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Why do most negatively charged phospholipids concentrate in the inner leaftlet?

Why do most negatively charged phospholipids concentrate in the inner leaftlet?


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Due to the asymmetry of the lipid membrane, negatively charged phospholipids such as phosphatidylserine, phosphatidylinositol are concentrated in the inner leaflet, creating a different charge distribution between the two leaflets.

Why do most of the negatively charged phospholipids concentrate in the inner leaflet?


A7. Molecular Basis of High Affinity Interactions

  • Contributed by Henry Jakubowski
  • Professor (Chemistry) at College of St. Benedict/St. John's University

What differentiates high and low affinity binding at the molecular level? Do high affinity interactions have lots of intramolecular H-bonds, salt bridges, van der Waals interactions, or are hydrophobic interactions most important? Recently, the crystal structures of a variety of antibody-protein complexes were determined in order to study the basis of affinity maturation of antibody molecules. It is well know that antibodies elicited on exposure to a foreign molecule (antigen) are initially of lower affinity than antibodies released later in the immune response. An incredible number of different antibodies can be made by antibody-producing B cells due to genetic mechanisms (combining different variable regions of antibody genes through splicing, imprecise splicing, and hypermutation of critical nucleotides in the genes of antigen binding regions of antibodies). Clones of antibody-producing cells with higher affinity are selected through binding and clonal expansion of these cells. Investigators studied the crystal structure of 4 different antibodies which bound to the same site (epitope) on the protein antigen lysozyme. Increased affinity was correlated with increased buried apolar surface area and not with increased numbers of H bonds or salt bridges.

Li,Y. et al. Nature: Structural Biology. 6, pg 484 (2003)

Electrostatic interactions between biological molecules are still very important interactions, even though we may consider them to be nonspecific. Witness the interaction of DNA binding proteins with positive domains with the negative polyanion, DNA. The initial encounter will be electrostatic in origin and obviously important to targeting the proteins to DNA where additional specific interactions may take place.

In a similar example (Yeung, T et al.), it was recently reported that moderately positively charged proteins are directed to endosomes and lysosomes through interactions with negatively charged membrane phosphatidylserine (PS), whereas more positively charged proteins are targeted to the inner surface of the plasma membrane, which is enriched in PS and phosphorylated phosphatidyl inositol derivatives (PIP2, PIP3), as shown below.

Figure: negatively charged phospholipids in biological membranes

To study this they used the C2 domain of lactadherin (Lact-C2) from milk that binds PS in the presence of calcium. The C2 domain was covalently linked to the green fluorescent protein, a protein which contains an internal fluorophore comprised of three amino acids (Ser65-Tyr66-Gly67) that cyclize spontaneously on folding to produce a fluorophore which emits green light. A fusion gene of Lact-C2 and GFP was introduced in wild type (WT) and mutant yeast lacking PS. It was bound to the inner leaflet in WT cells and to endosome and lysosome vesicles , but found diffused through cytoplasm in mutant cells. They also made cationic probes with farnesyl tails attached which could anchor the soluble probes to membranes. The most positively charged probes were recruited to the plasma membrane inner leaflet, while less charged ones were recruited to internal vesicles. The authors speculate that PS on cytoplasmic membrane layers can target signal transduction proteins to these regions.

Antibodies with Infinite Affinity. Chmura et al. PNAS. 98, pg 8480 (1998)


Pharmaceutical nanotechnology: Brief perspective on lipid drug delivery and its current scenario

Karthik Siram , . R. Hariprasad , in Biomedical Applications of Nanoparticles , 2019

2.4 Phospholipids

Phospholipids are an important class of membrane lipids that contain two categories of lipids, glycerophospholipids and sphingolipids. Glycerophospholipids are similar to triglycerides except that one hydroxyl group of glycerol is replaced by the ester of phosphoric acid and an amino alcohol, bonded through a phosphodiester bond. They form a very important part of cell membranes, which could be the possible reason behind the success of liposomes (which are made up of phospholipids). Several kinds of phospholipids exist in the body, including the brain. Lecithins are a kind of phospholipids that can be synthesized by the body, are found in the body cells, and also participate in the digestion ( Jannin et al., 2008 O’Driscoll and Griffin, 2008 ).

Sphingolipids (or brain lipids) are another kind of phospholipids that are esters of an 18-carbon alcohol called sphingosine. They are similar to phospholipids, but the glycerol backbone (in phospholipids) is replaced by sphingosine. They are essential to the structure of cell membranes and are abundant in brain tissues and nerve cell membranes. Sphingolipids include the sphingomyelins and cerebrosides that are based on the molecule sphingosine. As the name suggests, these lipids are affiliated with the myelin sheath surrounding the cells of the neurons. Sphingomyelins comprise about 25% of the lipids in the myelin sheath, and their key role is to transmit electric signals. Cerebrosides are not phospholipids, but contain sphingosine.


Introduction

One of the fundamental prerequisites for the function of epithelial cells is the polarization along their apical-basal axis. This apical-basal polarity is established and regulated by a set of highly conserved polarity determinants (Table 1), which act in antagonizing fashions in order to keep the balance between the apical and the basolateral plasma membrane domain. Notably, most key determinants of this epithelial apical-basal polarity also regulate anterior-posterior polarity in the zygote of Caenorhabditis elegans and the oocyte of Drosophila as well as front-rear polarity in migrating cells (for review see St Johnston and Ahringer, 2010 Tepass, 2012 Rodriguez-Boulan and Macara, 2014 Campanale et al., 2017).

Table 1. Summary of conserved polarity regulators and their reported phospholipid-binding capacity.

In order to achieve this balance, polarity determinants cluster in apical and basolateral polarity complexes: In particular, the PAR/aPKC-complex and the Crumbs complex determine the apical plasma membrane domain, whereas the Scribble (Scrb)/Discs Large (Dlg)/Lethal (2) Giant Larvae (Lgl) complex together with the kinases PAR-1 and LKB1 (PAR-4 in C. elegans) substantiate the (baso-) lateral domain (Figure 1). The core components of the PAR/aPKC complex are the scaffolding proteins PAR-3 (Bazooka in Drosophila) and PAR-6 (PAR-6α/β/γ in mammals) and the Serine/Threonine kinase aPKC (atypical protein kinase C, Protein Kinase Cζ and ι in mammals). Furthermore, PAR-6 is regulated by the small GTPase Cdc42, which dynamically associates with the PAR-complex too.

Figure 1. (A) Simplified scheme of a differentiated epithelial cell with apical-basal polarity regulators and their described phospholipid interaction. Apical-basal polarization starts with the recruitment and activation of PI3-Kinase to focal adhesions (Integrin-complexes) and to the Dystroglycan complex, determining the basal side of the epithelial cell. Subsequently, the PAR/aPKC-complex (PAR-3, PAR-6, aPKC) is localized to the Tight Junctions (TJ) by PAR-3. PAR-3 also recruits PTEN, which catalyzes the turn over from PI(3,4,5)P3 to PI(4,5)P2. Furthermore, PAR-3 binds the Rac1 activator Tiam1 and PAR-6/aPKC target Cdc42 to the complex. Together and partly in redundancy with the second TJ-associated complex, the Crb-complex (Crb, Pals1, and PATJ), the PAR/aPKC complex determines the PI(4,5)P2-enriched apical plasma membrane domain, which is counterbalanced by the basolateral cell polarity regulators. Here, the Lgl/Dlg/Scrb-module and the kinase LKB1/PAR-1 also exhibit phospholipid-binding capacities, which are essential for their localization and function. (B) Polarity complexes and phospholipids regulating cell migration. In contrast to apical-basal polarity, migrating cells exhibit a front-rear polarity with apical and basolateral polarity regulators localizing at the leading edge, regulating cell protrusions (lamellipodia in particular via Rac1 and filopodia via Cdc42) by modulating the actin cytoskeleton (gray fibers) or affecting microtubules (green fibers).

Within the Crumbs (Crb)-complex, the transmembrane protein Crb is stabilized in the plasma membrane by its adaptor protein Pals1 (Stardust in Drosophila), which in turn recruits the adaptor proteins Pals1-associated tight junction protein (PATJ) and Lin-7 to the complex (reviewed by Bulgakova and Knust, 2009). However, several studies demonstrated a crosstalk between these two apical complexes, indicating that their composition is highly dynamic and depends on the cell type, differentiation status and other stimuli of the epithelium (Hurd et al., 2003b Gao and Macara, 2004 Lemmers et al., 2004 Penkert et al., 2004 Sotillos et al., 2004 Wang et al., 2004 Kempkens et al., 2006 Krahn et al., 2010a Sen et al., 2015 Whitney et al., 2016). The basolateral localized Scrb, Dlg and Lgl are scaffolding proteins which function as a module to determine basolateral plasma membrane domain and to regulate the assembly of cell�ll contacts. Notably, deletion of these components results not only in polarity defects, but also in tissue overgrowth (in Drosophila and to some extent in vertebrates), leading to the identification of these proteins as tumor suppressors (reviewed by Stephens et al., 2018).

In order to mutually exclude apical and basolateral determinants, aPKC phosphorylates Lgl and PAR-1, which subsequently dissociate from the plasma membrane in the aPKC-active apical zone of epithelia and apical-basal polarized neural stem cells (neuroblasts) of Drosophila (Betschinger et al., 2003 Plant et al., 2003 Hurov et al., 2004 Suzuki et al., 2004 Wirtz-Peitz et al., 2008 Doerflinger et al., 2010). Conversely, PAR-1 phosphorylates PAR-3 and aPKC, displacing them from the basolateral cortex (Benton and St Johnston, 2003 Hurd et al., 2003a Krahn et al., 2009). In Drosophila neuroblasts, aPKC also excludes the adaptor protein Miranda and the Notch inhibitor Numb from the basal cortex by phosphorylation, thereby controlling asymmetric cell division (Smith et al., 2007 Atwood and Prehoda, 2009).

Phospholipids are a major component of biological membranes and not only responsible for dynamic membrane fluctuations but also function as signaling hubs (for review see Liu et al., 2013 Schink et al., 2016 Yang et al., 2018 Kay and Fairn, 2019). Phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS) and sphingomyelin are most frequent and constitute the framework of biological membranes, stabilized by cholesterol. However, the less abundant phosphatidic acid (PA) and phosphoinositides (PI) have been found to play crucial roles in recruiting membrane-associated proteins and function as signaling hubs. Moreover, the accumulation of distinct phospholipids (in particular of the PI family) is a characteristic feature of different cellular compartments, targeting phospholipid-binding proteins to these compartments. An overview of the generation and metabolism of the main phospholipids discussed in this review is given in Figure 2.

Figure 2. Metabolism of major phospholipids implicated in cell polarity. DGK, diacylglycerol kinase. CDP-DG, cytidine diphosphate diacylglycerol. CDS, CDP-diacylglycerol synthase. FIG4, FIG4 phosphoinositide 5-phosphatase. FYVE-type zinc finger containing. INPP4, inositol polyphosphate-4-phosphatase. OCRL, OCRL inositol polyphosphate 5-phosphatase. PIKfyve, phosphoinositide kinase. PIS, PI synthase. PTEN, phosphatase and tensin homolog. SHIP, Src homology 2 (SH2) domain containing inositol polyphosphate 5-phosphatase. TPTE, transmembrane phosphatase with tensin homology.


Vegetable Oils: Composition and Analysis

Phosphatides

Phosphatides are diglycerides that have been esterified with a phosphate group at the sn-3 position that itself can again be esterified with the hydroxyl group of compounds indicated as X: choline, ethanolamine, or inositol. The resulting phosphatides are called phosphatidylcholine (PC), phosphatidylethanolamine (PE), and phosphatidylinositol (PI). The structure in which R1 and R2 represent fatty acid chains is given in the succeeding text:

The phosphate group has a free hydroxyl group left that is quite acidic (pKa < 3.5). In crude oil, it may have a metal counterion (potassium, calcium, or magnesium) or hydrogen. This hydrogen is titrated when the acidity of the oil is measured. When the group X is a hydrogen atom, the phosphatide is called phosphatidic acid (PA). It is formed during the drying, conditioning, and extraction of the oilseeds. If PA is present as a free acid or potassium salt, it will be removed from the oil by hydration on water degumming. If PA is present as a calcium or magnesium salt, this salt will remain in the oil on water degumming as a so-called nonhydratable phosphatide.

Besides PC, PE, PI, and PA, there are some minor phosphatides such as phosphatidylserine in which the phosphate group has been esterified with the amino acid serine. There are the lysocompounds in which one of the fatty acids moieties has been eliminated. There is the acetylphosphatidylethanolamine in which the amino group has been acetylated. There is the phosphatidylglycerol in which the phosphate group has been esterified with a glycerol moiety.


Manipulating membrane asymmetry

The membranes in live cells are shown to be inherently asymmetric. Membrane models used for in vitro studies have been difficult to produce asymmetrical bilayers as in living cells [16]. Supported lipid bilayer (SLB) methodology was one of the first approaches aiming to manipulate membrane asymmetry. SLB consist on a lipid bilayer extended on a physical support (surface), which can be a solid, a polymer "cushion" or a self-assembled monolayer [17]. Additionally, SLB can be tethered to the surface, freely- suspended, or a solid-supported lipid bilayer can be the support for vesicular layers (Figure (PageIndex<9>)). The vesicles are initially placed on the surface, and they rupture after support-induced deformation. Ultimately the bilayers from the broken vesicles coalesce and form the SLB (Figure (PageIndex<1>)0). Studies with SLB allow to analyze molecular composition, molecular distribution and the curvature of membranes, which are the factors responsible for membrane asymmetry.

Figure (PageIndex<9>). Supported lipid bilayers: Basic types, and Figure (PageIndex<1>)0. SLB formation (From: Richter et al, Langmuir, Vol. 22 No. 8, 2006).

Issues such as bilayer-support unwanted interactions, and the fact that in some cases the supported bilayer becomes completely immobile, account for the difficulties to control membrane asymmetry [16]. Plus, a substantial part of research with SLBs have used vesicles with symmetrical compositions in both leaflets [18]. There are new ongoing approaches in recent years aiming to specifically manipulate membrane asymmetry. These methodologies include droplet-interface bilayers, inverted emulsion techniques, as well as methyl-beta-cyclodextrin catalyzed exchange. The latter approach basically consists of transporting phospholipids from the outer leaflet of a donor liposomes into the outer leaflet of an acceptor liposome [19]. This method is capable to produce asymmetric liposomes in a wide range of diameters, and is considered an easy method of transferring anionic phospholipids such as PS to an outer leaflet of a lipid bilayer, thus resembling membrane asymmetry of biological membranes. In recent years, it has been possible to conduct quantitative studies in lipid flip/flop rates, giving new possibilities for analysis of molecular composition and distribution of biological membranes [18].


Function

Phospholipids are very important molecules as they are a vital component of cell membranes. They help cell membranes and membranes surrounding organelles to be flexible and not stiff. This fluidity allows for vesicle formation, which enables substances to enter or exit a cell through endocytosis and exocytosis. Phospholipids also act as binding sites for proteins that bind to the cell membrane. Phospholipids are important components of tissues and organs including the brain and heart. They are necessary for the proper functioning of the nervous system, digestive system, and cardiovascular system. Phospholipids are used in cell to cell communications as they are involved in signal mechanisms that trigger actions such as blood clotting and apoptosis.


Structure of a Phospholipid Molecule

A phospholipid is an amphipathic molecule which means it has both a hydrophobic and a hydrophilic component. A single phospholipid molecule has a phosphate group on one end, called the “head,” and two side-by-side chains of fatty acids that make up the lipid “tails. ” The phosphate group is negatively charged, making the head polar and hydrophilic, or “water loving.” The phosphate heads are thus attracted to the water molecules in their environment.

The lipid tails, on the other hand, are uncharged, nonpolar, and hydrophobic, or “water fearing.” A hydrophobic molecule repels and is repelled by water. Some lipid tails consist of saturated fatty acids and some contain unsaturated fatty acids. This combination adds to the fluidity of the tails that are constantly in motion.


Treatment of infection in burns

James J. Gallagher , . David N. Herndon , in Total Burn Care (Third Edition) , 2007

Polymyxins

The polymyxins are amphipathic molecules that interact with the lipopolysaccharide (LPS) in the bacterial outer membrane . Entry into the cell is not necessary, since polymyxin B covalently attached to agarose beads retains the ability to alter membrane permeability and inhibit bacterial respiration. Initial binding to the outer membrane takes place when the polycationic portion of polymyxin B displaces Ca ++ and Mg ++ bridges that normally stabilize LPS molecules in the outer leaflet of the bacterial outer membrane. 77 Binding can be antagonized by high concentrations of divalent cations. Additional complexing with LPS is facilitated by hydrophobic interaction between the lipid A portion of LPS and the fatty acid of the antibiotic. Insertion of the antibiotic into the outer membrane disrupts the membrane and releases LPS into the surrounding milieu. They also have potent antiendotoxic properties and antibacterial activity against P. aeruginosa and many of the Enterobacteriaceae.

According to Storm et al., the polymyxins are bacteriostatic at low concentrations and bactericidal at high concentrations. Nord and Hoeprich reported that at a concentration of 0.01 m mol/mL, polymyxin B sulfate was bactericidal to 88% of the P. aeruginosa strains tested. 77 Bactericidal activity against P. aeruginosa is not seen with colistin until its concentration reaches 0.1 μ mol/mL. 77 Polymyxin B and colistin (polymyxin E) are usually given at doses of 1.5–2.5 and 5 mg/kg/day, respectively, in two divided doses. Dosing must be altered in renal failure since the kidney is the primary route of elimination. Distribution into pleural fluid, joints, and cerebrospinal fluid is poor.

Polymyxins are recommended for serious systemic infections caused by Gram-negative bacteria that are resistant to other agents and have a definite role in therapy of multi-drug-resistant Gram-negative bacterial infections. The pediatric burn hospital, Shriners Burns Hospital in Galveston, Texas reviewed the use of colistimethate sodium from 2000–2004 in 109 patients, 72 males and 37 females (median and mean age of 9 years) with a TBSA from 21% to 99% (median 60% and mean 62%). The overall survival rate was 80% in all 109 patients. Colistimethate sodium provided an important salvage option for burn patients with otherwise incompletely treated and life-threatening Gram-negative infections. In 2005 at SBH-G, A. baumannii/haemolyticus, E. cloacae, E. coli, and K. pneumoniae all showed 100% susceptibility to colistin and polymyxin B while P. aeruginosa showed 96% and 99% susceptibility to colistin and polymyxin B, respectively.

However, monitoring the dose-dependent nephrotoxicity and CNS toxicity associated with its systemic use is necessary to achieve a therapeutic outcome. When polymyxin B is given to animals or humans, it binds, via its free amino acid groups, to negatively charged phospholipids in tissues. Kunin and Bugg showed that binding is greatest to kidney and brain tissues, followed by liver, muscle, and lung tissues. 77 After repeated doses, the drug accumulates in tissues to concentrations four to five times higher than peak serum concentrations and persists in tissues for at least 5–7 days. 77 Removal of the drug by dialysis can be difficult due to extensive tissue binding. In our study, colistimethate sodium appears to proportionately increase the incidence of C. difficile-associated colitis, renal dysfunction, and neuropathies in relation to the length of its use.


Methods

Lipids and peptides/proteins

Rough mutant LPS from Salmonella enterica serovar Minnesota (S. minnesota) strains R595 (LPS R595), R4 (LPS R4), R7 (LPS R7), Rz (LPS Rz), R5 (LPS R5), R345 (LPS R345), and R60 (LPS R60) as well as deep rough mutant LPS from Escherichia coli strain F515 (LPS F515) and Proteus mirabilis strain R45 (LPS R45) were used [45–50]. LPS was extracted by the phenol/chloroform/petroleum ether method [51], purified, lyophylized, and transformed into the triethylamine salt form. The chemical structures are given in Fig. 1. The amounts of nonstoichiometric substitutions by fatty acids (data not shown), Ara4N, additional phosphates, and phosphoethanolamine were analyzed by mass spectrometry. In LPS R595, the Ara4N linked to the 4'-phosphate was present at a level of 40%. In LPS R45, approximately 50% of the 4'-phosphate of lipid A and 50% of the first Kdo were substituted with Ara4N. By taking into account the amounts of negatively charged phosphate groups and Kdo's and the positively charged Ara4Ns the net charges of the LPSs as summarized in Tab. 1, were calculated.

Phosphatidylcholine (PC) from egg yolk lecithin, phosphatidylglycerol (PG) from egg yolk lecithin (sodium salt), and synthetic diphosphatidylglycerol (DPG) were purchased from Avanti Polar Lipids (Alabaster, AL, USA) and used without further purification.

Polymyxin B (PMB) and its nonapeptide (PMBN), lactoferrin from human milk, lactalbumin from bovine milk, lysozyme from chicken egg white, and human hemoglobin were purchased from Sigma Aldrich (Deisenhofen, Germany), and recombinant human serum albumine (rHSA) from Welfide Corporation (Osaka, Japan). Recombinant BPI1–193 (rBPI21) was a kind gift of XOMA (Berkeley, CA, USA). The rabbit CAP18106–137 was a kind gift provided by J.W. Larrick (Palo Alto Institute of Molecular Medicine, Mountain View, CA, USA).

Determination of the surface potential

Fixed charges within the headgroups of lipid molecules cause an electric potential at the lipid bilayer surface with respect to the surrounding bathing solution, the surface potential [27, 52]. The ζ-potential is related to the surface potential of the lipid aggregates, and can be calculated from the electrophoretic mobility of the aggregates according to the Smoluchowski approximation [53]:

where ζ is the ζ-Potential, μ the electrophoretic mobility, η the viscosity (0.89·10 -3 kg m -1 s -1 ), ε0 the dielectric constant of vacuum (8.854·10 -12 A 2 s 4 kg -1 m -3 ) and εr the dielectric permittivity of water (78.54).

To study the surface potential of phospholipid and LPS aggregates, we determined their ζ-potentials. The measurements were performed on a ZetaSizer4 (Malvern Instruments GmbH, Herrsching, Germany) at 25°C and with a driving electric field of 19.2 V cm -1 .

Aggregates were prepared as 1 mM aqueous dispersions of lipid in buffer (10 mM Tris, 2 mM CsCl2, pH7). Briefly, the lipid dispersions were sonicated for 20 min at 60°C, cooled down to 4°C for 30 min and temperature-cycled twice between 60°C and 4°C (30 min each). The dispersions were equilibrated overnight at 4°C. Prior to ζ-potential measurements, lipid dispersions were diluted to a final concentration of 0.01 mM. Presented values (Fig. 3) are mean values with standard derivations resulting from 3 to 5 independent experiments.

Preparation of lipid monolayers used in film balance experiments

In general, two types of Langmuir-Pockels film balance experiments were utilized: (i) at a constant and (ii) at a variable film area. In both types of experiments the phospholipids were dissolved in chloroform and the LPSs in a 10:1 (v:v) mixture of chloroform and methanol at a concentration of 1 mM. All lipids were deposited in the given amounts on the subphase and the solvents were allowed to evaporate for 5 min.

Determination of calcium adsorption to and displacement from lipid monolayers

Calcium adsorption to lipid monolayers prepared from phospholipids or LPSs was determined by depositing 10 nmol of the respective lipid on 60 ml of an aqueous subphase buffered with 5 mM HEPES and adjusted to pH7. After equilibration of the monolayer, 5 aliquots of 200 μl of a 1.5 mM calcium solution adjusted to a relative β-activity of 24 kBq/ml with radioactive 45 Ca 2+ (Amersham Buchler, Braunschweig, Germany) were added to the subphase, resulting in a final calcium concentration of 25 μM. Calcium ions bind to the negatively charged headgroups of the lipids, and the low-energy β-radiation originating from the bound 45 Ca 2+ is not absorbed by the hydration shell anymore and, therefore, an increase in β-intensity was observed using a β-counter (gas ionization detector LB124, Berthold, Wildbad, Germany) (Fig 2, step 1 & 2). Thus, this method allows the determination of the relative amount of calcium bound to the monolayer [24]. To keep the number of lipid molecules and, therefore, the potential number of binding sites for calcium underneath the detection area of the β-counter constant, the experiments were performed in an acrylic glass trough with a total constant film area of 112 cm 2 .

The β-intensity I monooriginating from 45 Ca 2+ bound to the lipid monolayer at a given Ca 2+ concentration was calculated according to the equation:

(Eq. 5) I mono= I tot- I sub,

where I totis the β-intensity originating from the monolayer and the subphase and I subis the β-intensity of the pure subphase. These and the following experiments were performed at a subphase temperature of 20°C instead of 37°C to avoid condensation at the β-counter.

To investigate the capacity of various peptides/proteins to displace divalent Ca 2+ ions from LPS F515 monolayers, a subphase containing 12.5 μM Ca 2+ doped with radioactive 45 Ca 2+ (resulting in a relative β-activity of 250 Bq/ml), buffered with 5 mM HEPES at pH7 was used. Also in the displacement experiments, the number of LPS molecules was kept constant. The agents were added to the subphase at a constant total film area and relatively low lateral pressure of the monolayer (

10 mN m -1 ). This way, the final pressure after saturation of the intercalation of agents was still in the range of lateral pressures in biological membranes [41, 42]. The peptides/proteins were added to the subphase in different concentrations, and the equilibrium β-counting rates were recorded (Fig. 2, step 3). The relative I rel(c) in dependence on the peptide/protein concentration c was calculated from

where I tot(c) is the absolute β-intensity, I subthe β-intensity originating from the pure subphase, and I monothe β-intensity originating from the monolayer. From the displacement curves the concentrations at which 50% of the calcium were displaced (IC50 value) by the peptides/proteins were determined and summarized in Tab. 1.

All presented values are mean values with standard derivations resulting from 3 to 4 independent experiments.

Influence of the lateral pressure on the adsorption of calcium to LPS monolayers

A Langmuir-Pockels film balance equipped with a Wilhelmy system (Munitech, München, Germany) was used to determine the lipid pressure/area isotherms of LPS F515 monolayers. The lateral pressure, area, and the β-intensity were determined. For the experiments, 36 μl of the LPS F515 were spread on the buffer surfaces of 290 cm 2 . The solvent was allowed to evaporate at zero pressure for 5 min. Then the monolayers were isothermally compressed at a speed of 1.5 mm 2 s -1 to a lateral pressure of 40 mN m -1 .


Watch the video: Positive and Negative Charges. Science. iKen (May 2022).


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