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The aim of the study was to establish productivity of green bean, variety "Strike" irrigated at different pre-irrigation soil moisture. The field experiment was conducted during the period 2010 - 2012 on the experimental field of Agricultural University, Plovdiv. The tested variants are as follows: 1) no irrigation; variants 2) 3) 4) and 5) irrigated at soil moisture of 60,70,80 and 90% of FC. The irrigation rate for each of the variants is calculated to moisten the soil layer 0-60 cm. The type of irrigation is by gravity on short closed furrow. Summary data showed that without irrigation the average yield is 4248 kg/ha, with a range of 1144 kg/ha in dry years to 8393 kg/ha in medium wet years. Best results are obtained by maintaining soil moisture above/up to 80% of FC, and the yield was more than three times higher than that without irrigation and the mean value is 14805kg/ha, varying from 12046kg/ha to 16683kg/ha. [ABSTRACT FROM AUTHOR]

Melittin and phospholipase A2-activating protein (PLAP) are known as efficient activators of secretory phospholipase A2(sPLA2) types I, II, and III when phospholipid liposomes are used as substrate. The present study demonstrates that both peptides can either inhibit or activate sPLA2depending on the peptide/phospholipid ratio when erythrocyte membranes serve as a biologically relevant substrate. Low concentrations of melittin and PLAP were observed to inhibit sPLA2–triggered release of fatty acids from erythrocyte membranes. The inhibition was reversed at melittin concentrations above 1 μM. PLAP-induced inhibition of sPLA2persisted steadily throughout the used concentration range (0–150 nM). The two peptides induced a dose-dependent activation of sPLA2at low concentrations, followed by inhibition when model membranes were used as substrate. This opposite modulatory effect on biological membranes and model membranes is discussed with respect to different mechanisms the interaction of the regulatory peptides with the enzyme molecules and the substrate vesicles. [Copyright &y& Elsevier]

Investigations were performed on the influence of the phospholipid composition and physicochemical properties of the rat liver microsomal membranes on acyl-CoA synthetase and acyl-CoA:1-acyl-sn-glycero-3-phosphocholine O-acyltransferase activities. The phospholipid composition of the membranes was modified by incubation with different phospholipids in the presence of lipid transfer proteins or by partial delipidation with exogenous phospholipase C and subsequent enrichment with phospholipids. The results indicated that the incorporation of phosphatidylglycerol, phosphatidylserine and phosphatidylethanolamine induced a marked activation of acyl-CoA synthetase for both substrates used--palmitic and oleic acids. Sphingomyelin occurred as specific inhibitor for this activity especially for palmitic acid. Palmitoyl-CoA: and oleoyl-CoA: 1-acyl-sn-glycero-3-phosphocholine acyltransferase activities were found to depend on the physical state of the membrane lipids. The alterations in the membrane physical state were estimated using two different fluorescent probes--1,6-diphenyl-1,3,5-hexatriene and pyrene. In all cases of membrane fluidization this activity was elevated. On the contrary, in more rigid membranes obtained by incorporation of sphingomyelin and dipalmitoylphosphatidylcholine, acyltransferase activity was reduced for both palmitoyl-CoA and oleoyl-CoA. We suggest a certain similarity in the way of regulation of membrane-bound acyltransferase and phospholipase A2 which both participate in the deacylation-reacylation cycle. [ABSTRACT FROM AUTHOR]

Vesicle cycling, which is an important biological event, involves the interplay between membrane lipids and proteins, among which the enzyme phospholipase A2 (PLA2) plays a critical role. The capacity of PLA2 to trigger the budding and fission of liquid-ordered (L(o)) domains has been examined in palmitoyl-docosahexaenoylphosphatidylcholine (PDPC) and palmitoyl-oleoylphosphatidylcholine (POPC)/sphingomyelin/cholesterol membranes. They both exhibited a L(o)/liquid-disordered (L(d)) phase separation. We demonstrated that PLA2 was able to trigger budding in PDPC-containing vesicles but not POPC ones. The enzymatic activity, line tension, and elasticity of the membrane surrounding the L(o) domains are critical for budding. The higher line tension of Lo domains in PDPC mixtures was assigned to the greater difference in order parameters of the coexisting phases. The higher amount of lysophosphatidylcholine generated by PLA2 in the PDPC-containing mixtures led to a less-rigid membrane, compared to POPC. The more elastic L(d) membranes in PDPC mixtures exert a lower counteracting force against the L(o) domain bending.

The phase separation of aminophospholipids in glycerophospholipid matrix and the effect of cholesterol were studied by means of fluorescence microscopy of giant unilamellar vesicles (GUV). GUVs were composed of binary mixtures, egg yolk phosphatidylcholine (eggPC)/egg yolk phosphatidylethanolamine (eggPE) and egg yolk phosphatidylcholine (eggPC)/brain phosphatidylserine (brainPS), and ternary ones with both aminophospholipids (eggPC/eggPE/brainPS). Gel/liquid-disordered phase coexistence was detected in these mixtures, where aminophospholipids segregate in gel leaf-like domains. When cholesterol (CHOL) was added, the phase separation was shifted at lower temperatures. CHOL increases miscibility of aminophospholipids in PC matrix. Addition of PE and PS to the ternary mixtures (eggPC/eggSM/CHOL) induced liquid-ordered domain formation at higher temperatures. Based on these results, one can conclude that aminophospholipids promote the formation of Lo domains.

Investigations were performed on the influence of resveratrol on the lipid composition, metabolism, fatty acid and peroxide level in plasma membranes of hepatocytes, isolated from aged rats. Hepatocytes were chosen due to the central role of the liver in lipid metabolism and homeostasis. The obtained results showed that the level of sphingomyelin (SM) and phosphatidylserine (PS) was augmented in plasma membranes of resveratrol-treated senescent hepatocytes. The saturated/unsaturated fatty acids ratio of the two most abundant membrane phospholipids, phosphatidylcholine (PC) and phosphatidylethanolamine (PE), was decreased as a result of resveratrol treatment. The neutral sphingomyelinase was found to be responsible for the increase of SM and the decrease of ceramide in plasma membranes of resveratrol-treated senescent hepatocytes. Using labeled acetate as a precursor of lipid synthesis we demonstrated, that resveratrol treatment resulted in inhibition mainly of phospholipid synthesis, followed by fatty acids synthesis. Resveratrol induced reduction of specific membrane-associated markers of apoptosis such as localization of PS in the external plasma membrane monolayer and ceramide level. Finally, the content of lipid peroxides was investigated, because the unsaturated fatty acids, which were augmented as a result of resveratrol treatment, are an excellent target of oxidative attack. The results showed that the lipid peroxide level was significantly lower, ROS were slightly reduced and GSH was almost unchanged in resveratrol-treated hepatocytes. We suggest, that one possible biochemical mechanism, underlying the reported resveratrol-induced changes, is the partial inactivation of neutral sphingomyelinase, leading to increase of SM, the latter acting as a native membrane antioxidant. In conclusion, our studies indicate that resveratrol treatment induces beneficial alterations in the phospholipid and fatty acid composition, as well as in the ceramide and peroxide content in plasma membranes of senescent hepatocytes. Thus, the presented results imply that resveratrol could improve the functional activity of the membrane lipids in the aged liver by influencing specific membrane parameters, associated with the aging process., (Copyright © 2013. Published by Elsevier Ireland Ltd.)

Complementary biophysical approaches were used to study the structural organization of plasma membrane lipids obtained from fibroblasts cultured as two-dimensional (2D) monolayer and in tissue-like three-dimensional (3D) conditions. Fluorescence microscopy experiments demonstrated different domain patterns for 2D and 3D plasma membrane lipid extracts. ESR demonstrated that 3D lipid extract is characterized with lower order parameter than 2D in the deep hydrophobic core of the lipid bilayer. Higher cholesterol and sphingomyelin content in 3D extract, known to increase the order in the glycerophospholipid matrix, was not able to compensate higher fatty acid polyunsaturation of the phospholipids. The interfacial region of the bilayer was probed by the fluorescent probe Laurdan. A higher general polarization value for 3D extract was measured. It is assigned to the increased content of sphingomyelin, cholesterol, phosphatidylethanolamine and phosphatidylserine in the 3D membranes. These results demonstrate that cells cultured under different conditions exhibit compositional heterogeneity of the constituent lipids which determine different structural organization of the membranes., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Sphingosine is a bioactive molecule which is known to participate in the regulation of a number of cellular processes such as apoptosis, cell differentiation, growth, etc. Sphingosine was observed to exhibit different domain morphology depending on the surrounding lipid matrix in biomimetic systems such as giant vesicles. Our current results showed that in a glycerophospholipid matrix sphingosine segregated in gel leaf-like domains whereas cholesterol presence increased its miscibility by melting gel domains in a concentration-dependent manner. Sphingosine and cholesterol did not form merging liquid domains on the micron scale as observed for sphingomyelin and cholesterol. However, we were able to visualize that sphingosine appears as a stabilizer and amplifier of domains in liquid-ordered phase by increasing the temperature of their formation and fraction. These results imply that sphingosine acts as a modulator of the lipid domain formation and thus it could exert its biological role, not only through direct binding to proteins, but also indirectly by influencing their sorting in membranes and modulating the processes of signal transduction., (Copyright 2010 Elsevier Inc. All rights reserved.)

The three-dimensional (3D) cell culture approach offers a means to study cells under conditions that mimic an in vivo environment, thus avoiding the limitations imposed by the conventional two-dimensional (2D) monolayer cell cultures. By using this approach we demonstrated significant differences in the plasma membrane phospholipid composition and susceptibility to oxidation in cells cultured in three-dimensional environment compared to conventional monolayer cultures. The plasma membrane sphingomyelin (SM), which is a functionally active membrane phospholipid, was markedly increased in plasma membranes of 3D cells. To analyze the mechanisms underlying SM accumulation, we determined the activities of sphingolipid-metabolizing enzymes like neutral sphingomyelinase and ceramidase, which are also related to cellular redox homeostasis and to oxidative stress. Fibroblasts cultured in three-dimensional environment showed different redox potential and lower lipid susceptibility to oxidative damage compared to monolayer cells. The relative content of unsaturated fatty acids, which serve as targets of oxidative attack, was observed to be higher in major phospholipids, such as phosphatidylcholine and phosphatidylethanolamine, in plasma membranes of 3D cells. The possibility that the higher level of SM, might be responsible for the lower degree of oxidation of 3D phospholipids was tested by selective reduction of SM through treatment with exogenous sphingomyelinase. The results showed that the decrease of plasma membrane SM was accompanied by an increase of the lipid peroxides in both 2D and 3D cells. We presume that culturing as a monolayer is stressful for the cells and leads to activation of certain stress-related enzymes, resulting in reduction of the SM level. Our results show that the lower content of plasma membrane SM in cells cultured as a monolayer renders the phospholipid molecules more susceptible to oxidative stress.

Free-standing giant unilamellar vesicles were used to visualize the complex lateral heterogeneity, induced by ceramide in the membrane bilayer at micron scale using C(12)-NBD-PC probe partitioning under the fluorescence microscope. Ceramide gel domains exist as leaf-like structures in glycerophospholipid/ceramide mixtures. Cholesterol readily increases ceramide miscibility with glycerophospholipids but cholesterol-ceramide interactions are not involved in the organization of the liquid-ordered phase as exemplified by sphingomyelin/cholesterol mixtures. Sphingomyelin stabilizes the gel phase and thus decreases ceramide miscibility in the presence of cholesterol. Gel/liquid-ordered/liquid-disordered phase coexistence was visualized in quaternary phosphatidylcholine/sphingomyelin/ceramide/cholesterol mixtures as occurrence of dark leaf-like and circular domains within a bright liquid phase. Sphingomyelin initiates specific ceramide-sphingomyelin interactions to form a highly ordered gel phase appearing at temperatures higher than pure ceramide gel phase in phosphatidylcholine/ceramide mixtures. Less sphingomyelin is engaged in formation of liquid-ordered phase leading to a shift in its formation to lower temperatures. Sphingomyelinase activity on substrate vesicles destroys micron L(o) domains but induces the formation of a gel-like phase. The activation of phospholipase A(2) by ceramide on heterogeneous membranes was visualized. Changes in the phase state of the membrane bilayer initiates such morphological processes as membrane fragmentation, budding in and budding out was demonstrated.

The structure, thermotropic phase behavior, dynamic motion and order parameters of bilayer dispersions of egg phosphatidylcholine, egg sphingomyelin, egg ceramide and cholesterol have been determined. The coexistence of gel, liquid-ordered and liquid-disordered structure has been determined by peak fitting analysis of synchrotron X-ray powder patterns. Order parameters and extent of distribution of 16-doxyl-stearic acid spin probe between ordered and disordered environments has been estimated by ESR spectral simulation methods. The presence of ceramide in proportions up to 20 mol% in phosphatidylcholine is characterized by gel-fluid phase coexistence at temperatures up to 46 degrees C depending on the amount of ceramide. Cholesterol tends to destabilize the ceramide-rich domains formed in phosphatidylcholine while sphingomyelin, by formation of stable complexes with ceramide, tends to stabilize these domains. The stability of sphingomyelin-ceramide complexes is evident from the persistence of highly ordered structure probed by ESR spectroscopy and appearance of a sharp wide-angle X-ray reflection at temperatures higher than the gel-fluid transition of ceramide alone in egg phosphatidylcholine bilayers. The competition between ceramide and cholesterol for interaction with sphingomyelin is discussed in terms of control of lipid-mediated signaling pathways by sphingomyelinase and phospholipase A2.

The effect of detergents on giant unilamellar vesicles (GUVs) composed of phosphatidylcholine, sphingomyelin and cholesterol and containing liquid-ordered phase (l(o)) domains was investigated. Such domains have been used as models for the lipid rafts present in biological membranes. The studied detergents included lyso-phosphatidylcholine, the product of phospholipase A2 activity, as well as Triton X-100 and Brij 98, i.e. detergents used to isolate lipid rafts as DRMs. Local external injection of each of the three detergents at subsolubilizing amounts promoted exclusion of l(o) domains from the GUV as small vesicles. The budding and fission processes associated with this vesiculation were interpreted as due to two distinct effects of the detergent. In this framework, the budding is caused by the initial incorporation of the detergent in the outer membrane leaflet which increases the spontaneous curvature of the bilayer. The fission is related to the inverted-cone molecular shape of the detergent which stabilizes positively curved structures, e.g. pores involved in vesicle separation. On the other hand, we observed in GUVs neither domain formation nor domain coalescence to be induced by the addition of detergents. This supports the idea that isolation of DRM from biological membranes by detergent-induced extraction is not an artifact. It is also suggested that the physico-chemical mechanisms involved in l(o) domain budding and fission might play a role in rafts-dependant endocytosis in cells.

The phase behaviour of mixed molecular species of phosphatidylethanolamine, phosphatidylserine and sphingomyelin of biological origin were examined in aqueous co-dispersions using synchrotron X-ray diffraction. The co-dispersions of phospholipids studied were aimed to model the mixing of lipids populating the cytoplasmic and outer leaflets in the resting or "scrambled" activated cell membrane. Mixtures enriched with phosphatidylethanolamine and phosphatidylserine were characterized by a phase separation of non-lamellar phases (cubic and inverted hexagonal) with a lamellar gel phase comprising the most saturated molecular species. Inclusion of sphingomyelin in the mixture resulted in a suppression of the hexagonal-II phase in favour of lamellar phases at temperatures where a proportion of the phospholipid was fluid. The effect was also dependent on the total amount of sphingomyelin in ternary mixtures, and the lamellar phase dominated in mixtures containing more than 30 mol%, irrespective of the relative proportions of phosphatidylserine/sphingomyelin. A transition from gel to liquid-crystal phase was detected by wide-angle scattering during heating scans of ternary mixtures enriched in sphingomyelin and was shown by thermal cycling experiments to be coupled with a hexagonal-II phase to lamellar transition. In such samples there was evidence of a coexistence of non-lamellar phases with a lamellar gel phase. A transition of the gel phase to the fluid state on heating from 35 to 41 degrees C was evidenced by a progressive increase in the lamellar d-spacing. The presence of calcium enhanced the phase separation of a lamellar gel phase from a hexagonal-II phase in mixtures enriched in phosphatidylserine. This effect was counteracted by charge screening with 150 mM NaCl. The effect of sphingomyelin on stabilizing the lamellar phase is discussed in the context of an altered composition in the cytoplasmic/outer leaflets of the plasma membrane resulting from scrambling of the phospholipid distribution. The results suggest that a lamellar structure can be retained by the inward translocation of sphingomyelin in biological membranes. The presence of monovalent cations serves also to stabilize the bilayer in activated cells where a translocation of aminoglycerophospholipids and an influx of calcium occur simultaneously.

Cellular processes involving membrane vesiculation are related to cellular transport and membrane components trafficking. Endocytosis, formation of caveolae and caveosomes, as well as Golgi membranes traffic have been linked to the existence and dynamics of particular types of lipid/protein membrane domains, enriched in sphingolipids and cholesterol, called rafts [Nature 387 (1997) 569; Trends Cell Biol. 12 (2002) 296; Biochemistry 27 (1988) 6197]. In addition, the participation of phospholipases in the vesiculation of Golgi and other membranes has been already established [Traffic 1 (2000) 504] essentially in their role in the production of second messenger molecules. In this work we illustrate with raft-containing giant lipid vesicles a mechanism for raft-vesicle expulsion from the membrane due to the activity of a single enzyme-phospholipase A(2) (PLA(2)). This leads to the hypothesis that the PLA(2), apart from its role in second messenger generation, might play a direct and general role in the vesiculation processes underlying the intermembrane transport of rafts through purely physicochemical mechanisms. These mechanisms would be: enzyme adsorption leading to membrane curvature generation (budding), and enzyme activity modulation of the line tension at the raft boundaries, which induces vesicle fission.

Madin-Darby canine kidney type II cells were shown to release low amounts of AA (arachidonic acid) and prostaglandin E2 in response to various stimuli when analysed after cell confluence. In contrast, non-confluent Madin-Darby canine kidney type II cells released much higher amounts of AA and prostaglandin E2. In both stationary and non-confluent cells, AA was released by type IV cPLA2 (cytosolic phospholipase A2), as shown by the use of specific inhibitors and by analysis of the profile of fatty acids released. This confluence-dependent cPLA2 activation was not due to a difference in expression, or in phosphorylation of the enzyme, or in the amount of its substrate. To find out the mechanism by which cPLA2 activation may be regulated as a function of cell confluence, immunofluorescence and co-immunoprecipitation experiments were performed using cPLA2, p11, a natural inhibitor of the enzyme, and annexin II, the natural ligand of p11. These three proteins were expressed at a constant level, regardless of the cell confluence. In contrast, whereas annexin II and cPLA2 interacted at a constant rate, p11 and cPLA2 interacted more strongly in stationary cells, thus indicating that cPLA2 activation is regulated by its accessibility to p11, independent of their expression level. Our results indicate that, in epithelial cells, the cell confluence, i.e. the establishment of cell-cell contacts, rather than cell proliferation directly controls cPLA2 activation by changing the stoichiometry of p11/cPLA2 interaction.

Hydrolysis of surfactant phospholipids by secreted phospholipases A(2) (sPLA(2)) contributes to surfactant dysfunction in acute respiratory distress syndrome. The present study demonstrates that sPLA(2)-IIA, sPLA(2)-V, and sPLA(2)-X efficiently hydrolyze surfactant phospholipids in vitro. In contrast, sPLA(2)-IIC, -IID, -IIE, and -IIF have no effect. Since purified surfactant protein A (SP-A) has been shown to inhibit sPLA(2)-IIA activity, we investigated the in vitro effect of SP-A on the other active sPLA(2) and the consequences of sPLA(2)-IIA inhibition by SP-A on surfactant phospholipid hydrolysis. SP-A inhibits sPLA(2)-X activity, but fails to interfere with that of sPLA(2)-V. Moreover, in vitro inhibition of sPLA(2)-IIA-induces surfactant phospholipid hydrolysis correlates with the concentration of SP-A in surfactant. Intratracheal administration of sPLA(2)-IIA to mice causes hydrolysis of surfactant phosphatidylglycerol. Interestingly, such hydrolysis is significantly higher for SP-A gene-targeted mice, showing the in vivo inhibitory effect of SP-A on sPLA(2)-IIA activity. Administration of sPLA(2)-IIA also induces respiratory distress, which is more pronounced in SP-A gene-targeted mice than in wild-type mice. We conclude that SP-A inhibits sPLA(2) activity, which may play a protective role by maintaining surfactant integrity during lung injury.

Detection of immiscible lipid domains in biological membranes offers an alternative support to protein sorting. Liquid ordered domains ("rafts") comprising cholesterol and saturated sphingolipids incorporate saturated glycosyl-phosphatidylinositol (GPI)-anchored or acylated (palmitoyl- and myristoyl-) proteins or particular transmembrane protein sequences. These lipid domains can be isolated in the form of Detergent resistant membranes (DRM) from biological plasma membrane preparations. Caveolae appear to be a differentiated fraction of plasma membranes comprising such numerous cross-linked microdomains associated with caveolin in different cell types. While the biological relevance of such membrane domains is evidenced in vivo by co-patching of proteins sharing the identical affinity for sphingolipids and by the disruption of co-patching following cell cholesterol depletion, only a few physical studies confort the principle of membrane heterogeneity. Results are now presented where cholesterol addition in a tertiary lipid mixture forces outphase-separation, as a realistic model where the lipid segregation can promote protein sorting to the segregated Lo phase. A lipid mixture comprising phosphatidylserine, phosphatidylethanolamine and sphingomyelin of natural origin in the ratio (1/4/3: mole/mole) has been rendered neatly heterogeneous after the addition of cholesterol (27 mole%). Xray diffraction (Small angle Xray scattering) showed the splitting of two neatly resolved lamellar diffractions in the presence of cholesterol. Above 37 degrees C the heterogeneity was traceable by a broadened diffraction spot up to the complete get-to-liquid transition of sphingomyelin at temperatures > 40 degrees C where the spot became again symmetrical and narrow. The large temperature range where the immiscible lamellar phases are detected, the specific requirement for cholesterol association with sphingomyelin, the positive influence of calcium and the reversibility of domain formation support the occurrence for such domains at the inner side of the plasma membrane whereon lipids-bound proteins concentrate.

Secretory type II phospholipase A2 (sPLA2) is inhibited by sphingomyelin (SPH); cholesterol either mixed with the model glycerophospholipid substrate or added to the assay medium as separated liposomes counteracts this inhibition efficiently. The inhibition of fatty acid release assayed by quantitative gas chromatography-MS is observed when SPH is added to erythrocyte membranes as the substrate instead of a readily hydrolysable phosphatidylethanolamine/phosphatidylserine model mixture. Hydrolysis of SPH by Staphylococcus aureus sphingomyelinase suppresses its inhibitory potency. The addition of cholesterol to SPH liposomes with a 1:1 stoichiometry relieves completely the inhibition of sPLA2 exerted by SPH. The mechanism of inhibition suggested by the binding assay is that sPLA2 binds with affinity to the SPH interface, after either phase segregation at the assay temperature or on the pure SPH liposomes added to the incubation medium. Cholesterol is shown to suppress the binding affinity of the enzyme for the SPH interface. A model for inhibition is suggested in which binding of the sphingosine moiety is competitive for sPLA2 (inhibition) or for cholesterol (release of the enzyme).

Oxidative damage, which plays a major role in the early stages of atherosclerosis, is associated with arachidonic acid (AA) release in vascular smooth muscle cells (VSMC) as in other cell types. In this study, H2O2 was used to investigate mechanisms of AA release from VSMC on oxidative stress. Cell treatment with H2O2 inhibited AA incorporation in an inverse relationship to prolonged H2O2-induced AA release. Identical kinetics of inhibition of AA incorporation and AA release were observed after cell treatment with AlF4-, a process not involving phospholipase A2 (PLA2) activation as recently described (A. Cane, M. Breton, G. Béréziat, and O. Colard. Biochem. Pharmacol. 53: 327-337, 1997). AA release was not specific, since oleic acid also increased in the extracellular medium of cells treated with H2O2 or AlF4- as measured by gas chromatography-mass spectrometry. In contrast, AA and oleic acid cell content decreased after cell treatment. Oleoyl and arachidonoyl acyl-CoA synthases and acyltransferases, assayed using a cell-free system, were not significantly modified. In contrast, a good correlation was observed between decreases in AA acylation and cell ATP content. The decrease in ATP content is only partially accounted for by mitochondrial damage as assayed by rhodamine 123 assay. We conclude that oxidant-induced arachidonate release results from impairment of fatty acid esterification and that ATP availability is probably responsible for free AA accumulation on oxidative stress by preventing its reesterification and/or transmembrane transport.

Conjectural results have been reported on the capacity of inflammatory secreted phospholipase A2 (sPLA2) to hydrolyse mammalian membrane phospholipids. Development of an assay based on the release of non-esterified fatty acids by the enzyme acting on the organized phospholipid mixture constituting the membrane matrix has led to the identification of two prominent effectors, sphingomyelin (SPH) and annexin. Recombinant human type II sPLA2 hydrolyses red-cell membrane phospholipids with a marked preference for the inner leaflet. This preference is apparently related to the high content of SPH in the outer leaflet, which inhibits sPLA2. This inhibition by SPH is specific for sPLA2. Cholesterol counteracts the inhibition of sPLA2 by SPH, suggesting that the SPH-to-cholesterol ratio accounts in vivo for the variable susceptibility of cell membranes to sPLA2. Different effects were observed of the presence of the non-hydrolysable D-alpha-dipalmitoyl phosphatidylcholine (D-DPPC), which renders the membranes rigid but does not inhibit sPLA2. Annexin VI was shown, along with other annexins, to inhibit sPLA2 activity by sequestering the phospholipid substrate. The present study has provided the first evidence that annexin VI, in concentrations that inhibit hydrolysis of purified phospholipid substrates, stimulated the hydrolysis of membrane phospholipids by sPLA2. The activation requires the presence of membrane proteins. The effect is specific for type II sPLA2 and is not reproducible with type I PLA2. The activation by annexin VI of sPLA2 acting on red cell membranes results in the preferential release of polyunsaturated fatty acids. It suggests that type II sPLA2, in conjunction with annexin VI, might be involved in the final step of endocytosis and/or exocytosis providing the free polyunsaturated fatty acids acting synergistically to cause membrane fusion.

1-Acyl lysophosphatidylcholine prepared from egg yolk has been chemically reacylated to form decanoyl, dodecanoyl, myristoyl and palmitoyl derivatives of phosphatidylcholine. The liposomes formed by these semi-synthetic phospholipids have been characterized by calorimetry, X-ray diffraction and fluorescence probe methods. Asymmetric phosphatidylcholines tend to promote formation of excimers of a codispersed fluorescent phospholipid (1-palmitoyl-sn-2-(1-pyrenedecanoyl)-L-alpha-phosphatidic acid) (2 mol%). Excimer formation is correlated with the rate of hydrolysis of the fluorescent anionic phospholipid by Crotalus venom phospholipase A2. Codispersion with the semi-synthetic phosphatidylcholine of cholesterol or unsaturated fluid lecithin modulated both excimer formation and the susceptibility of the fluorescent probe to hydrolysis by venom phospholipase A2 at 22 degrees C. Similar results were obtained with hydrolysis of a radiolabelled substrate, 1-palmitoyl-sn-2-[1-14C]linoleoylphosphatidylethanolamine, codispersed with the semi-synthetic phosphatidylcholine. Enrichment of rat hepatocyte plasma membranes with semi-synthetic asymmetric phosphatidylcholines was mediated by incubation of membranes with phospholipid dispersions in the presence of a phospholipid exchange protein. Enrichment of the membranes with semi-synthetic phosphatidylcholines of between 30 and 60% of the membrane phosphatidylcholine was achieved. The resulting alteration of the biomembrane is associated with a decreased activity of endogenous membrane phospholipase A2 acting on extramembranous radiolabelled substrate vesicles. By contrast, the activity of acyl-CoA:lysophospholipid acyltransferase is increased in membranes enriched with highly asymmetric phospholipids.

The dependence of acyl-CoA synthetase on the lipid composition of rat liver plasma membranes has been investigated. For this purpose the composition of the membranes was modified by incorporation of different phospholipids in the presence of partially purified lipid transfer proteins. Another approach to the modification of the membrane phospholipid composition was treatment with exogenous phospholipase C and subsequent enrichment with different phospholipids. The experiments performed in vitro indicated that the presence of certain phospholipids such as phosphatidylnositol, phosphatidylethanolamine, phosphatidylglycerol and phosphatidylserine was essential for the activation of long chain fatty acids by acyl-CoA synthetase. However, some differences were observed when oleate and palmitate were used as substrates. Sphingomyelin was found to inhibit this activity especially when oleic acid served as substrate. In addition, we tried to modify in vivo the membrane lipid composition by treatment with D-galactosamine, which is known to induce acute hepatitis and cause biochemical and biophysical alterations in liver membranes. The results thus obtained confirmed the idea that the augmentation of the membrane lipids and especially of PI, PE and PG was accompanied by acyl-CoA synthetase activation. The presence of two different enzymes, activating the saturated and unsaturated fatty acids is discussed.

Investigations were carried out on the influence of rat liver plasma membranes phospholipid composition on phospholipase C activity using PIP, PIP2, PC and PE as substrates. The membrane phospholipids were modified by incorporation of definite phospholipids with the aid of lipid transfer proteins or after partial delipidation with exogenous phospholipases A2 and C. The results indicated that sphingomyelin inhibited all phospholipase C activities. The incorporation of two different molecular species of phosphatidylcholine did not alter significantly the investigated phospholipase C activities, indicating that membrane fluidity was not essential in this case. Phosphatidylglycerol, phosphatidylserine, phosphatidylinositol and phosphatidylethanolamine served as specific activators of plasma membrane-bound phospholipase C when PIP, PIP2 and PC were used as substrates. However, these four phospholipids inhibited phospholipase C activity towards PE. The role of phosphoinositide-specific phospholipase C in the production of second messengers as well as the eventual biological significance of PC and PE as substrates for phospholipase C is discussed.