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PubMed2005

Lipids, lipid rafts and caveolae: their importance for GPCR signaling and their centrality to the endocannabinoid system.

Scientific views of cell membrane organization are presently changing. Rather than serving only as the medium through which membrane proteins diffuse, lipid bilayers have now been shown to form compartmentalized domains with different biophysical properties (rafts/caveolae). For membrane proteins such as the G protein coupled receptors (GPCRs), a raft domain provides a platform for the assembly of signaling complexes and prevents cross-talk between pathways. Lipid composition also has a strong influence on the conformational activity of GPCRs. For certain GPCRs, such as the cannabinoid receptors, the lipid bilayer has additional significance. Endocannabinoids such as anandamide (AEA) are created in a lipid bilayer from lipid and act at the membrane embedded CB1 receptor. Endocannabinoids exiting the CB1 receptor are transported either by a carrier-mediated or a simple diffusion process to the membrane of the postsynaptic cell. Following cellular uptake, perhaps via caveolae/lipid raft-related endocytosis, AEA is rapidly metabolized by a membrane-associated enzyme, fatty acid amide hydrolase (FAAH) located in the endoplasmic reticulum. The entry point for AEA into FAAH appears to be from the lipid bilayer. This review explores the importance of lipid composition and lipid rafts to GPCR signaling and then focuses on the intimate relationship that exists between the lipid environment and the endocannabinoid system.

Amidohydrolases↗
PubMed2002

Characterization of the inhibitory effect of PEG-lipid conjugates on the intracellular delivery of plasmid and antisense DNA mediated by cationic lipid liposomes.

Poly(ethylene glycol)-lipid (PEG-lipid) conjugates are widely used in the field of liposomal drug delivery to provide a polymer coat that can confer favorable pharmacokinetic characteristics on particles in the circulation. More recently these lipids have been employed as an essential component in the self-assembly of cationic and neutral lipids with polynucleic acids to form small, stable lipid/DNA complexes that exhibit long circulation times in vivo and accumulate at sites of disease. However, the presence of a steric barrier lipid might be expected to inhibit the transfection activity of lipid/DNA complexes by reducing particle-membrane contact. In this study we examine what effect varying the size of the hydrophobic anchor and hydrophilic head group of PEG-lipids has on both gene and antisense delivery into cells in culture. Lipid/DNA complexes were made using unilamellar vesicles composed of 5 mole% PEG-lipids in combination with equimolar dioleoylphosphatidylethanolamine and the cationic lipid dioleyldimethylammonium chloride. Using HeLa and HepG2 cells we show that under the conditions employed PEG-lipids had a minimal effect on the binding and subsequent endocytosis of lipid/DNA complexes but they severely inhibited active gene transfer and the endosomal release of antisense oligodeoxynucleotides into the cytoplasm. Decreasing the size of the hydrophobic anchor or the size of the grafted hydrophilic PEG moiety enhanced DNA transfer by the complexes.

Cell Nucleus↗
PubMed2002

A two-photon view of an enzyme at work: Crotalus atrox venom PLA2 interaction with single-lipid and mixed-lipid giant unilamellar vesicles.

We describe the interaction of Crotalus atrox-secreted phospholipase A2 (sPLA2) with giant unilamellar vesicles (GUVs) composed of single and binary phospholipid mixtures visualized through two-photon excitation fluorescent microscopy. The GUV lipid compositions that we examined included 1-palmitoyl-2-oleoyl-phosphatidylcholine, 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), and 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) (above their gel-liquid crystal transition temperatures) and two well characterized lipid mixtures, 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE):DMPC (7:3) and 1,2-dilauroyl-sn-glycero-3-phosphocholine (DLPC)/1,2-diarachidoyl-sn-glycero-3-phosphocholine (DAPC) (1:1) equilibrated at their phase-coexistence temperature regime. The membrane fluorescence probes, 6-lauroyl-2-(dimethylamino) napthalene, 6-propionyl-2-(dimethylamino) naphthalene, and rhodamine-phosphatidylethanolamine, were used to assess the state of the membrane and specifically mark the phospholipid domains. Independent of their lipid composition, all GUVs were reduced in size as sPLA2-dependent lipid hydrolysis proceeded. The binding of sPLA2 was monitored using a fluorescein-sPLA2 conjugate. The sPLA2 was observed to associate with the entire surface of the liquid phase in the single phospholipid GUVs. In the mixed-lipid GUV's, at temperatures promoting domain coexistence, a preferential binding of the enzyme to the liquid regions was also found. The lipid phase of the GUV protein binding region was verified by the introduction of 6-propionyl-2-(dimethylamino) naphthalene, which partitions quickly into the lipid fluid phase. Preferential hydrolysis of the liquid domains supported the conclusions based on the binding studies. sPLA2 hydrolyzes the liquid domains in the binary lipid mixtures DLPC:DAPC and DMPC:DMPE, indicating that the solid-phase packing of DAPC and DMPE interferes with sPLA2 binding, irrespective of the phospholipid headgroup. These studies emphasize the importance of lateral packing of the lipids in C. atrox sPLA2 enzymatic hydrolysis of a membrane surface.

2-Naphthylamine↗
PubMed1999

Binding of peripheral proteins to mixed lipid membranes: effect of lipid demixing upon binding.

Binding isotherms have been determined for the association of horse heart cytochrome c with dioleoyl phosphatidylglycerol (DOPG)/dioleoyl phosphatidylcholine (DOPC) bilayer membranes over a range of lipid compositions and ionic strengths. In the absence of protein, the DOPG and DOPC lipids mix nearly ideally. The binding isotherms have been analyzed using double layer theory to account for the electrostatics, either the Van der Waals or scaled particle theory equation of state to describe the protein surface distribution, and a statistical thermodynamic formulation consistent with the mass-action law to describe the lipid distribution. Basic parameters governing the electrostatics and intrinsic binding are established from the binding to membranes composed of anionic lipid (DOPG) alone. Both the Van der Waals and scaled particle equations of state can describe the effects of protein distribution on the DOPG binding isotherms equally well, but with different values of the maximum binding stoichiometry (13 lipids/protein for Van der Waals and 8 lipids/protein for scaled particle theory). With these parameters set, it is then possible to derive the association constant, Kr, of DOPG relative to DOPC for surface association with bound cytochrome c by using the binding isotherms obtained with the mixed lipid membranes. A value of Kr (DOPG:DOPC) = 3.3-4.8, depending on the lipid stoichiometry, is determined that consistently describes the binding at different lipid compositions and different ionic strengths. Using the value of Kr obtained it is possible to derive the average in-plane lipid distribution and the enhancement in protein binding induced by lipid redistribution using the statistical thermodynamic theory.

Animals↗
PubMed2003

Solid lipid microparticle formulations of the pyrethroid gamma-cyhalothrin-incompatibility of the lipid and the pyrethroid and biological properties of the formulations.

Pyrethroids such as gamma-cyhalothrin (GCH) are valuable insecticides that possess a high and unwanted toxicity towards aquatic organisms. The aim of the present study was to test the ability of the solid lipid nanoparticle technology to reduce the aquatic toxicity and concurrently retain the insecticidal activity of GCH. Applying the lipid Compritol) 888 ATO and homogenising the crude o/w emulsions of melted GCH and compritol at different pressures, 150-1500 bar, solid lipid microparticle formulations were produced having average particle diameters between 0.3 and 100 microm. GCH had limited solubility in the solid lipid phase and probably the lipid showed transition from the alpha to the beta' crystal form upon storage. This resulted in expulsion of GCH from the lipid and appearance of GCH crystals in the water phase of the formulations. By using as surfactant polyvinyl alcohol instead of a mixture of Synperonic PE/F68 and sodium lauryl sulphate the appearance of GCH crystals was delayed. This delay was probably due to the fact that the GCH solubility was significantly lower in aqueous polyvinyl alcohol solutions than in solutions of the above-mentioned surfactant mixture. Compared with a traditional emulsifiable concentrate formulation of GCH the solid lipid microparticle formulations reduced the toxicity towards fish (Brachydanio rerio) and daphnia (Daphnia magna) by a factor 10 and 63, respectively. The solid lipid microparticle formulations and the emulsifiable concentrate formulation had about the same insecticidal activity on both Dysdercus cingulatus nymphs and Spodoptera littoralis larvae. Surprisingly the particle size of the solid lipid microparticle formulations only affected the biological activity slightly.

Animals↗
PubMed1996

Stimulation of splenocytes in C3H/HeJ mice with Porphyromonas gingivalis lipid A in comparison with enterobacterial lipid A.

Porphyromonas gingivalis 381 lipid A induced strong mitogenic response in splenic B cells separated from LPS-non-responsive C3H/HeJ mice as well as LPS-responsive C3H/HeN mice, by using a magnetic cell sorting system. The lipid A also exhibited mitogenic activity in splenic T cells. P. gingivalis lipid A induced lower production of interleukin-1 beta (IL-1 beta) in splenic macrophage cultures and exhibited a comparable IL-6 producing activity in splenic B cells of C3H/HeN mice as compared to Escherichia coli-type synthetic lipid A (compound 506) and monophosphoryl lipid A from Salmonella minnesota Re 595 (MLA). Furthermore, P. gingivalis lipid A, but not compound 506 nor MLA, induced low IL-1 beta and high IL-6 production in C3H/HeJ mice. C3H/HeJ T cells in response to P. gingivalis lipid A stimulation resulted in definite IL-2 mRNA and its production, whereas IL-4 response was scarcely active in both C3H/HeN and C3H/HeJ T cells. P. gingivalis lipid A increased tyrosine, serine/threonine phosphorylation of proteins with various major induced bands in splenic B cells of both C3H/HeN and C3H/HeJ mice. Thus, P. gingivalis lipid A stimulated the splenic immunocytes of C3H/HeJ as well as C3H/HeN mice.

Adjuvants, Immunologic↗
PubMed1995

Interaction of alpha-lactalbumin with phosphatidylglycerol. Influence of protein binding on the lipid phase transition and lipid acyl chain mobility.

The mobility of spin-labeled lipids has been studied in dioleoyl and dimyristoyl phosphatidylglycerol bilayers and in their complexes with alpha-lactalbumin at pH 4.0, by using electron spin resonance (ESR) spectroscopy. The ESR spectra of phosphatidylglycerol spin-labeled at position 5 of the sn-2 chain indicate that association of alpha-lactalbumin with dimyristoyl phosphatidylglycerol bilayers increases the chain mobility at temperatures in the lipid gel phase, restricts the chain mobility at temperatures corresponding to the lipid fluid phase, and abolishes the cooperative lipid chain-melting transition. The ESR spectra of phosphatidylglycerols spin-labeled at eight different positions in the sn-2 chain show that binding of alpha-lactalbumin to dioleoyl phosphatidylglycerol bilayers at pH 4.0 causes a motional restriction throughout the full length of the lipid acyl chain. For phosphatidylglycerols spin-labeled at the terminal methyl end of the chains, a population of motionally restricted lipids that directly contacts membrane penetrant portions of the protein is detected. This population corresponds to 6.3 +/- 0.7 lipids/alpha-lactalbumin at saturation binding, and the high degree of motional restriction (maximum hyperfine splitting approximately 60 G) suggests that the protein may traverse the lipid bilayer. A small selectivity of phosphatidylglycerol over zwitterionic phospholipids for interaction with alpha-lactalbumin is found at subsaturating levels of binding at pH 4.0. Binding of alpha-lactalbumin also strongly restricts the motion of lipids spin-labeled in the polar head group region. These results are of direct relevance to the insertion and translocation of a protein in the molten globule state across lipid membranes.

Electron Spin Resonance Spectroscopy↗
PubMed1988

Penetration of the signal sequence of Escherichia coli PhoE protein into phospholipid model membranes leads to lipid-specific changes in signal peptide structure and alterations of lipid organization.

In order to obtain more insight in the initial steps of the process of protein translocation across membranes, biophysical investigations were undertaken on the lipid specificity and structural consequences of penetration of the PhoE signal peptide into lipid model membranes and on the conformation of the signal peptide adopted upon interaction with the lipids. When the monolayer technique and differential scanning calorimetry are used, a stronger penetration is observed for negatively charged lipids, significantly influenced by the physical state of the lipid but not by temperature or acyl chain unsaturation as such. Although the interaction is principally electrostatic, as indicated also by the strong penetration of N-terminal fragments into negatively charged lipid monolayers, the effect of ionic strength suggests an additional hydrophobic component. Most interestingly with regard to the mechanism of protein translocation, the molecular area of the peptide in the monolayer also shows lipid specificity: the area in the presence of PC is consistent with a looped helical orientation, whereas in the presence of cardiolipin a time-dependent conformational change is observed, most likely leading from a looped to a stretched orientation with the N-terminus directed toward the water. This is in line also with the determined peptide-lipid stoichiometry. Preliminary 31P NMR and electron microscopy data on the interaction with lipid bilayer systems indicate loss of bilayer structure.

Bacterial Outer Membrane Proteins↗
PubMed1997

Spin-label studies on the anchoring and lipid-protein interactions of avidin with N-biotinylphosphatidylethanolamines in lipid bilayer membranes.

The specific binding of hen egg white avidin to phosphatidylcholine lipid membranes containing spin-labeled N-biotinylphosphatidylethanolamines (biotin-PESLs) was investigated by using ESR spectroscopy. Spin-labeled biotin-PEs were prepared with the nitroxide group at position C-5, C-8, C-10, C-12, or C-14 of the sn-2 chain and were incorporated at 1 mol % in lipid bilayer membranes of dimyristoylphosphatidylcholine. Binding of avidin produced a strong and selective restriction of the biotin-PE lipid mobility at all positions of chain labeling, as shown by the ESR spectra recorded in the fluid lipid phase. The spectral components of the fraction of the biotin-PESLs that were not complexed by avidin indicated that the mobility of the bulk membrane lipids was unperturbed by binding avidin, as demonstrated by difference spectroscopy. Comparison of the positional profiles and temperature dependences of the outer hyperfine splittings from the biotin-PESLs suggests that the C-12 and C-14 positions of the avidin-bound biotin-PEs are in register with the C-5 and C-7/C-6 positions, respectively, of the chains of the bulk membrane lipids. This indicates that the biotin-PEs are partially withdrawn from the membrane, with a vertical displacement of ca. 7-8 A, on complexation with avidin. In addition, the specific lipid-protein interaction with avidin results in a selective reduction in the rates of lipid chain motion, as shown by the increased ESR line widths. These data define the way in which avidin is anchored to lipid membranes containing biotin-PEs.

Avidin↗
PubMed2001

Formation of polycyclic aromatic hydrocarbons in the smoke from heated model lipids and food lipids.

The contents of polycyclic aromatic hydrocarbons (PAHs) in the smoke from model lipids and food lipids during heating were determined and the mechanism of PAH formation was studied. A Rancimat oil stability analyzer was used as a model system for heating model lipids and food lipids at 220 degrees C for 2 h and for adsorption of smoke. The various lipid degradation products and PAHs in the smoke were identified and quantified by a GC/MS technique. Results showed that model lipids were more susceptible to smoke formation than food lipids during heating, but the PAH levels were lower for the former than latter. Methyl linolenate produced the highest amount of PAHs, followed by methyl linoleate, methyl oleate, and methyl stearate. Also, soybean oil generated a larger amount of PAHs than canola oil or sunflower oil. Benzene-like compounds were found to be possible precursors for PAHs formation. Several PAH derivatives were also present in heated model lipids and food lipids.

Adsorption↗
PubMed1999

Differences in the lipoprotein distribution of halofantrine are regulated by lipoprotein apolar lipid and protein concentration and lipid transfer protein I activity: in vitro studies in normolipidemic and dyslipidemic human plasmas.

The purpose of these studies was to determine the distribution of a lipophilic antimalarial agent, halofantrine hydrochloride (Hf), in fasted plasma from hypo-, normo-, and hyperlipidemic patients that displayed differences in lipoprotein concentration and lipid transfer protein I (LTP I) activity. To assess the influence of modified lipoprotein concentrations and LTP I activity on the plasma distribution of Hf, Hf at a concentration of 1000 ng/mL was incubated in either hypo-, normo-, or hyperlipidemic human plasma for 1 h at 37 degreesC. Following incubation, the plasma samples were separated into their lipoprotein and lipoprotein-deficient plasma (LPDP) fractions by density gradient ultracentrifugation and assayed for Hf by high-pressure liquid chromatography. The activity of LTP I in the dyslipidemic plasma samples was determined in terms of its ability to transfer cholesteryl ester from low-density lipoproteins (LDL) to high-density lipoproteins (HDL). Total plasma and lipoprotein cholesterol (esterified and unesterified), triglyceride, and protein levels in the dyslipidemic plasma samples were determined by enzymatic assays. When Hf was incubated in normolipidemic plasma for 1 h at 37 degreesC, the majority of drug was found in the LPDP fraction. When Hf was incubated in human plasma of varying total lipid, lipoprotein lipid, and protein concentrations and LTP I activity, the following relationships were observed. As the triglyceride-rich lipoprotein (TRL) lipid and protein concentration increased from hypolipidemia through to hyperlipidemia, the proportion of Hf associated with TRL increased (r > 0.90). As the HDL lipid and protein concentration increased, the proportion of Hf associated with HDL decreased (r > 0.70). As the total and lipoprotein lipid levels increased, the LTP I activity of the plasma also proportionally increased (r > 0.85). Furthermore, with the increase in LTP I activity, the proportion of Hf associated with the TRL fraction increased (r > 0.70) and the proportion of Hf associated with the HDL fraction decreased (r > 0.80). In addition, a positive correlation between the proportion of apolar lipid and Hf recovered within each lipoprotein fraction was observed within hypo- (r > 0.80), normo- (r = 0.70), and hyperlipidemic (r > 0.90) plasmas. These findings suggest that changes in the HDL and TRL lipid and protein concentrations, LTP I activity, and the proportion of apolar lipid within each lipoprotein fraction may influence the plasma lipoprotein distribution of Hf in dyslipidemia.

Antimalarials↗
PubMed2003

Physicochemical investigations on solid lipid nanoparticles and on oil-loaded solid lipid nanoparticles: a nuclear magnetic resonance and electron spin resonance study.

PURPOSE: Recently, colloidal dispersions made of mixtures from solid and liquid lipids have been described to combine controlled-release characteristics with higher drug-loading capacities than solid lipid nanoparticles (SLNs). It has been proposed that these nanostructured lipid carriers (NLCs) are composed of oily droplets that are embedded in a solid lipid matrix. The present work investigates the structure and performance of NLCs. METHODS: Colloidal lipid dispersions were produced by high-pressure homogenization and characterized by laser diffraction, photon correlation spectroscopy, wide-angle x-ray scattering, and differential scanning calorimetry. Proton nuclear magnetic resonance spectroscopy and electron spin resonance experiments were performed to investigate the mobility of the components and the molecular environment of model drugs. Furthermore, a nitroxide reduction assay with ascorbic acid was conducted to explore the accessibility of the lipid model drug from the outer aqueous phase. RESULTS: Proton nuclear magnetic resonance spectra clearly demonstrate that NLC nanoparticles differ from nanoemulsions and from SLNs by forming a liquid compartment that is in strong interaction to the solid lipid. The electron spin resonance model drug was found to be accommodated either on the particle surface with close water contact (SLN) or additionally in the oil (NLC). The oil compartment must be localized on the particle surface, because it can be easily reached by ascorbic acid. CONCLUSION: Neither SLN nor NLC lipid nanoparticles showed any advantage with respect to incorporation rate or retarded accessibility to the drug compared with conventional nanoemulsions. The experimental data let us conclude that NLCs are not spherical solid lipid particles with embedded liquid droplets, but they are rather solid platelets with oil present between the solid platelet and the surfactant layer.

Calorimetry, Differential Scanning↗
PubMed2000

Mechanisms of biliary lipid secretion and their role in lipid homeostasis.

Bile secretion serves different important functions. First, it is one of the main mechanisms for the disposition of many endogenous and exogenous amphipatic compounds, including drugs, toxins, and waste products. Second, it supplies bile salts to the intestine, which is of crucial importance for the emulsification of dietary lipids. In the last decade considerable progress has been achieved in the elucidation of the process of bile formation. Several key transporters in the canalicular membrane have been identified and characterized. This also holds for the mechanism of biliary lipid secretion, where the lipid translocating function of a P-glycoprotein was found to be indispensable for phospholipid secretion. Concomitantly, it became clear that bile salt-induced lipid secretion is an extremely complex process, in which several steps remain elusive. The production of mice with a specific defect in biliary lipid secretion and the identification of an analogous inherited human disease have made it possible to study the integrated function of biliary lipid secretion in whole body lipid homeostasis. In this review we discuss our current understanding of hepatocanalicular lipid secretion in this context. The pathologic consequences of defects in biliary lipid secretion are discussed in another review in this issue.

ATP Binding Cassette Transporter, Subfamily B↗
PubMed2000

Nitric oxide reaction with lipid peroxyl radicals spares alpha-tocopherol during lipid peroxidation. Greater oxidant protection from the pair nitric oxide/alpha-tocopherol than alpha-tocopherol/ascorbate.

The reactions of nitric oxide ((.)NO) and alpha-tocopherol (alpha-TH) during membrane lipid oxidation were examined and compared with the pair alpha-TH/ascorbate. Nitric oxide serves as a more potent inhibitor of lipid peroxidation propagation reactions than alpha-TH and protects alpha-TH from oxidation. Mass spectrometry, oxygen and (.)NO consumption, conjugated diene analyses, and alpha-TH fluorescence determinations all demonstrated that (.)NO preferentially reacts with lipid radical species, with alpha-TH consumption not occurring until (.)NO concentrations fell below a critical level. In addition, alpha-TH and (.)NO cooperatively inhibit lipid peroxidation, exhibiting greater antioxidant capacity than the pair alpha-TH/ascorbate. Pulse radiolysis analysis showed no direct reaction between (.)NO and alpha-tocopheroxyl radical (alpha-T(.)), inferring that peroxyl radical termination reactions are the principal lipid-protective mechanism mediated by (.)NO. These observations support the concept that (.)NO is a potent chain breaking antioxidant toward peroxidizing lipids, due to facile radical-radical termination reactions with lipid radical species, thus preventing alpha-TH loss. The reduction of alpha-T(.) by ascorbate was a comparatively less efficient mechanism for preserving alpha-TH than (.)NO-mediated termination of peroxyl radicals, due to slower reaction kinetics and limited transfer of reducing equivalents from the aqueous phase. Thus, the high lipid/water partition coefficient of (.)NO, its capacity to diffuse and concentrate in lipophilic milieu, and a potent reactivity toward lipid radical species reveal how (.)NO can play a critical role in regulating membrane and lipoprotein lipid oxidation reactions.

Amidines↗
PubMed2000

Membrane lipid domains distinct from cholesterol/sphingomyelin-rich rafts are involved in the ABCA1-mediated lipid secretory pathway.

Efflux of excess cellular cholesterol mediated by lipid-poor apolipoproteins occurs by an active mechanism distinct from passive diffusion and is controlled by the ATP-binding cassette transporter ABCA1. Here we examined whether ABCA1-mediated lipid efflux involves the selective removal of lipids associated with membrane rafts, plasma membrane domains enriched in cholesterol and sphingomyelin. ABCA1 was not associated with cholesterol and sphingolipid-rich membrane raft domains based on detergent solubility and lack of colocalization with marker proteins associated with raft domains. Lipid efflux to apoA-I was accounted for by decreases in cellular lipids not associated with cholesterol/sphingomyelin-rich membranes. Treating cells with filipin, to disrupt raft structure, or with sphingomyelinase, to digest plasma membrane sphingomyelin, did not impair apoA-I-mediated cholesterol or phosphatidylcholine efflux. In contrast, efflux of cholesterol to high density lipoproteins (HDL) or plasma was partially accounted for by depletion of cholesterol from membrane rafts. Additionally, HDL-mediated cholesterol efflux was partially inhibited by filipin and sphingomyelinase treatment. Apo-A-I-mediated cholesterol efflux was absent from fibroblasts with nonfunctional ABCA1 (Tangier disease cells), despite near normal amounts of cholesterol associated with raft domains and normal abilities of plasma and HDL to deplete cholesterol from these domains. Thus, the involvement of membrane rafts in cholesterol efflux applies to lipidated HDL particles but not to lipid-free apoA-I. We conclude that cholesterol and sphingomyelin-rich membrane rafts do not provide lipid for efflux promoted by apolipoproteins through the ABCA1-mediated lipid secretory pathway and that ABCA1 is not associated with these domains.

ATP Binding Cassette Transporter 1↗
PubMed2003

Salt modulates the stability and lipid binding affinity of the adipocyte lipid-binding proteins.

Adipocyte lipid-binding protein (ALBP or aP2) is an intracellular fatty acid-binding protein that is found in adipocytes and macrophages and binds a large variety of intracellular lipids with high affinity. Although intracellular lipids are frequently charged, biochemical studies of lipid-binding proteins and their interactions often focus most heavily on the hydrophobic aspects of these proteins and their interactions. In this study, we have characterized the effects of KCl on the stability and lipid binding properties of ALBP. We find that added salt dramatically stabilizes ALBP, increasing its Delta G of unfolding by 3-5 kcal/mol. At 37 degrees C salt can more than double the stability of the protein. At the same time, salt inhibits the binding of the fluorescent lipid 1-anilinonaphthalene-8-sulfonate (ANS) to the protein and induces direct displacement of the lipid from the protein. Thermodynamic linkage analysis of the salt inhibition of ANS binding shows a nearly 1:1 reciprocal linkage: i.e. one ion is released from ALBP when ANS binds, and vice versa. Kinetic experiments show that salt reduces the rate of association between ANS and ALBP while simultaneously increasing the dissociation rate of ANS from the protein. We depict and discuss the thermodynamic linkages among stability, lipid binding, and salt effects for ALBP, including the use of these linkages to calculate the affinity of ANS for the denatured state of ALBP and its dependence on salt concentration. We also discuss the potential molecular origins and potential intracellular consequences of the demonstrated salt linkages to stability and lipid binding in ALBP.

Adipocytes↗
PubMed2003

Ceramide selectively displaces cholesterol from ordered lipid domains (rafts): implications for lipid raft structure and function.

Ceramide is a membrane lipid involved in a number of crucial biological processes. Recent evidence suggests that ceramide is likely to reside and function within lipid rafts; ordered sphingolipid and cholesterol-rich lipid domains believed to exist within many eukaryotic cell membranes. Using lipid vesicles containing co-existing raft domains and disordered fluid domains, we find that natural and saturated synthetic ceramides displace sterols from rafts. Other raft lipids remain raft-associated in the presence of ceramide, showing displacement is relatively specific for sterols. Like cholesterol-containing rafts, ceramide-rich "rafts" remain in a highly ordered state. Comparison of the sterol-displacing abilities of natural ceramides with those of saturated diglycerides and an unsaturated ceramide demonstrates that tight lipid packing is critical for sterol displacement by ceramide. Based on these results, and the fact that cholesterol and ceramides both have small polar headgroups, we propose that ceramides and cholesterol compete for association with rafts because of a limited capacity of raft lipids with large headgroups to accommodate small headgroup lipids in a manner that prevents unfavorable contact between the hydrocarbon groups of the small headgroup lipids and the surrounding aqueous environment. Minimizing the exposure of cholesterol and ceramide to water may be a strong driving force for the association of other molecules with rafts. Furthermore, displacement of sterol from rafts by ceramide is very likely to have marked effects upon raft structure and function, altering liquid ordered properties as well as molecular composition. In this regard, certain previously observed physiological processes may be a result of displacement. In particular, a direct connection to the previously observed sphingomyelinase-induced displacement of cholesterol from plasma membranes in cells is proposed.

Cell Membrane↗
PubMed1959

The effects of bacterial endotoxin on lipide metabolism. I. The responses of the serum lipides of rabbits to single and repeated injections of Shear's polysaccharide.

Single intravenous injections of Shear's polysaccharide in varying dosages invariably produced an elevation in the levels of the total serum lipides 24 hours after injection of endotoxin. The total serum cholesterol and lipide phosphorus were also affected, although they did not change with smaller doses of endotoxin and were rarely elevated to the same degree as were the total serum lipides. The degree of elevation of the serum lipides was apparently related to the amount of endotoxin injected up to a certain point, beyond which there was no further increase. There were two types of response to endotoxin by the serum lipides, a moderate increase and an uncontrolled increase. Higher dosages of endotoxin and fasting apparently increased the incidence of the latter response. No direct correlation could be made between serum lipide responses and histologic evidence typical of the generalized Shwartzman reaction following this regimen of endotoxin injection. The Shwartzman reaction did occur with greater frequency and with lower dosages of endotoxin in fasted animals. Animals given repeated injections of endotoxin showed an initial increase in serum lipides followed by a progressive decrease to normal levels as tolerance to the febrile action of endotoxin appeared. The febrile tolerance as well as the unresponsiveness of the serum lipides to endotoxin was abolished by thorium dioxide (thorotrast) in these animals. In similar experiments a "breakthrough" of lipide unresponsiveness to endotoxin was obtained by increasing the amount of endotoxin injected. Some of the implications of these results for the metabolic alterations produced by bacterial endotoxins are discussed.

Animals↗