Phosphatidylcholine Derivatives Research Articles

Oxidized Phosphatidylcholines That Modify Proteins: ANALYSIS BY MONOCLONAL ANTIBODY AGAINST OXIDIZED LOW DENSITY LIPOPROTEIN

Oxidatively modified low density lipoprotein (OxLDL) is known to be involved in atherogenesis. We have previously developed a murine monoclonal antibody, FOH1a/DLH3, which recognized oxidatively modified lipoproteins as well as foam cells in human atherosclerotic lesions (Itabe, H., Takeshima, E., Iwasaki, H., Kimura, J., Yoshida, Y., Imanaka, T., and Takano, T. (1994) J. Biol. Chem. 269, 15274-15279). The antigen of this monoclonal antibody was formed by peroxidation of phosphatidylcholine (PC), and the antigenic oxidized PC (OxPC) derivatives are thought to form complexes with polypeptides including apolipoproteins. OxLDL was measured by a sensitive sandwich enzyme-linked immunosorbent assay using the monoclonal antibody and anti-human apolipoprotein B antibody, in which antigenic OxPC competed with OxLDL. When antigenic activities of PC analogs were tested by the competition assay, 1-palmitoyl-2-(9-oxononanoyl) PC (9-CHO PC) and the hydroperoxide of egg PC potently inhibited the detection of OxLDL. 1-Palmitoyl-2-linoleoyl PC was oxidized with ferrous ion and ascorbic acid, and the antigenic products were purified from the OxPC extracts on high pressure liquid chromatography columns and subsequently analyzed by laser desorption mass spectrometry. Molecular weight determination and retention times of high pressure liquid chromatography suggest that one of these products was 9-CHO PC. Other products are thought to be 8-carbon aldehyde, dihydroxy, and ketohydroxy derivatives of PC. When a C-terminal 16-mer synthetic peptide of the 70-kDa peroxisomal membrane protein was simply incubated with 9-CHO PC, it was found to be reactive in a sandwich enzyme-linked immunosorbent assay using FOH1a/DLH3 and an anti-peptide antiserum. These results suggest that the anti-OxLDL monoclonal antibody FOH1a/DLH3 reacts with several oxidized products of PC including aldehyde derivatives of PC, which covalently modify polypeptides.

The effects of phospholipids on the percutaneous penetration of indomethacin through the dorsal skin of guinea pig in vitro. 2. The effects of the hydrophobic group in phospholipids and a comparison with general enhancers

The enhancing effects of phospholipids on the in vitro percutaneous penetration of indomethacin (IM) was investigated using a dorsal skin of guinea pigs mounted on a Franz-type diffusion chamber. The phospholipids were (1) six phosphatidylglycerol (PG) derivatives comprising PGE (from egg yolk), PGS (from soybean), DMPG (dimyristyl PG), DPPG (dipalmityl PG), DSPG (distearyl PG) and DOPG (dioleoyl PG); (2) five phosphatidylcholine (PC) derivatives comprising PCS (from soybean), PLE (from egg yolk), DOPC (dioleoyl PC), DLPC (dilinoleoyl PC) and HPC (hydrogenated PC, from soybean); (3) two phosphatidylethanolamine (PE) derivatives comprising PE (from egg yolk) and DOPE (dioleoyl PE). The enhancing effects of PG derivatives on the percutaneous penetration of IM were in the order of DOPG > PGE > PGS > DMPG > control > DPPG = DSPG. The enhancing effects of PC derivatives on the percutaneous penetration of IM were in the order of DOPC > DLPC > PCS > PLE > control > HPC and the effects of PE derivatives on the penetration were DOPE > PE > control. The enhancement of percutaneous penetration of IM by azone, oleic acid (Δ9, C18:F1), asclepic acid (Δll, C18: F1), and palmitoleic acid (Δ9, C16:F1), which are known to be penetration enhancers, were compared with that of phospholipids. DOPG, PGE, DOPC, DLPC and PCS showed significantly superior effects to these three unsaturated fatty acids on the percutaneous penetration of IM. Moreover, PGE, PGS, DOPG, PLE, PCS, DOPC, DLPC, PE and DOPE were better than azone on enhancing percutaneous penetration of IM. Differences in enhancement by phospholipids may be due to differences in hydrophobic groups rather like the effects of hydrophilic groups shown in a previous study. It was observed that hydrophobic groups in phospholipids had to be unsaturated fatty acids in order to promote percutaneous penetration of IM. These results suggest that phospholipids containing unsaturated fatty acids in the hydrophobic group are strong penetration enhancers of the percutaneous delivery of some topically-applied drugs.

Activation of phospholipase A2 by amyloid beta-peptides in vitro.

Amyloid beta-peptides (A beta) are centrally involved in the pathogenesis of Alzheimer's disease. Using secretory phospholipase A2 (PLA2) from porcine pancreas as a model and in the presence of a limiting Ca2+ concentration of approximately 50 nM, the synthetic peptide A beta 1-42 activates the hydrolysis of the pyrene-labeled acidic phospholipid analog 1-palmitoyl-2-[(pyren-1-yl)]hexanoyl-sn-glycero-3-phosphoglycerol (PPHPG) maximally 2.3-fold, whereas an inhibition of PLA2 action by 50% on the corresponding phosphatidylcholine derivative (PPHPC) was observed. The above effects were evident at 0.24 nM A beta 1-42 corresponding to A beta 1-42:phospholipid and A beta 1-42:PLA2 molar ratios of 1:10 650 and 1:7.6, respectively. The presence of 10 mol % 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (POPG) in PPHPC reversed the inhibitory effect of A beta 1-42 peptide and for these vesicles the hydrolytic activity of PLA2 toward the fluorescent phosphatidylcholine was enhanced approximately 1.8-fold by A beta 1-42. In contrast, inclusion of 10 mol % POPG into PPHPG did not influence either the hydrolytic rate toward the latter lipid or the activating effect of A beta 1-42. Ca2+ concentrations exceeding 15 microM abolished the enhancing effect of A beta 1-42 on the hydrolysis of PPHPG whereas a slight activation of PPHPC hydrolysis now became evident. With limiting [Ca2+] preaggregated A beta 1-42 enhanced the hydrolysis of both PPHPG as well as PPHPC but the peptide concentrations required were higher by 3-4 orders of magnitude. The synthetic peptide A beta 25-35 corresponding to the hydrophobic membrane-spanning segment of the beta amyloid precursor protein activated PLA2 when using PPHPG as a substrate; however, compared to A beta 1-42 the extent of activation was less (approximately 2-fold) and required higher (1 nM) peptide. A beta 25-35 did not affect the hydrolysis of the phosphatidylcholine derivative. The hydrophilic peptide A beta 1-28 had no effect on PLA2-catalyzed hydrolysis of either PPHPG or PPHPC under the conditions used in the present study. Interestingly, the above activating effects of A beta 1-42 and A beta 25-35 on PLA2-catalyzed hydrolysis of the acidic phospholipid substrate parallel their toxicity on cultured neurons whereas A beta 1-28 had no influence either on cultured cells or on PLA2 activity.

Soluble phospholipids enhance factor Xa-catalyzed prothrombin activation in solution.

Acidic phospholipids play an important but incompletely understood role in prothrombin activation. Here we report the effect of short-chain phosphatidylserine (dicaproylphosphatidylserine, C6PS) and the corresponding phosphatidylglycerol (C6PG) and phosphatidylcholine (C6PC) derivatives on the rate of prothrombin activation by factor Xa. The critical micellar concentrations of these short-chained phospholipids have been determined under a variety of conditions that we used for kinetic and structural studies. Under conditions for which these lipids exist in a soluble form, the results demonstrate that: (i) the rate of human prothrombin activation by human factor Xa was enhanced in a calcium-dependent fashion up to 60-fold by addition of C6PS, roughly 20% of the optimal enhancement seen with bovine phosphatidylserine/palmitoyloleoylphosphatidylcholine (25/75 PS/POPC) membranes; (ii) C6PS inhibited the rate of hydrolysis of synthetic factor Xa substrate (S-2765), an effect that was mimicked, but at much lower lipid concentrations, by PS/POPC membranes; (iii) there was no enhancement of prothrombin activation and much less inhibition of hydrolysis of S-2765 by factor Xa in the presence of C6PG or C6PC; and (iv) the thermal denaturation of prothrombin was altered in a calcium-independent but dose-dependent fashion by either C6PS or C6PG. These results have been interpreted in terms of the existence of (a) specific PS binding site(s) on factor Xa (Kd approximately 73 microM) that regulate(s) the activity of this serine protease. Our results do not rule out the possibility that the rate of prothrombin activation is also influenced by a weaker, calcium-independent, and less specific acidic lipid binding site on prothrombin, the occupancy of which results in conformational changes in this protein. The results clearly suggest that PS binding regulates the rate of prothrombin activation.

Separation of phospholipid derivatives by microcolumn liquid chromatography

Packed, fused silica capillaries were employed, at high chromatographic resolution, to separate complex mixtures of phospholipids derived from plant and animal sources. Benzoylation of phosphatidylcholine derivatives and dansylation of phosphatidylethanolamines were used in conjunction with miniaturized spectroscopic detectors. Under the conditions of stepwise gradient elution, detection limits for both derivatives were on the order of picomoles. © 1996 John Wiley & Sons, Inc.

Phospholipids with a short acyl chain modulate phospholipase and acyltransferase activities

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-α-phosphatidic acid) (2 mol%). Excimer formation is correlated with the rate of hydrolysis of the fluorescent anionic phospholipid by Crotalus venom phospholipase A 2. 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 A 2 at 22°C. Similar results were btained 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 A 2 acting on extramembranous radiolabelled substrate vesicles. By contrast, the activity of acylCoAaysophospholipid acyltransferase is increased in membranes enriched with highly asymmetric phospholipids.

Characterization of complexes formed in fully hydrated dispersions of dipalmitoyl derivatives of phosphatidylcholine and diacylglycerol

The phase diagram of fully hydrated binary mixtures of dipalmitoylphosphatidylcholine (DPPC) with 1,2-dipalmitoylglycerol (DPG) published recently by López-García et al. identifies regions where stoichiometric complexes of 1:1 and 1:2 DPPC:DPG, respectively, are formed. In this study, the structural parameters of the 1:1 complex in the presence of pure DPPC was characterized by synchrotron low angle and static x-ray diffraction methods. Structural changes upon transitions through phase boundaries were correlated with enthalpy changes observed by differential scanning calorimetry in mixtures of DPPC with 5, 7.5, 10, and 20 mol% DPG dispersed in excess water. Phase separation of a complex in gel phase could be detected by calorimetry in the mixture containing 5 mol% DPG but was not detectable by synchrotron low angle x-ray diffraction. Static x-ray measurements show evidence of phase separation, particularly in the reflections indexing chain packing. In the mixture containing 7.5 mol% DPG, two distinct lamellar repeat spacings could be seen in the temperature range from 25 to 34 degrees C. The lamellar spacing of about 6.6 nm was assigned to pure gel phase DPPC because the change in the spacing corresponds with thermal transition of the pure phospholipid, and a longer repeat spacing of about 7.2 nm was assigned to domains of the 1:1 complex of DPPC-DPG. In the temperature range from 34 to 420C, i.e., in the region of coexistence of the ripple phase of DPPC and the gel phase of the complex, a single, rather broad lamellar reflection appears because of superposition of two reflections of DPPC and the complex; the lamellar spacing of DPPC in the ripple phase is similar to that of the gel phase of complex. In the coexistence region of the liquid-crystalline phase of DPPC and the gel phase of complex (-42-480C), the lamellar reflections of the both phases are present. The fluidus boundary lies between the coexistence region and the fluid region.In the fluid region (-48-550C), the gel state of complex persists up to the fluidus boundary, whereupon the liquid-crystalline state of complex replaces the gel state of the complex. This indicates that the complex is also immiscible with DPPC even above the fluidus boundary at least in the temperature range close to the phase boundary. For mixtures comprising 10 and 20 mol%DPG in DPPC, complex formation is clearly detectable in both the gel region and the coexistence region by x-ray diffraction.Synchrotron x-ray measurements indicate phase separation between pure DPPC and liquid-crystalline complex just above thefluidus boundary. Static, wide angle x-ray measurements also suggest phase separations of the 1:1 complex not only from the gel phase but also the liquid-crystalline phase of pure DPPC. Two distinct diffraction peaks were detected for the mixture of DPPC with 5, 10, and 20 mol% DPG. One is due to the chain spacing of the complex, and the other is due to that of the pure DPPC. In the coexistence region of the liquid-crystalline phase of DPPC and the gel phase of complex, two kinds of diffraction peaks of the hydrocarbon chain of the gel phase complex and the broad scattering profile for the chain melting of DPPC were observed in the wide angle region. Electron density reconstructed from the lamellar reflections indicates that the thicknesses of both the bilayer and the water layer of the gel phase complex are greater than those of the respective thicknesses of gel phase DPPC.

Platelet-Activating Factor Produced by Endothelial Cells. A Molecule with Autocrine and Paracrine Properties

Endothelial cells (EC) participate in microenvironment homeostasis by regulating the trafficking of cells and molecules from the bloodstream to the tissues. The 'area codes' used to control the trafficking are adhesion molecules, receptors which pick up external signals, and mediators of cell-to-cell communication. Platelet-activating factor (PAF) is a mediator that belongs to this class of 'area-code' molecules. PAF is an acetylated derivative of phosphatidylcholine which is produced by EC and may act in an autocrine manner. Several stimuli may induce the synthesis of PAF by EC with different time courses. Thrombin, elastase and tumor necrosis factor (TNF) are prototypic molecules inducing very-early, early and delayed PAF synthesis, respectively. These stimuli activate the 'remodelling pathway' which requires the activation of phospholipase A2 and acetyl CoA:1-alkyl-2-lyso-sn-glycero-3-phosphate acetyltransferase. The effects of very-early and early stimuli are mediated by a direct stimulation of this pathway, whereas the action of TNF is mediated by the new synthesis of a serine protease. Stimulated EC produce PAF molecules with both an ether and an ester bond at the sn-1 position, but the former is predominant. The PAF produced is partially exposed on the external membranes and participates in adhesion or migration of neutrophils. PAF released can also stimulate EC themselves, by interacting with a specific receptor. PAF stimulates the migration and the shape change of EC by activating calcium influx and several serine/threonine and tyrosine kinases which regulate the cytoskeleton.

レシチン化ＳＯＤ 臨床応用可能なＳＯＤ製剤を目指して

Superoxide dismutase (SOD), an enzyme that scavenges superoxide anion (O2-), has been suggested as a possible therapeutic tool, for treating diseases related to active oxygen spiecies. However, its low cell membrane affinity and rapid metabolic clearance limit its clinical application. We synthesized SOD, in which cytotropic phosphatidylcholine derivatives were covalently bound to recombinant human SOD, in order to target SOD to cell membrane. Lecithinized SOD was shown to have a high cell membrane affinity using laser confocal imaging technique, showed a longer plasma half-life, and efficiently scavenged O2- produced by phorbol myristate acetate(PMA). Lecithinized SOD showed more potent inhibitory effect on human endothelial cells injury by O2- produced by PMA stimulated neutrophils and on ischemia-reperfusion mouse paw edema than, that of unmodified SOD. Moreover, other potent pharmacological activities of lecithinized SOD has been reported, and toxicological and antigenic findings of lecithinized SOD had no particullar in comparison with unmodified SOD. Accordingly, lecithinized SOD seems to have the potential for clinical applications.

Study of plasma membrane heterogeneity using a phosphatidylcholine derivative of 1,6-diphenyl-1,3,5-hexatriene [2-(3-(diphenylhexatriene)propanoyl)-3-palmitoyl-l-α-phosphatidylcholine

The fluorescent probe 1,6-diphenyl-1,3,5-hexatriene (DPH) and DPH derivatives have been used to characterize structural and physicochemical properties of specific membrane domains. Steady-state and fluorescence decay measurements of three probes, DPH (1,6-diphenyl-1,3,5-hexatriene), TMA-DPH [1-(4-trimethyl-ammonium-phenyl)-6-phenyl-1,3,5-hexatriene], and a phosphatidylcholine derivative of DPH, DPH-pPC [2-(3-(diphenylhexatriene)propanoyl)-3-pamitoyl-L-α-phosphatidyl choline], have been performed in erythrocyte membranes and in lymphocyte plasma membranes. The steady-state fluorescence polarization of the three probes showed a similar trend in both membranes. In fact either in erythrocyte or in lymphocyte plasma membranes the fluorescence polarization values of DPH-pPC and TMA-DPH were similar, but significantly higher with respect to DPH. A better characterization of erythrocyte and lymphocyte plasma membranes was possible by using fluorescence decay measurements. The data suggest the possible use of different DPH derivatives to characterize specific domains in biological membranes.

The effect of the cryoprotective sugar, trehalose on the phase behaviour of mixed dispersions of dioleoyl derivatives of phosphatidylethanolamine and phosphatidylcholine

Abstract The phase behaviour of a mixed dispersion of dioleoyl derivatives of phosphatidylethanolamine and phosphatidylcholine (3:1, by weight) in an excess of water was compared with that in 1·8 M trehalose using dynamic X-ray diffraction, differential scanning calorimetry and freeze-fracture electron microscopy. The phase transitions of the fully-hydrated dispersion observed upon heating, proceeded with lamellar-gel→lamellar liquid crystalline→inverse cubic→inverse hexagonal phase at temperatures of −10°C, 49°C, and 67°C, respectively. Our results confirm the existence of an inverse cubic phase of this system and support the model mechanism for the lamellar/non-lamellar phase transition previously suggested by Siegel (1986, Biophys. J. 49, 1155, 1171). Dispersion of the binary lipid mixture in a 1·8 M solution of the naturally occurring cryoprotective disaccharide, trehalose, inhibited the formation of both lamellar liquid crystalline and inverse cubic phases and resulted in a direct transition from lamellar-gel to inverse hexagonal phase at about −6°C. The effect of trehalose is discussed in terms of a ‘water replacement’ model and Hofmeister effects on water structure. Trehalose is regarded as a kosmotropic agent that may also interact directly with the lipid polar groups. Comparison of the relationship between temperature and the dimensions of the inverse hexagonal phase formed in the presence and absence of trehalose suggests that the osmotic effect of the impermeant trehalose prevents water molecules from being taken up by the hexagonal mesophase which is normally more hydrated that the lamellar phase.

A Fluorescence-Based Assay for Human Type II Phospholipase A 2

A fluorescence assay for quantitation of human Type II Phospholipase A 2 activity is described. Hydrolysis of 1-Acyl-2-( N-4-nitrobenzo-2-oxo-1,5-diazole)aminododecanoyl Phosphatidylethanolamine is accompanied by an increase in fluorescence intensity that is linearly proportional to enzyme activity. Substrate is prepared in the absence of detergents as a sonicated dispersion in aqueous buffer. Hydrolysis of the corresponding phosphatidylcholine derivative is more than an order of magnitude slower under identical assay conditions. A plot of initial rate versus substrate concentration could be fit to a simple Michaelis-Menten relationship with K m = 13 μM. In contrast to commonly used radiochemical assays for this enzyme, the method described here is continuous and allows estimation of enzyme activity without separation of substrate from product. Thus, the method is suitable for both kinetic analysis and large-scale screening using automated readers for 96-well tissue culture plates. The fluorescence-based assay displays advantages over other continuous assays for human Type II Phospholipase A 2 based on (a) high sensitivity and (b) the use of a commercially available substrate.

Design, Synthesis, and Evaluation of Phospholipid Analogs as Inhibitors of the Bacterial Phospholipase C from Bacillus cereus

Enzymes belonging to the phospholipase C (PLC) family hydrolyze the phosphodiester bond of phospholipids to give a diacylglycerol and a phosphorylated head group. The bacterial phospholipase C from Bacillus cereus (PLC Bc ) has been studied extensively, and there is a wealth of information regarding those structural features that are important for substrate activity. In contrast, there is virtually no data available regarding structure-activity relationships for inhibitors of this enzyme. To address this shortcoming, a series of optically pure analogues of 1,2-dihexanoyl-sn-glycero-3-phosphocholine (2) containing different replacements of the phosphate group were first synthesized including the phosphoramidates 4 and 8, the phosphonate 5, the (difluoromethylene)phosphonate 6, the thiophosphate 7, the diastereomeric phosphorothioates 9 and 10, and the phosphorodithioate 11. Each of these phosphatidylcholine derivatives was tested for inhibitor or substrate activity with PLC Bc using the water-soluble phosphatidylcholine 2 as the monomeric substrate. The measurements were conducted below the critical micellar concentrations of both 2 and the inhibitor. Of the analogues, only 7 and 9 underwent observable enzymatic hydrolysis under the assay conditions used. The k cat of the (S P )-phosphorothioate 9 was approximately one-fifth that of 2, and when compared to 2,7 was hydrolyzed only very slowly by the enzyme. Kinetic studies indicated that the phospholipid analogues tested were competitive inhibitors with increasing K i 's

The phase behavior of mixed aqueous dispersions of dipalmitoyl derivatives of phosphatidylcholine and diacylglycerol

The phases and transition sequences for aqueous dispersions of mixtures of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and 1,2-dipalmitoyl-sn-glycerol (1,2-DPG) have been studied by differential scanning calorimetry, dynamic x-ray diffraction, freeze-fracture electron microscopy, 31P-nuclear magnetic resonance spectroscopy, and Fourier-transform infrared spectroscopy. The results have been used to construct a dynamic phase diagram of the binary mixture as a function of temperature over the range 20 degrees-90 degrees C. It is concluded that DPPC and 1,2-DPG form two complexes in the gel phase, the first one with a DPPC/1,2-DPG molar ratio of 55:45 and the second one at a molar ratio of approximately 1:2, defining three different regions in the phase diagram. Two eutectic points are postulated to occur: one at a very low 1,2-DPG concentration and the other at a 1,2-DPG concentration slightly higher than 66 mol%. At temperatures higher than the transition temperature, lamellar phases were predominant at low 1,2-DPG concentrations, but nonlamellar phases were found to be predominant at high proportions of 1,2-DPG. A very important aspect of these DPPC/1,2-DPG mixtures was that, in the gel phase, they showed a ripple structure, as seen by freeze-fracture electron microscopy and consistent with the high lamellar repeat spacings seen by x-ray diffraction. Ripple phase characteristics were also found in the fluid lamellar phases occurring at concentrations up to 35.6 mol% of 1,2-DPG. Evidence was obtained by Fourier transform infrared spectroscopy of the dehydration of the lipid-water interface induced by the presence of 1,2-DPG. The biological significance of the presence of diacylglycerol in membrane lipid domains is discussed.

Conductimetric Assays for the Hydrolase and Transferase Activities of Phospholipase D Enzymes

Measurement of solution electrical conductance (conductimetry) is a simple direct assay method for the protogenic, hydrolytic reactions catalyzed by all phospholipase enzymes. The technique is especially suitable for assay of phospholipase D (PLD) enzymes where cleavage of zwitterionic substrates reinforces the pH dependent conductance change and allows the method to be used over a much wider pH range than the equivalent titrimetric assay. The ability to detect zwitterion cleavage enables the method to assay reactions in which phospholipase D transfers neutral, or anionic, alcohol species to the zwitterionic substrates phosphatidyl choline and phosphatidyl ethanolamine. The method can follow the sequential attack by different phospholipases and provides a simple technique for investigating the effect of substrate structure on susceptibility to various phospholipase enzymes. The results confirm that PLD from Streptomyces chromofuscus can attack lysophospholipids, but cannot transfer primary alcohols to the phosphatidyl residue, while the PLD from savoy cabbage is an efficient transferase, but cannot attack lysophospholipids. The data suggest that the bacterial PLD fails to act as a transferase because it hydrolyzes the transphosphatidylation products. Some phosphatidyl alcohols are more highly susceptible to PLA2 attack than the parent phosphatidyl choline derivatives.

1,2-dipalmitoyl phosphatidylcholine, 1,2-dipalmitoyl phosphatidic acid or 1,2-dipalmitoyl- sn-glycerol inhibit sporangia formation and promote exponential growth of various frankia isolates from the casuarinaceae family

Frankia strains from Casuarina (BR, S21, Thr), Allocasuarina (Allo2) and Gymnostoma (G80) genera were found to grow exponentially in stirred, propionate-containing BAP medium supplemented with 1.2 or 2.4 μ m synthetic 1,2-dipalmitoyl phosphatidylcholine, 1,2-dipalmitoyl phosphatidic acid or 1,2-dipalmitoyl- sn-glycerol. Phosphatidylcholines containing C15:0 or C17:0 acyl residues instead of C16:0 (palmitoyl) residues were equally beneficial but stearoyl (C18:0) residues favored the formation of small sporangia. Oleoyl residues (C18:1) appeared to be toxic as they produced a progressive post-exponential decrease in biomass. Our results suggest that phospholipids containing palmitoyl residues are part of the beneficial compounds present in the complex egg yolk phosphatidyl choline mixture which promote exponential growth of Frankia strains, inhibit sporangia formation and delay post-exponential biomass degradation. Phosphatidic acids do not appear to mediate the enhancing effect of phosphatidylcholine derivatives. The fact that 1,2-dipalmitoyl- sn-glycerol alone is beneficial, suggests that the phosphate and choline residues of the phosphatidylcholine molecule are not essential for balanced growth of Frankia under our conditions. Taken together our results suggest that a primary target of the beneficial compounds may be the plasma membrane of Frankia cells.

A Fluorescent Assay for Agonist-Activated Phospholipase D in Mammalian Cell Extracts

Phospholipase D (PLD) is activated in mammalian cells in response to a wide variety of stimuli. A rapid assay for agonist-activated PLD activity in cell extracts was developed, utilizing a fluorescent derivative of phosphatidylcholine as substrate. Utilization of the substrate was assessed following thin-layer chromatography of the reaction mixture. Hydrolysis products generated by phospholipases D, C, and A 2 could be visualized in the same reaction. Phorbol ester and vasopressin increased PLD activity in intact A7r5 vascular smooth muscle cells, as measured by an isotopic labeling method. Using the in vitro fluorescent assay, enhanced PLD activity was detected in membranes prepared from A7r5 cells that had been treated with phorbol ester or vasopressin. The agonist-activated activity was independent of phosphorylation occurring during the course of the assay. PLD activity was detected, in varying amounts, in membranes prepared from a variety of different mouse tissues. These results show that a fluorescent assay can be used to rapidly assess the activity of PLD and other phosphatidylcholine-utilizing phospholipases in cell and tissue extracts. The effects of agonists on PLD activity can be retained and quantitated in a broken cell preparation, permitting characterization of the agonist-activated form of the enzyme.

Interaction of a phosphatidylcholine derivative of 1,6-diphenyl-1,3,5-hexatriene (DPH) with intact living cells: Steady-state fluorescence polarization and phase fluorometry studies

The potential interest of DPH-PC was checked with a macrophagic cell line (P388D1). The uptake of DPH-PC was associated with a rapid increase in both fluorescence intensity and a slow decrease in anisotropy values. A flow cytometry comparative study with DPH revealed in both cases the existence of two cell subpopulations with different labeling levels. The analysis of fluorescence decay of DPH-PC showed two components. The fractional intensity of the main component (9.7 ns) is higher than 92%. The Lorentzian distribution of the main lifetime presents an important homogeneity. The observation that an increase in temperature induced a decrease in steady state anisotropy values but did not affect the lifetime suggests that the anisotropy variations effectively reflect modifications in the cohesion of probe micro-surroundings. A transmembrane diffusional phenomenon of a fraction of fluorescent phospholipids (205) was suggested by a study with a nonpermeant membrane quencher. The transmembrane diffusion was confirmed by extraction of the phospholipid analog with fatty acid free BSA. The use of inhibitors of endogenous phospholipase A2 showed a progressive hydrolysis of the fluorescent phospholipid. Nevertheless, the hydrolysis can be neglected in the case of short term interactions with cells (<30 min). Therefore, it can be assumed that DPH-PC can be used as a membrane probe.

Pyrularia thionin binding to and the role of tryptophan-8 in the enhancement of phosphatidylserine domains in erythrocyte membranes.

Pyrularia thionin is a small, strongly basic peptide which interacts readily with cellular and synthetic membranes. With cells it induces hemolysis, depolarizes the cellular membrane with an accompanying influx of Ca2+, and activates an endogenous phospholipase A2. Evidence points toward a binding site involving phosphatidylserine (PS). This study shows that addition of the peptide to erythrocyte membranes as well as to vesicles formed from phospholipids isolated from erythrocyte membranes causes an enhancement of phospholipid domains which are made visible by the use of fluorescence digital imaging microscopy with fluorescent derivatives of PS (NBD-PS) and phosphatidylcholine (NBD-PC). Addition of thionin caused a large increase in NBD-PS domains, with an accompanying enrichment of NBD-PC in another separate domain. Double-labeling experiments performed with a Texas Red derivative of thionin show that the peptide binds to the domain enriched in NBD-PS. P thionin inactivated by modification of Trp-8 with N-bromosuccinimide lost the ability to enhance PS domains, although it bound to the membrane with the same affinity as native P thionin. This shows that binding to the membrane is not in itself sufficient to cause the NBD-PS and NBD-PC redistribution into domains.

Photoinduced electron transfer reactions oftrans-stilbene surfactants in Langmuir-Blodgett assemblies and phospholipid bilayers

The photochemistry and photophysics of a series of stilbene-functionalized fatty acids (SFA) have been examined in homogeneous solutions, supported Langmuir-Blodgett assemblies, and as guests in phospholipid bilayers and micellar dispersions. The spectroscopic characteristics of stilbene-functionalized derivatives of phosphatidyl choline (SFA-PC) have also been examined in organic solvents, aqueous dispersions and aqueous solution containing an excess of dipalmitoyl choline (DPPC), as well as in aqueous-methanol solutions containing γ-cyclodextrin. Langmuir-Blodgett assemblies of individual SFAs, as well as mixtures of various SFAs, exhibit spectroscopic properties (blue-shifted absorbance and red-shifted fluorescence relative to those observed in organic solvents) consistent with the formation of an “H” aggregate. The same effect is observed for SFA-PCs in aqueous dispersions and in aqueous-methanol solutions containing γ-cyclodextrin. The “H” aggregate is found to be the preferred geometric orientation of the stilbene chromophores which may correspond to an energy minimum for the systems investigated. Preliminary studies with SFA-PC in methanol-water solutions containing γ-cyclodextrin suggest that an association of only two stilbene chromophores is required to form the “H” aggregate.