specialized functions and a characteristic set of membrane proteins. While this compartmentalized membrane organization is essential for neuronal functions, it remains controversial how neurons establish these domains on the laterally fluid membrane. Here, using immunostaining, lipid-MS analysis and gene ablation with the CRISPR/Cas9 system, we report that the pancreatic lipase-related protein 2 (PLRP2), a phospholipase A1 (PLA1), is a key organizer of membrane protein localization at the neurite tips of PC12 cells. PLRP2 produced local distribution of 1-oleoyl-2-palmitoyl-PC at these sites through acyl-chain remodeling of membrane phospholipids. The resulting lipid domain assembled the syntaxin 4 (Stx4) protein within itself by selectively interacting with the transmembrane domain of Stx4. The localized Stx4, in turn, facilitated the fusion of transport vesicles that contained the dopamine transporter with the domain of the plasma membrane, which led to the localized distribution of the transporter to that domain. These results revealed the pivotal roles of PLA1, specifically PLRP2, in the formation of functional domains in the plasma membrane of neurons. In addition, our results suggest a mode of membrane organization in which the local acyl-chain remodeling of membrane phospholipids controls the selective localization of membrane proteins by regulating both lipid-protein interactions and the fusion of transport vesicles to the lipid domain.