Diacyl Lipids Research Articles

Molecular Dynamics Simulation Studies of Cardiolipin Bilayers

Molecular dynamics (MD) simulations of tetramyristoyl cardiolipin (TMCL) and tetraoleoyl cardiolipin (TOCL) were carried out with the newly developed CHARMM lipid force field (FF), C36, and with head group charges q= −1 and −2. The surface areas per lipid, AL, for q=-1 are 126 ± 0.1 Å2 for TOCL and 111 ± .1 Å2 for TMCL. These are 1.8 times than those of the diacyl equivalents: 63 Å2 for dimyristoylphosphatidylcholine (DMPC) at 328K, and 69 Å2 for dioleoylphosphatidylcholine (DOPC) at 310K. Area compressibility, Ka, of TOCL equals 340 ± 40 dyn/cm, approximately 50% higher than experimentally obtained for DOPC (and most diacyl lipids); an experimental value for Ka for cardiolipins is not presently available. The areas and compressibilities for TOCL from the present simulation differ substantially from those obtained by Dahlberg and Maliniak using the FF of Berger et al. under the same conditions (AL= 99 Å2; Ka = 1100 dyn/cm). The origin of the differences appears to be in the ion binding to the surfaces of the cardiolipin bilayer. Under FF of Berger et al., ions bind closer to the carbonyl group in the lipid chain region whereas C36 CHARMM FF results ion binding closer to the negatively charged phosphate groups of the head group. Deuterium order parameter measurements are underway to determine which FF yields areas more representative of the fluid state of cardiolipin bilayers.

Effect of Cholesterol Nanodomains on the Targeting of Lipid-Based Gene Delivery in Cultured Cells

Targeted gene delivery offers immense potential for clinical applications. Liposomes decorated with targeting ligands have been extensively used for both in vitro and in vivo gene delivery. Lipoplexes with high cholesterol content that result in cholesterol domain formation within the complexes have been shown to exhibit enhanced transfection in vitro and resistance to serum-induced aggregation. In the present study, folate was employed as a targeting ligand that was conjugated with either cholesterol or a diacyl lipid (DSPE), and these conjugates were incorporated into lipoplexes formulated with DOTAP/cholesterol (wt/wt: 31/69) that are known to possess cholesterol nanodomains. Cellular uptake and transfection of these lipoplexes in the presence of 50% serum were examined when the ligand was located within or excluded from the cholesterol nanodomain. Lipoplexes with folate-cholesterol exhibited a 50-fold increase in transfection compared to those with folate-DSPE, while the cellular uptake level is only 40% of that with folate-DSPE. These results indicate that the presence of the ligand within the cholesterol domain promotes more productive transfection in cultured cells, and intracellular trafficking of the lipoplexes after entry into cells plays a crucial role in gene delivery.

Interactions of lipid-based liquid crystalline nanoparticles with model and cell membranes

Lipid-based liquid crystalline nanoparticles (LCNPs) are interesting candidates for drug delivery applications, for instance as solubilizing or encapsulating carriers for intravenous (i.v.) drugs. Here it is important that the carriers are safe and tolerable and do not have, e.g. hemolytic activity. In the present study we have studied LCNP particles of different compositions with respect to their mixing behavior and membrane destabilizing effects in model and cell membrane systems. Different types of non-lamellar LCNPs were studied including cubic phase nanoparticles (Cubosome ®) based on glycerol monooleate (GMO), hexagonal phase nanoparticles (Hexosome ®) based on diglycerol monooleate (DGMO) and glycerol dioleate (GDO), sponge phase nanoparticles based on DGMO/GDO/polysorbate 80 (P80) and non-lamellar nanoparticles based on soy phosphatidylcholine (SPC)/GDO. Importantly, the LCNPs based on the long-chain monoacyl lipid, GMO, were shown to display a very fast and complete lipid mixing with model membranes composed of multilamellar SPC liposomes as assessed by a fluorescence energy transfer (FRET) assay. The result correlated well with pronounced hemolytic properties observed when the GMO-based LCNPs were mixed with rat whole blood. In sharp contrast, LCNPs based on mixtures of the long-chain diacyl lipids, SPC and GDO, were found to be practically inert towards both hemolysis in rat whole blood as well as lipid mixing with SPC model membranes. The LCNP dispersions based on a mixture of long-chain monoacyl and diacyl lipids, DGMO/GDO, displayed an intermediate behavior compared to the GMO and SPC/GDO-based systems with respect to both hemolysis and lipid mixing. It is concluded that GMO-based LCNPs are unsuitable for parenteral drug delivery applications (e.g. i.v. administration) while the SPC/GDO-based LCNPs exhibit good properties with limited lipid mixing and hemolytic activity. The correlation between results from lipid mixing or FRET experiments and the in vitro hemolysis data indicates that FRET assays can be one useful screening tool for parenteral drug delivery systems. It is argued that the hemolytic potential is correlated with chemical activity of the monomers in the mixtures.

Characterization of Oxidized Phosphatidylethanolamine Derived from RAW 264.7 Cells Using 4-(Dimethylamino) Benzoic Acid Derivatives

Recently, a derivative of PE, namely the 4-(dimethylamino)benzoic acid derivative has been developed with various isotope labeled variants that provided a universal precursor ion scan for diacyl, ether and plasmalogen PE lipids that can not be accomplished otherwise. This derivative was further investigated as a means to facilitate characterization of various oxidized phosphatidylethanolamine lipids by collision activation. Phospholipids derived from RAW 264.7 cells were treated with a free radical generating system to generate a complex mixture of oxidized and non-oxidized lipids that were separated by reversed-phase high-performance liquid chromatography and detected using a precursors of m/z 191 scan for the d(0)-DMABA-labeled control sample and a precursor of m/z 197 scan for the d(6)-DMABA-labeled oxidized sample. Collisional activation of the corresponding [M - H](-) ions permitted the identification of several chain shortened omega-aldehydes, as well as direct oxygen addition products including isoprostane PE and monohydroxy PE oxidized phospholipids that were not easily detected without the use of the DMABA derivatives. The stable isotope labeled derivatives permitted assessment of relative quantitative changes in oxidized lipids based upon ion abundance.

Stable Isotope Labeled 4-(Dimethylamino)benzoic Acid Derivatives of Glycerophosphoethanolamine Lipids

A set of four (D(0), D(4), D(6), and D(10)) deuterium enriched 4-(dimethylamino)benzoic acid (DMABA) N-hydroxysuccinimide (NHS) ester reagents was developed that react with the primary amine group of glycerophosphoethanolamine (PE) lipids to create derivatives where all subclasses of DMABA labeled PE are detected by a common precursor ion scan. The positive ion collision induced dissociation data from (D(0), D(4), D(6), and D(10))-DMABA labeled PE standards indicated that a precursor ion scan of m/z 191.1, 195.1, 197.1, and 201.1 could be used to selectively detect (D(0), D(4), D(6), and D(10))-DMABA modified PE, respectively, in a complex biological mixture. The PE lipids from a time course (0, 30, 60, and 300 min) of 2,2'-azobis-(2-amidinopropane) hydrochloride (AAPH) treatment of liposomes made of RAW 264.7 cell phospholipids were each labeled with the D(0)-, D(4)-, D(10)-, and D(6)-DMABA NHS ester reagents, respectively. The DMABA derivatives revealed loss of endogenous PE lipids and an increase in oxidized PE lipid throughout the time course of AAPH treatment. These DMABA NHS ester reagents provide a universal scan for diacyl, ether, and plasmalogen PE lipids that cannot be readily observed otherwise, enable differential labeling, and provide an internal standard for each PE lipid.

Photodynamic therapy of experimental B-16 melanoma in mice with tumor-targeted 5,10,15,20-tetraphenylporphin-loaded PEG-PE micelles

Polyethylene glycol (PEG)-diacyl lipid micelles have been prepared by loading with the hydrophobic meso-5,10,15,20-tetraphenyl-21H,23H-porphine (TPP) and used for the photodynamic treatment of B-16 melanoma cells in vitro and in vivo. The use of PEG-PE micelles allowed for a 150-fold increased the solubilization of TPP, compared with the native drug. The average size of the PEG-PE micelles was in the range of 10–12 nm with a narrow size distribution. At 50 μg/ml of TPP in micelles with an irradiation intensity of 4.5–21.5 mW/cm2, the viability of B-16 melanoma cells in vitro decreased in a fluence-dependent manner. A highly effective outcome of photodynamic therapy (PDT) with TPP-loaded PEG-PE micelles can be further increased by modifying such micelles with cancer-specific monoclonal antibody 2C5 to TPP-loaded micelles to tumor cells. TPP-containing 2C5-modified micelles provided the strongest phototoxic effect against B-16 cells in vitro compared with TPP-loaded plain micelles at the same TPP concentration. The association of TPP-loaded immuno-targeted micelles with melanoma cells was also studied by flow cytometry. An increase in cell association was found for 2C5-targeted micelles compared with non-targeted micelles. In vivo, the PDT treatment of subcutaneous melanoma-bearing C57BL/6 mice with 100 mW/cm2 of 630 nm laser light 9 h after the administration of the micellar TPP (1 mg/kg of TPP) resulted in a significant inhibition of tumor growth. Compared with controls, the weight of postmortem tumors was approx. 3.5- and 7.5-fold smaller with TPP-loaded PEG-PE micelles and TPP-loaded PEG-PE 2C5-immunomicelles, respectively.

Effects of Cholesterol on Dry Bilayers: Interactions between Phosphatidylcholine Unsaturation and Glycolipid or Free Sugar

Cholesterol and other sterols are important components of biological membranes and are known to strongly influence the physical characteristics of lipid bilayers. Although this has been studied extensively in fully hydrated membranes, little is known about the effects of cholesterol on the stability of membranes in the dry state. Here, we present a Fourier transform infrared spectroscopy study on the effects of cholesterol on the phase behavior of dry liposomes composed of phosphatidylcholines with different degrees of fatty acid unsaturation or of mixtures of phosphatidylcholine with a plant galactolipid. In addition, we have analyzed the H-bonding of cholesterol, galactose, and a combination of the two additives to the P=O and C=O groups in dry phosphatidylcholine bilayers. The data indicate a complex balance of interactions between the different components in the dry state and a strong influence of fatty acid unsaturation on the interactions of the diacyl lipids with both cholesterol and galactose.

A Novel Type of Membrane Based on Cholesteryl Phosphocholine, Cholesteryl Phosphate, or Sitosteryl Phosphate, and Dimyristoylglycerol

Mixtures of amphiphilic cholesteryl phosphate (CP), sitosteryl phosphate (SP), or cholesteryl phosphocholine (CPC) with the nonphosphoryl diacyl lipid dimyristoylglycerol (DMG) or with cholesterol give self-organized systems (giant vesicles) in a wide range of pH, as demonstrated by fluorescence microscopy, differential scanning calorimetry, and small-angle X-ray scattering. The water permeability of a 1 : 1 molar mixture of CPC and DMG was also measured by a stopped-flow/light-scattering method. The novel self-organized systems are akin to natural eukaryotic ones, the only difference being the site of the phosphate-containing head-group, located on cholesterol instead of DMG. They might be present in some organisms not yet studied for the composition of their membranes.

Letters to www-finishing-com/Letters

Cyanobacteria are photoautotrophic prokaryotes with a plant-like photosynthetic machinery. Because of their short generation times, the ease of their genetic manipulation, and the limited size of their genome and proteome, cyanobacteria are popular model organisms for photosynthetic research. Although the principal mechanisms of photosynthesis are well-known, much less is known about the biogenesis of the thylakoid membrane, hosting the components of the photosynthetic, and respiratory electron transport chain in cyanobacteria. Here we present a detailed proteome analysis of the important model and host organism Synechocystis sp. PCC 6803 under light-activated heterotrophic growth conditions. Because of the mechanistic importance and severe changes in thylakoid membrane morphology under light-activated heterotrophic growth conditions, a focus was put on the analysis of the membrane proteome, which was supported by a targeted lipidome analysis. In total, 1528 proteins (24.5% membrane integral) were identified in our analysis. For 641 of these proteins quantitative information was obtained by spectral counting. Prominent changes were observed for proteins associated with oxidative stress response and protein folding. Because of the heterotrophic growth conditions, also proteins involved in carbon metabolism and C/N-balance were severely affected. Although intracellular thylakoid membranes were significantly reduced, only minor changes were observed in their protein composition. The increased proportion of the membrane-stabilizing sulfoqinovosyl diacyl lipids found in the lipidome analysis, as well as the increased content of lipids with more saturated acyl chains, are clear indications for a coordinated synthesis of proteins and lipids, resulting in stabilization of intracellular thylakoid membranes under stress conditions.

Dynamics in Diether Lipid Bilayers and Interdigitated Bilayer Structures Studied by Time-Resolved Emission Spectra, Decay Time and Anisotropy Profiles

We present a comparative fluorescence spectroscopic investigation of diacyl and diether phosphatidylcholine vesicles using different probes with well-defined localization within either the hydrophilic headgroup region or the hydrophobic part of the bilayer. Time-resolved emission spectra have been used to characterize the solvent relaxation behavior in both symmetric and asymmetric diether and diacyl phosphatidylcholines. It is shown that time-resolved emission spectra of Prodan (6-propionyl-2-(dimethylamino)-naphthalene) and its long-alkyl chain derivative Patman (6-palmitoyl-2-[[trimethylammoniumethyl]methylamino]-naphthalene chloride) are a sensitive tool for the detection of differences in the micropolarities and viscosities at the hydrophobic/hydrophilic membrane interface of diether and diacyl lipids, respectively. Moreover, a new approach for the detection of interdigitated bilayers is discussed. It relies on the construction of anisotropy and decay time profiles for the set of n-anthroyloxy fatty acids and is compared with an older fluorescence assay based on intensity measurements only. The shape of plots of the fluorescence steady-state anisotropy versus the position of the chromophore (anthracene-9-carboxylic acid) combined with fluorescence lifetime measurements can be used to differentiate among non-fully, and mixed interdigitated gel phase structures and to predict structures for new lipid species.

Electron density modeling and reconstruction of infinite periodic minimal surfaces (IPMS) based phases in lipid-water systems. II. Reconstruction of D surface based phases

This is the second of two papers dealing with the structure of lipid-water phases based on Infinite Periodic Minimal Surfaces (IPMS). The first paper describes mathematical modeling of such phases. In this paper, a new reconstruction method, called the methyl trough search, is described and used to solve the structures based on powder pattern X-ray diffraction data. Structures are derived for both a single chain lipid-water system (mono-olein) and a diacyl phospholipid-water system (2-2 methyl butyl 16:0 phosphatidylcholine). The methyl trough search uses the low electron density of the lipid methyl tails to determine the correct phasing for the electron density reconstruction. The data are consistent with a structure based on the IPMS D surface. The results are compared to other methods used to solve the mono-olein structure; the structure of the diacyl lipid has never before been solved. We discuss the subtleties involved in reconstruction of D surface based phases and the substantial artifacts that arise in low-resolution reconstructions of hydrocarbon lipids lacking heavy-atom sites.

Thermodynamic Analysis of Chain-Melting Transition Temperatures for Monounsaturated Phospholipid Membranes: Dependence on cis-Monoenoic Double Bond Position

Unsaturated phospholipid is the membrane component that is essential to the dynamic environment needed for biomembrane function. The dependence of the chain-melting transition temperature, T t, of phospholipid bilayer membranes on the position, n u, of the cis double bond in the glycerophospholipid sn-2 chain can be described by an expression of the form T t = T t c(1 + h′ c| n u − n c|)/(1 + s′ c| n u − n c|), where n c is the chain position of the double bond corresponding to the minimum transition temperature, T t c, for constant diacyl lipid chain lengths. This implies that the incremental transition enthalpy (and entropy) contributed by the sn-2 chain is greater for whichever of the chain segments, above or below the double-bond position, is the longer. The critical position, n c, of the double bond is offset from the center of the sn-2 chain by an approximately constant amount, δn c ≈ 1.5 C-atom units. The dependence of the parameters T t c, h′ c, and s′ c on sn-1 and sn-2 chain lengths can be interpreted consistently when allowance is made for the chain packing mismatch between the sn-1 and sn-2 chains. The length of the sn-2 chain is reduced by ∼0.8 C-atom units by the cis double bond, in addition to a shortening by ∼1.3 C-atom units by the bent configuration at the C-2 position. Based on this analysis, a general thermodynamic expression is proposed for the dependence of the chain-melting transition temperature on the position of the cis double bond and on the sn-1 and sn-2 chain lengths. The above treatment is restricted mostly to double-bond positions close to the center of the sn-2 chain. For double bonds positioned closer to the carboxyl or terminal methyl ends of the sn-2 chain, the effects on transition enthalpy can be considerably larger. They may be interpreted by the same formalism, but with different characteristic parameters, h′ c and s′ c, such that the shorter of the chain segments makes a considerably smaller contribution to the calorimetric properties of the chain-melting transition.

Time-resolved emission spectra and anisotropy profiles for symmetric diacyl- and dietherphosphatidylcholines

The solvent relaxation behavior of Patman (6-palmitoyl-2-[[2-(trimethylammonium) ethyl]methylamino]naphthalene chloride) was investigated in small unilamellar vesicles composed of symmetric diacyl( 1,2-dipalmitoylphosphatidylcholine; DPPC) and diether lipids (l,2-dihexadecylphosphatidylcholine; DHPC), calculating time-resolved emission spectra (TRES) and correlation functions. Both the steady-state spectra as a function of temperature and excitation wavelength and the TRES of Patman in DPPC are blue-shifted compared to those in DHPC. The solvent relaxation at three temperatures above and below the phase transition is considerably faster in DHPC than in DPPC. As the steady-state anisotropies of Patman and TMA-DPH [l-(4-trimethylammoniumphenyl)-6-phenyl-l,3,5-hexatriene] are similar in both lipids as a function of both temperature and emission wavelength, we conclude that the introduction of ether linkages allows more efficient water penetration in the glycerol region, leading to a more polar environment and therefore faster solvent relaxation of the incorporated dyes. Using a series ofn-(9-anthroyloxy) fatty acids (n = 2, 3, 6, 9, 12; 16-AP), we show that anisotropy profiles can be used to distinguish between noninterdigitated (DPPC) and fully interdigitated (DHPC) gel-phase structures. 16-(9Antroyloxy) palmitic acid (16-AP) is an especially useful probe exhibiting pronounced differences in the steady-state anisotropies in non- and fully interdigitated gel phases.

Effect of hypoxia on steady-state arachidonic acid metabolism in bovine aortic endothelial cells.

At the onset of acute hypoxia, eicosanoid synthesis by bovine aortic endothelial cells (BAEC) markedly decreases, reflecting a decreased release of arachidonic acid from endogenous stores. To determine the cause of decreased arachidonic acid release, we pulse-labeled BAEC with [14C]arachidonic acid for 5 min under normoxic conditions and chased cells for 1 h under normoxic or hypoxic conditions. The 14C incorporation and specific activity (disintegrations per minute per nanomole) of three major arachidonyl molecular species (16:0-20:4, 18:1-20:4, and 18:0-20:4) of each phospholipid class were determined in cells chased under either of the two conditions. There was no relevant difference between normoxic and hypoxic cells in the metabolism of any of the arachidonyl molecular species of diacyl lipids. However, there was a marked decrease (approximately 40%) in the turnover of arachidonyl alkenylacyl phosphatidylethanolamine in the hypoxic cells. From these results, it appears that the source of arachidonic acid supporting constitutive eicosanoid synthesis in BAEC is alkenylacyl phosphatidylethanolamine and that the limiting enzyme activity determining the rate of eicosanoid synthesis is a plasmalogen-specific phospholipase A2.

Plasma factors affecting the in vitro conversion of high-density lipoproteins labeled with a non-transferable marker

We studied the in vitro conversion of HDL 3 labeled with a radioiodinated diacyl lipid associating peptide (diLAP). DiLAP was previously shown to be nontransferable, which permitted its' use as a reliable marker of HDL particles. DiLAP-labeled HDL 3 was incubated for 23 h at 37°C in human or rat plasma or in reconstituted media containing delipidated plasma and/or lipoproteins and/or partially purified CETP. At the end of the incubations, the samples were adjusted to a density of 1.125 g/ml and ultracentrifuged. The two resulting fractions containing HDL 2 and HDL 3, respectively, were analyzed by gradient gel electrophoresis. Depending upon experimental conditions, diLAP-labeled HDL 3 was converted into HDL 2b- and/or small HDL 3c-like particles. LCAT inhibition and to a lesser extent CETP promoted the formation of small HDL 3c. Reactivation of LCAT led to the disappearance of small HDL 3c. No HDL 3c formed from HDL 2 even in the absence of LCAT activity. When the incubations were performed in the presence of 100 mM thimerosal, which inhibited PLTP but not CETP activity, the conversion of diLAP-labeled HDL 3 into HDL 2 was almost completely blocked. Collective consideration of these data indicates that the formation of small HDL is moderately facilitated by CETP; that small HDL are converted to larger HDL species by LCAT and that the transformation of HDL 3 into HDL 2 is a process which largely depends upon PLTP activity.

Interbilayer transfer of phospholipid-anchored macromolecules via monomer diffusion.

A series of conjugates has been prepared by linking various hydrophilic macromolecules [poly-(ethylene glycols), polylysine, aminodextrans, or apotransferrin] to synthetic phosphatidylethanolamines via linker moieties incorporating a fluorescent bimane group. Using a fluorescence energy transfer-based assay, the rate of transfer of these species between phospholipid vesicles has been monitored as a function of the nature and size of the coupled macromolecule and of the acyl chain composition of the lipid anchor. Conjugates in which the phospholipid anchor is linked to a small hydrophilic terminal residue (e.g., ethanolamine or ethylenediamine) transfer between large unilamellar vesicles of egg phosphatidylcholine with half-times ranging from tens of minutes (for dimyristoyl lipid conjugates) to a few tens of hours (for dipalmitoyl and 1-palmitoyl-2-oleoyl lipid conjugates), in agreement with previous results for unlabeled phospholipids. Conjugation of these same lipid anchors to larger hydrophilic molecules markedly accelerates their rates of intermembrane transfer, by factors ranging from 5-7-fold (for conjugates with apotransferrin and aminodextrans of molecular weight 10,000-70,000) to over 25-fold [for conjugates with poly(ethylene glycol)-5000]. In all cases the observed transfer appears to reflect the diffusion of lipid monomers through the aqueous phase. Our results suggest that substantial intermembrane transfer can occur, on a time scale of several hours or less, for hydrophilic macromolecules conjugated to diacyl(/alkyl) lipids with 14- to 18-carbon chains unless portions of the conjugate other than the lipid anchor also interact strongly with the membrane.

The platelet-activating factor precursor of the injured cornea is selectively implicated in arachidonate and eicosanoid release.

The purpose of this study was to isolate the platelet-activating factor (PAF) precursor and other choline phosphoglycerides (GPC) i.e. the alkenylacyl and diacyl lipids from the rabbit cornea, to analyze their fatty acid content and to determine which pool was the most susceptible to arachidonate depletion when activated corneal tissue released arachidonic acid (AA) and metabolites. Rabbit iridal GPC was also analyzed for comparative purposes. The fatty acid methyl esters of the GPC components extracted from the rabbit cornea and iris-ciliary body, isolated by high performance liquid chromatography (HPLC), were determined by capillary gas liquid chromatography. Rabbit corneas were labelled in vivo by intracameral injection of 3H-AA (1 microCi, specific activity = 218 Ci/mmol) and cryogenically injured 18 h later. Corneas were incubated in vitro and the AA and eicosanoids released into the medium were extracted and separated by HPLC. The GPC was extracted from the tissues and the labeling of the three GPC constituents was quantified by liquid scintillation counting. The corneal and iridal PAF precursor represented 4.1 +/- 0.2% and 2.9 +/- 0.2% respectively of total GPC in those tissues. On a mole basis, the alkyl arachidonoyl species constituted 12.7 +/- 0.7% of the corneal and 38 +/- 0.6% of the iridal PAF precursors respectively. The release of AA and prostaglandins by the cornea was linear until 15 min; whereas 12-HETE levels continuously increased until 60 min. All GPC components lost label but 1-O-alkyl-2-arachidonoyl was the most affected, with its labeled content 50% less than the non-injured control.(ABSTRACT TRUNCATED AT 250 WORDS)

Secondary electron transfer reactions of the isolated Photosystem II reaction centre after reconstitution with plastoquinone-9 and diacylglycerolipids

A Photosystem II (PS II) reaction centre preparation isolated with Triton X-100 and stabilised by transfer to dodecylmaltoside has been used in a reconstitution procedure with diacyl lipids and the naturally occurring PS II quinone, plastoquinone-9 (PQ9). As shown previously, this isolated protein complex consists of the D1, D2, b -559 α and β polypeptides, plus the psb I gene product. Before reconstitution there was only about 0.004 mol PQ9 and 0.2 mol diacyl lipid per mole chlorophyll a associated with the isolated complex. After reconstitution and precipitation by centrifugation the complex was found to have an associated lipid matrix of 1.3 mol PQ9 and 3.7 mol diacyl lipid for each mole of chlorophyll a . After reconstitution, the presence of a functional quinone in the PS II reaction centre was indicated by a strong thermoluminescence signal with a peak emission at −60°C. This was recognised as the previously characterised Z v signal on the basis of the relationship between emission and excitation temperatures and a complete quenching by ethanol. This signal was essentially unaltered by addition of 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) and was the same after reconstitution with phosphatidylcholine or a thylakoid lipid extract. Calculation of the activation free energy for the charge separation reaction which gave rise to this signal suggested that this was 0.48 ± 0.01 eV and was the same as for a smaller Z v signal detected in the isolated complex before reconstitution with PQ9. When duroquinone was used this value was not significantly different, although a much smaller signal was observed. With decylplastoquinone the thermoluminescence peak was as large as with PQ9 but the difference in shape indicated that the activation free energy was significantly higher at 0.55 ± 0.02 eV. Photosynthetic electron transfer in the reconstituted complex was assayed by reduction of 2,6-dichlorophenolindophenol (DCPIP) with diphenylcarbazide (DPC) as an artificial electron donor. A low residual rate was strongly enhanced by reconstitution with PQ9. This activity was greater when reconstitution was with a thylakoid lipid extract than with phosphatidylcholine and was inhibited by addition of DCMU ( I 50 = 0.2 mM).

Activation of phospholipase A2 by adriamycin in vitro. Role of drug-lipid interactions.

To probe adriamycin-phospholipid interactions, the effects of this cytotoxin on the hydrolysis of a pyrene-labeled acidic alkyl-acyl phospholipid analog 1-octa-cosanyl-2-(6-pyren-1-yl)hexanoyl-sn-glycero-3-phos p hatidylmethanol (C28-O-PHPM) by porcine pancreatic phospholipase A2 (PLA2) were studied. In the absence of added Ca2+ adriamycin caused a 3-4-fold activation of hydrolysis of this pyrenelipid whereas an inhibition of action of PLA2 on the corresponding phosphatidylcholine derivative C28-O-PHPC was observed. Under similar conditions adriamycin also enhanced the rate of hydrolysis of the pyrene-labeled diacyl lipid 1-palmitoyl-2-(pyren-1-yl)hexanoyl-sn-glycero-3-phosphatidylgly cer ol and inhibited the hydrolysis of PLA2 on the phosphatidylcholine derivative. Increasing calcium concentrations abolished the activating and most of the inhibitory effects of adriamycin with the above phospholipid substrates. Quenching of pyrene excimer fluorescence by adriamycin revealed efficient binding of the drug to acidic lipids. Addition of 1 mM calcium reduced fluorescence quenching by adriamycin maximally by approximately 90%. In comparison, quenching by adriamycin of pyrene-labeled phosphatidylcholine was much weaker and calcium had only an insignificant effect. Monolayer experiments at an air/water interface showed a rapid and surface pressure-dependent penetration of the drug into a film of C28-O-PHPM. Increase in surface pressure was reversed by 80% by the inclusion of 1 mM Ca2+ into the subphase. Penetration of adriamycin into a monolayer of C28-O-PHPC was much weaker. In agreement with earlier studies two types of binding of adriamycin to C28-O-PHPM are proposed.

Intermolecular interactions in lipid/carotenoid monolayers.

The collapse pressure at the air-water interface of monomolecular films of 1,2-distearoyl derivatives of phosphatidylcholine and digalactosyldiacylglycerol containing various proportions of the carotenoid astaxanthin was related to the composition of the monolayer. The results were analysed by using a regular-association approximation by which it is assumed that there is a stepwise formation of ABi-type associations where A and B represent the diacyl lipid and astaxanthin respectively and 1 less than or equal to i less than or equal to 6. This treatment provides an adequate description of the experimental data and permits calculation of equilibrium constants for the steps in complex-formation; each step is said to have the same equilibrium constant. The value for the collapse-surface-pressure increment associated with formation of ABi complexes was also derived. Calculated values of equilibrium constants and collapse-surface-pressure increments are greater for phosphatidylcholine/astaxanthin mixed monolayers than for digalactosyldiacylglycerol/astaxanthin mixed monolayers. These differences are discussed in terms of intermolecular interactions between components in the two systems.