ABSTRACT
The interaction of L-Phe with the membrane components, i.e., lipids and proteins, has been discussed in the current literature due to the interest to understand the effect of single amino acids in relation to the formation of amyloid aggregates. In the present work, it is shown that L-Phe interacts with 9:1 DMPC (1,2-dimyristoyl-sn-glycero-3 phosphocholine)/DPPC (1,2-dipalmitoyl-sn-glycero-3 phosphocholine) mixtures but not in the 1:9 one. An important observation is that the interaction disappears when DPPC is replaced by diether PC (2-di-O-hexadecyl-sn-glycero-3-phosphocholine) a lipid lacking carbonyl groups (CO). This denotes that CO groups may interact specifically with L-Phe in accordance with the appearance of a new peak observed by Infrared spectroscopy (FTIR-ATR). The interaction of L-Phe affects the compressibility pattern of the 9:1 DMPC/DPPC mixture which is congruent with the changes observed by Raman spectra. The specific interaction of L-Phe with CO, propagates to phosphate and choline groups in this particular mixture as analyzed by FTIR-ATR spectroscopy and is absent when DMPC is dopped with diether PC.
Subject(s)
Dimyristoylphosphatidylcholine , Phenylalanine , Phenylalanine/chemistry , Phenylalanine/metabolism , Dimyristoylphosphatidylcholine/chemistry , Spectroscopy, Fourier Transform Infrared , Lipid Bilayers/chemistry , Lipid Bilayers/metabolism , 1,2-Dipalmitoylphosphatidylcholine/chemistry , 1,2-Dipalmitoylphosphatidylcholine/metabolism , Membrane Lipids/chemistry , Membrane Lipids/metabolismABSTRACT
Research on lipid/drug interactions at the nanoscale underpins the emergence of synergistic mechanisms for topical drug administration. The structural understanding of bio-mimetic systems employing 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) as a lung surfactant model mixed with antibiotics, as well as their biophysical properties, is of critical importance to modulate the effectiveness of therapeutic agents released directly to the airways. In this paper, we investigate the structural details of the interaction between Levofloxacin, 'a respiratory quinolone', and the macrolide Clarithromycin, with DPPC monolayers at the air-water interface, using a combination of Brewster angle microscopy, polarization modulation-infrared reflection-adsorption spectroscopy (PM-IRRAS), surface pressure isotherms and neutron reflectometry (NR) to describe the structural details of this interaction. The results allowed association of changes in the π-A isotherm profile with changes in the molecular organization and the co-localization of the antibiotics within the lipid monolayer by NR measurements. Overall, both antibiotics are able to increase the thickness of the acyl tails in DPPC monolayers with a corresponding reduction in tail tilt as well as to interact with the phospholipid headgroups as shown by PM-IRRAS experiments. The effects on the DPPC monolayers are correlated with the physical-chemical properties of each antibiotic and dependent on its concentration.
Subject(s)
1,2-Dipalmitoylphosphatidylcholine/analogs & derivatives , Clarithromycin/chemistry , Levofloxacin/chemistry , 1,2-Dipalmitoylphosphatidylcholine/chemistry , 1,2-Dipalmitoylphosphatidylcholine/metabolism , Adsorption , Air , Clarithromycin/metabolism , Levofloxacin/metabolism , Lipid Bilayers/metabolism , Lipid Metabolism , Lipids , Membranes, Artificial , Microscopy/methods , Models, Biological , Phospholipids/chemistry , Pulmonary Surfactants/chemistry , Spectrophotometry, Infrared/methods , Surface Properties/drug effects , Water/chemistryABSTRACT
Mineralization of the skeleton starts within cell-derived matrix vesicles (MVs); then, minerals propagate to the extracellular collagenous matrix. Tissue-nonspecific alkaline phosphatase (TNAP) degrades inorganic pyrophosphate (PPi), a potent inhibitor of mineralization, and contributes Pi (Phosphate) from ATP to initiate mineralization. Compared to the plasma membrane, MVs are rich in Cholesterol (Chol) (â¼32%) and TNAP, but how Chol influences TNAP activity remains unclear. We have reconstituted TNAP in liposomes of dipalmitoylphosphatidylcholine (DPPC) or dioleoylphosphatidylcholine (DOPC) combined with Chol or its derivatives Cholestenone (Achol) and Ergosterol (Ergo). DPPC plus 36% sterols in liposome increased the catalytic activity of TNAP toward ATP. The presence of Chol also increased the propagation of minerals by 3.4-fold. The catalytic efficiency of TNAP toward ATP was fourfold lower in DOPC proteoliposomes as compared to DPPC proteoliposomes. DOPC proteoliposomes also increased biomineralization by 2.8-fold as compared to DPPC proteoliposomes. TNAP catalyzed the hydrolysis of ATP more efficiently in the case of the proteoliposome consisting of DOPC with 36% Chol. The same behavior emerged with Achol and Ergo. The organization of the lipid and the structure of the sterol influenced the surface tension (γ), the TNAP phosphohydrolytic activity in the monolayer, and the TNAP catalytic efficiency in the bilayers. Membranes in the Lα phase (Achol) provided better kinetic parameters as compared to membranes in the Lo phase (Chol and Ergo). In conclusion, the physical properties and the lateral organization of lipids in proteoliposomes are crucial to control mineral propagation mediated by TNAP activity during mineralization.
Subject(s)
Alkaline Phosphatase/metabolism , Calcification, Physiologic , Cellular Microenvironment , Cholesterol/chemistry , Minerals/metabolism , 1,2-Dipalmitoylphosphatidylcholine/chemistry , 1,2-Dipalmitoylphosphatidylcholine/metabolism , Adenosine Triphosphate/metabolism , Animals , Cells, Cultured , Cholestenones/chemistry , Cholestenones/metabolism , Cholesterol/metabolism , Diphosphates/chemistry , Diphosphates/metabolism , Ergosterol/chemistry , Ergosterol/metabolism , Liposomes/chemistry , Liposomes/metabolism , Male , Osteoblasts/cytology , Osteoblasts/metabolism , Phosphates/chemistry , Phosphates/metabolism , Phosphatidylcholines/chemistry , Phosphatidylcholines/metabolism , Rats, Wistar , Surface PropertiesABSTRACT
The cyclic ketones, thujone and dihydrocarvone, are lipophilic components of essential oils extracted from different plants, which have proven insecticidal activity. The GABAA receptor is activated by the neurotransmitter GABA and is the action site of widely used neurotoxic pesticides. Many compounds that regulate GABAA receptor function interact with membrane lipids, causing changes in their physical properties and consequently, in the membrane dynamic characteristics that modulate receptor macromolecules. In the present study, the biophysical effects of thujone (a gabaergic reference compound) and dihydrocarvone (structurally very similar) were explored by using monomolecular films of DPPC as a model membrane system, to gain insight into membrane-drug interaction. The compression isotherms showed that both ketones expand the DPPC isotherms and increase membrane elasticity. They penetrate the monolayer but their permanence depends on the possibility of establishing molecular interactions with the film component, favored by defects present in the membrane at the phase transition. Finally, by using Brewster angle microscopy (BAM) as a complementary technique for direct visualization of the study films, we found that incorporating ketone seems to reduce molecular repulsion among phospholipid headgroups. Our results reinforce the notion that changes in membrane mechanics may be occurring in the presence of the assayed ketones, suggesting that their interaction with the receptor's surrounding membrane may modulate or affect its functionality, possibly as part of the mechanism of the bioactivity described for thujone and DHC.
Subject(s)
Cell Membrane/chemistry , Cell Membrane/drug effects , Monoterpenes/pharmacology , 1,2-Dipalmitoylphosphatidylcholine/chemistry , 1,2-Dipalmitoylphosphatidylcholine/metabolism , Adsorption , Bicyclic Monoterpenes , Cell Membrane/metabolism , Cyclohexane Monoterpenes , Monoterpenes/chemistry , RheologyABSTRACT
Tissue-nonspecific alkaline phosphatase (TNAP) plays a crucial role during skeletal mineralization, and TNAP deficiency leads to the soft bone disease hypophosphatasia. TNAP is anchored to the external surface of the plasma membranes by means of a GPI (glycosylphosphatidylinositol) anchor. Membrane-anchored and solubilized TNAP displays different kinetic properties against physiological substrates, indicating that membrane anchoring influences the enzyme function. Here, we used Electron Spin Resonance (ESR) measurements along with spin labeled phospholipids to probe the possible dynamic changes prompted by the interaction of GPI-anchored TNAP with model membranes. The goal was to systematically analyze the ESR data in terms of line shape changes and of alterations in parameters such as rotational diffusion rates and order parameters obtained from non-linear least-squares simulations of the ESR spectra of probes incorporated into DPPC liposomes and proteoliposomes. Overall, the presence of TNAP increased the dynamics and decreased the ordering in the three distinct regions probed by the spin labeled lipids DOPTC (headgroup), and 5- and 16-PCSL (acyl chains). The largest change was observed for 16-PCSL, thus suggesting that GPI-anchored TNAP can give rise to long reaching modifications that could influence membrane processes halfway through the bilayer.
Subject(s)
1,2-Dipalmitoylphosphatidylcholine/metabolism , Alkaline Phosphatase/metabolism , Liposomes/metabolism , Animals , CHO Cells , Cricetulus , Electron Spin Resonance Spectroscopy , Humans , Spin LabelsABSTRACT
A detailed molecular description of the mechanism of action of the antimalarial drug amodiaquine (AQ) is still an open issue. To gain further insights on that, we studied the interactions of AQ with lipid model membranes composed of dipalmitoylphosphatidylcholine (DPPC) and dipalmitoylphosphatidylserine (DPPS) by spin labeling electron spin resonance (ESR) and differential scanning calorimetry (DSC). Both techniques indicate a coexistence of an ordered DPPS-rich domain with a disordered DPPC-rich domain in the binary DPPC/DPPS system. We found that AQ slightly lowered the melting transition temperatures associated to both domains and significantly increased the enthalpy change of the whole DPPC/DPPS phase transition. DSC and ESR data also suggest that AQ increases the number of DPPC molecules in the DPPC-rich domains. AQ also causes opposing ordering effects on different regions of the bilayer: while the drug increases the ordering of the lipid acyl chains from carbon 7 to 16, it decreases the order parameter of the lipid head group and of carbon 5. The gel phase was mostly affected by the presence of AQ, suggesting that AQ is able to influence more organized lipid domains. Moreover, the effects of AQ and cholesterol on lipid acyl chain ordering and mobility were compared at physiological temperature and, in a general way, they are similar. Our results suggest that the quinoline ring of AQ is located completely inside the lipid bilayers with its phenol ring and the tertiary amine directed towards the head group region. The nonspecific interaction between AQ and DPPC/DPPS bilayers is a combination of electrostatic and hydrophobic interactions.
Subject(s)
1,2-Dipalmitoylphosphatidylcholine/metabolism , Amodiaquine/metabolism , Antimalarials/metabolism , Lipid Bilayers/metabolism , Phosphatidylserines/metabolism , Electron Spin Resonance SpectroscopyABSTRACT
In this work, we developed a biosystem based on Concanavalin A (ConA) and lipid membranes to recognize glycoproteins from the serum of patients contaminated with dengue serotypes 1, 2 and 3 (DENV1, DENV2 and DENV3). The modified gold electrode was characterized using cyclic voltammetry (CV), electrochemical impedance spectroscopy (EIS) and atomic force microscopy. Morphological analyses of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), DPPC-ConA, DPPC-ConA-DENV1, DPPC-ConA-DENV2 and DPPC-ConA-DENV3 revealed the existence of a non-uniform covering and large globules. EIS and CV measurements have shown that redox probe reactions on the modified gold electrodes were partially blocked due to the adsorption of lipid-ConA system and reveal the interaction response of the immobilized ConA to the presence of glycoproteins of dengue serum. The biosystem exhibited a wide linear response to different concentrations of sera of dengue serotypes 1, 2 and 3. A higher impedimetric response to glycoproteins present in dengue serotype 3 was observed. Our results demonstrate the applicability of lectin and lipid membranes to the development of biosensors for dengue infections.
Subject(s)
1,2-Dipalmitoylphosphatidylcholine/metabolism , Biosensing Techniques/methods , Cell Membrane , Concanavalin A/metabolism , Dengue/blood , Glycoproteins/blood , Biosensing Techniques/instrumentation , Dengue/diagnosis , Electrochemistry , Electrodes , Gold/chemistry , Humans , Liposomes/metabolism , Point-of-Care SystemsABSTRACT
The key challenges for producing devices based on nanostructured films with control over the molecular architecture are to preserve the catalytic activity of the immobilized biomolecules and to provide a reliable method for determining the intermolecular interactions and the accommodation of molecules at very small scales. In this work, the enzymes cellulase and alcohol dehydrogenase (ADH) were coimmobilized with dipalmitoylphosphatidylcholine (DPPC) as Langmuir-Blodgett (LB) films, and their biological activities were assayed by accommodating the structure formed in contact with cellulose. For this purpose, the polysaccharide was dissolved in an ionic liquid, 1-buthyl-3-methylimidazolium chloride (BMImCl), and dropped on the top of the hybrid cellulase-ADH-DPPC LB film. The interactions between cellulose and ethanol, which are the catalytic substrates of the enzymes as well as important elements in the production of second-generation fuels, were then investigated using polarization-modulation infrared reflection-absorption spectroscopy (PM-IRRAS). Investigation of the secondary structures of the enzymes was performed using PM-IRRAS, through which the presence of ethanol and cellulose was observed to highly affect the structures of ADH and cellulase, respectively. The detection of products formed from the catalyzed reactions as well as the changes of secondary structure of the enzymes immobilization could be carried out, which opens the possibility to produce a means for producing second-generation ethanol using nanoscale arrangements.
Subject(s)
1,2-Dipalmitoylphosphatidylcholine/metabolism , Alcohol Dehydrogenase/metabolism , Cellulase/metabolism , Cellulose/metabolism , Enzymes, Immobilized/metabolism , Ethanol/metabolism , 1,2-Dipalmitoylphosphatidylcholine/chemistry , Alcohol Dehydrogenase/chemistry , Cellulase/chemistry , Cellulose/chemistry , Enzyme Activation , Enzymes, Immobilized/chemistry , Ethanol/chemistry , Spectrophotometry, Infrared , Surface TensionABSTRACT
Amaranth seeds are one of the more promising food ingredients, due to their high protein content, among which the most important are storage proteins known as globulins. However, little is known about the physicochemical of the globulin proteins. In this work, we study the physicochemical behavior of films made of amaranth 7S globulin and its interaction with a model membrane made of L-α-dipalmitoylphosphatidylcholine (L-α-DPPC) at the air-liquid interface. The study was done by means of Langmuir balance, Brewster angle microscopy (BAM), fluorescence microscopy, and atomic force microscopy (AFM). We found that isotherms of pure 7S globulin directly deposited on either water or buffer subphases behave similarly and globulin forms a condensed film made of globular and denature structures, which was confirmed by BAM observations. Good mixtures of the protein with L-α-DPPC are formed at low surface pressure. However, they phase separate from moderate to high surface pressure as observed by BAM. Isotherms detect the presence of the protein in the mixture with L-α-DPPC, but we were unable to detect it through BAM or AFM. We show that fluorescence microscopy is a very good technique to detect the presence of the protein when it is well-mixed within the LE phase of the lipid. AFM images clearly show the formation of protein mono- and multilayers, and in phase mode, we detected domains that are formed by protein and LE lipid phase, which were corroborated by fluorescence microscopy. We have shown that globulin 7S mix well with lipid phases, which could be important in food applications as stabilizers or emulsifiers, but we also show that they can phase separate with a moderate to high surface pressure.
Subject(s)
1,2-Dipalmitoylphosphatidylcholine/metabolism , Amaranthus/metabolism , Globulins/metabolism , Plant Proteins/metabolism , 1,2-Dipalmitoylphosphatidylcholine/chemistry , Air , Amaranthus/chemistry , Globulins/chemistry , Microscopy, Atomic Force , Microscopy, Fluorescence , Plant Proteins/chemistry , Pressure , Seeds/chemistry , Seeds/metabolism , Water/chemistryABSTRACT
A better understanding of the structural effects induced by thyroid hormones in model membranes is attained by Raman spectroscopy. The interactions of T3 and T4 with multilamellar vesicles of dipalmytoylphosphatidylcholine (DPPC) in the gel phase are characterized by analyzing the spectral behavior of the C-H and C-C stretching vibrations of the acyl chains. The spectra evidence an increase in the relative number of gauche conformation, which indicates the hormones are able to penetrate into the hydrophobic region of the bilayer and partially alter the lipid structure. In addition, the density packing of the acyl chains appears increased and the rotational mobility of the terminal methylene groups is slightly reduced in the iodothyronine/DPPC mixtures. These effects are interpreted in terms of the transition to an interdigitated phase due to the hormone incorporation to the membrane. The polar heads of the lipids also interact with the hormone, as evidenced by the PO2(-) symmetric stretching band.
Subject(s)
1,2-Dipalmitoylphosphatidylcholine/metabolism , Liposomes/metabolism , Thyronines/metabolism , Thyroxine/metabolism , 1,2-Dipalmitoylphosphatidylcholine/chemistry , Liposomes/chemistry , Spectrum Analysis, Raman/methodsABSTRACT
Combined effects of flunitrazepam (FNZ) and lidocaine (LDC) were studied on the thermotropic equilibrium of dipalmitoyl phosphatidylcholine (dpPC) bilayers. This adds a thermodynamic dimension to previously reported geometric analysis in the erythrocyte model. LDC decreased the enthalpy and temperature for dpPC pre- and main-transitions (ΔHp, ΔHm, Tp, Tm) and decreased the cooperativity of the main-transition (ΔT(1/2,m)). FNZ decreased ΔHm and, at least up to 59 µM, also decreased ΔHp. In conjunction with LDC, FNZ induced a recovery of ∆T(1/2,m) control values and increased ΔHm even above the control level. The deconvolution of the main-transition peak at high LDC concentrations revealed three components possibly represented by: a self-segregated fraction of pure dpPC, a dpPC-LDC mixture and a phase with a lipid structure of intermediate stability associated with LDC self-aggregation within the lipid phase. Some LDC effects on thermodynamic parameters were reverted at proper LDC/FNZ molar ratios, suggesting that FNZ restricts the maximal availability of the LDC partitioned into the lipid phase. Thus, beyond its complexity, the lipid-LDC mixture can be rationalized as an equilibrium of coexisting phases which gains homogeneity in the presence of FNZ. This work stresses the relevance of nonspecific drug-membrane binding on LDC-FNZ pharmacological interactions and would have pharmaceutical applications in liposomal multidrug-delivery.
Subject(s)
Cell Membrane/drug effects , Cell Membrane/metabolism , Flunitrazepam/pharmacology , Lidocaine/pharmacology , Light , Lipid Bilayers/metabolism , Scattering, Radiation , 1,2-Dipalmitoylphosphatidylcholine/metabolism , Calorimetry, Differential Scanning , Cell Membrane/chemistry , Drug Interactions , Lipid Bilayers/chemistry , ThermodynamicsABSTRACT
The interaction between the antimicrobial peptide gramicidin (Gr) and dipalmitoylphosphatidylcholine (DPPC)/dioctadecyldimethylammonium bromide (DODAB) 1:1 large unilamellar vesicles (LVs) or bilayer fragments (BFs) was evaluated by means of several techniques. The major methods were: 1) Gr intrinsic fluorescence and circular dichroism (CD) spectroscopy; 2) dynamic light scattering for sizing and zeta-potential analysis; 3) determination of the bilayer phase transition from extrinsic fluorescence of bilayer probes; 4) pictures of the dispersions for evaluation of coloidal stability over a range of time and NaCl concentration. For Gr in LVs, the Gr dimeric channel conformation is suggested from: 1) CD and intrinsic fluorescence spectra similar to those in trifluoroethanol (TFE); 2) KCl or glucose permeation through the LVs/Gr bilayer. For Gr in BFs, the intertwined dimeric, non-channel Gr conformation is evidenced by CD and intrinsic fluorescence spectra similar to those in ethanol. Both LVs and BFs shield Gr tryptophans against quenching by acrylamide but the Stern-Volmer quenching constant was slightly higher for Gr in BFs confirming that the peptide is more exposed to the water phase in BFs than in LVs. The DPPC/DODAB/Gr supramolecular assemblies may predict the behavior of other antimicrobial peptides in assemblies with lipids.
Subject(s)
1,2-Dipalmitoylphosphatidylcholine/metabolism , Gramicidin/metabolism , Lipid Bilayers/metabolism , Quaternary Ammonium Compounds/metabolism , 1,2-Dipalmitoylphosphatidylcholine/chemistry , Gramicidin/chemistry , Lipid Bilayers/chemistry , Lipids/chemistry , Molecular Conformation , Phase Transition , Quaternary Ammonium Compounds/chemistryABSTRACT
Miltefosine (MT) is an alkylphospholipid approved for breast cancer metastasis and visceral leishmaniasis treatments, although the respective action mechanisms at the molecular level remain poorly understood. In this work, the interaction of miltefosine with the lipid component of stratum corneum (SC), the uppermost skin layer, was studied by electron paramagnetic resonance (EPR) spectroscopy of several fatty acid spin-labels. In addition, the effect of miltefosine on (i) spherical lipid vesicles of 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine (DPPC) and (ii) lipids extracted from SC was also investigated, by EPR and time-resolved polarized fluorescence methods. In SC of neonatal Wistar rats, 4% (w/w) miltefosine give rise to a large increase of the fluidity of the intercellular membranes, in the temperature range from 6 to about 50°C. This effect becomes negligible at temperatures higher that ca. 60°C. In large unilamelar vesicles of DPPC no significant changes could be observed with a miltefosine concentration 25% molar, in close analogy with the behavior of biomimetic vesicles prepared with bovine brain ceramide, behenic acid and cholesterol. In these last samples, a 25 mol% molar concentration of miltefosine produced only a modest decrease in the bilayer fluidity. Although miltefosine is not a feasible skin permeation enhancer due to its toxicity, the information provided in this work could be of utility in the development of a MT topical treatment of cutaneous leishmaniasis.
Subject(s)
Antineoplastic Agents/pharmacology , Membrane Lipids/metabolism , Phosphorylcholine/analogs & derivatives , Skin/drug effects , 1,2-Dipalmitoylphosphatidylcholine/metabolism , Animals , Animals, Newborn , Antineoplastic Agents/administration & dosage , Biomimetic Materials , Cattle , Ceramides/metabolism , Cholesterol/metabolism , Electron Spin Resonance Spectroscopy , Fatty Acids/metabolism , Fluorescence Polarization , Membrane Fluidity/drug effects , Phosphorylcholine/administration & dosage , Phosphorylcholine/pharmacology , Rats , Rats, Wistar , Skin/metabolism , Spin Labels , TemperatureABSTRACT
The interaction of the tryptophan-containing variant of microcin J25, MccJ25 I13W, with phosphatidylcholine membranes was studied by fluorescence spectroscopy techniques. The peptide was able to interact with dimiristoylphophatidylcholine and dipalmitoylphosphatidylcholine liposomes only when the membranes were in gel phase, as was demonstrated by the blue shift of the intrinsic fluorescence of MccJ25 I13W. The binding isotherm showed a cooperative partition of the peptide toward the membrane and the binding constant increased as the temperature decreased and the order parameter increased. No interaction with liquid crystalline membranes was observed. Studies of dynamic quenching of the fluorescence indicated that the peptide penetrated the lipid bilayer and was located primarily in the interfacial region. Our results suggest that MccJ25 I13W interacts with gel phase phospholipids and increases both its own affinity for the bilayer and the membrane permeability of small ions.
Subject(s)
Bacteriocins/genetics , Bacteriocins/metabolism , Cell Membrane/metabolism , Mutation , 1,2-Dipalmitoylphosphatidylcholine/chemistry , 1,2-Dipalmitoylphosphatidylcholine/metabolism , Amino Acid Sequence , Amino Acid Substitution , Bacteriocins/chemistry , Binding Sites/genetics , Binding, Competitive , Cell Membrane/chemistry , Dimyristoylphosphatidylcholine/chemistry , Dimyristoylphosphatidylcholine/metabolism , Gels/chemistry , Kinetics , Lipid Bilayers/chemistry , Lipid Bilayers/metabolism , Models, Molecular , Molecular Sequence Data , Phase Transition , Phosphatidylcholines/chemistry , Phosphatidylcholines/metabolism , Protein Binding , Protein Structure, Secondary , Protein Structure, Tertiary , Spectrometry, Fluorescence , Thermodynamics , Unilamellar Liposomes/chemistry , Unilamellar Liposomes/metabolismABSTRACT
Frutalin is a homotetrameric alpha-d-galactose (d-Gal)-binding lectin that activates natural killer cells in vitro and promotes leukocyte migration in vivo. Because lectins are potent lymphocyte stimulators, understanding the interactions that occur between them and cell surfaces can help to the action mechanisms involved in this process. In this paper, we present a detailed investigation of the interactions of frutalin with phospho- and glycolipids using Langmuir monolayers as biomembrane models. The results confirm the specificity of frutalin for d-Gal attached to a biomembrane. Adsorption of frutalin was more efficient for the galactose polar head lipids, in contrast to the one for sulfated galactose, in which a lag time is observed, indicating a rearrangement of the monolayer to incorporate the protein. Regarding ganglioside GM1 monolayers, lower quantities of the protein were adsorbed, probably due to the farther apart position of d-galactose from the interface. Binary mixtures containing galactocerebroside revealed small domains formed at high lipid packing in the presence of frutalin, suggesting that lectin induces the clusterization and the forming of domains in vitro, which may be a form of receptor internalization. This is the first experimental evidence of such lectin effect, and it may be useful to understand the mechanism of action of lectins at the molecular level.
Subject(s)
Plant Lectins/chemistry , Plant Lectins/metabolism , 1,2-Dipalmitoylphosphatidylcholine/chemistry , 1,2-Dipalmitoylphosphatidylcholine/metabolism , Adsorption , Artocarpus/chemistry , Binding Sites , Cell Line, Tumor , Cell Membrane/chemistry , Cell Membrane/metabolism , Elasticity , G(M1) Ganglioside/chemistry , G(M1) Ganglioside/metabolism , Galactose/metabolism , Humans , In Vitro Techniques , Membrane Fluidity , Membrane Lipids/chemistry , Membrane Lipids/metabolism , Membranes, Artificial , Models, Biological , Protein BindingABSTRACT
Oligonucleotides have unique molecular recognition properties, being involved in biological mechanisms such as cell-surface receptor recognition or gene silencing. For their use in human therapy for drug or gene delivery, the cell membrane remains a barrier, but this can be obviated by grafting a hydrophobic tail to the oligonucleotide. Here we demonstrate that two oligonucleotides, one consisting of 12 guanosine units (G(12)), and the other one consisting of five adenosine and seven guanosine (A(5)G(7)) units, when functionalized with poly(butadiene), namely PB-G(12) and PB-A(5)G(7), can be inserted into Langmuir monolayers of dipalmitoyl phosphatidyl choline (DPPC), which served as a cell membrane model. PB-G(12) and PB-A(5)G(7) were found to affect the DPPC monolayer even at high surface pressures. The effects from PB-G(12) were consistently stronger, particularly in reducing the elasticity of the DPPC monolayers, which may have important biological implications. Multilayers of DPPC and nucleotide-based copolymers could be adsorbed onto solid supports, in the form of Y-type LB films, in which the molecular-level interaction led to lower energies in the vibrational spectra of the nucleotide-based copolymers. This successful deposition of solid films opens the way for devices to be produced which exploit the molecular recognition properties of the nucleotides.
Subject(s)
Cell Membrane/chemistry , Cell Membrane/metabolism , Models, Biological , Oligonucleotides/chemistry , Oligonucleotides/metabolism , 1,2-Dipalmitoylphosphatidylcholine/chemistry , 1,2-Dipalmitoylphosphatidylcholine/metabolism , Adenosine/chemistry , Adenosine/metabolism , Butadienes/chemistry , Elasticity , Elastomers/chemistry , Guanosine/chemistry , Guanosine/metabolism , Spectrophotometry, Ultraviolet , Spectroscopy, Fourier Transform Infrared , Surface Tension , Time FactorsABSTRACT
The probes C(12)-NBD-FA (12-[(7-nitrobenz-2-oxa-1,3-diazol-4-yl) amino)] dodecanoic acid) and C(18)-R (octadecyl rhodamine B chloride) have been used as donor and acceptor, respectively, in FRET studies on liposomes subjected to pancreatic PLA(2) action. Neither of these fluorophores is a substrate for the enzyme but one of them, C(12)-NBD-FA, is an analog of the fatty acid reaction product. The fluorophores were incorporated into 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine (DPPC) liposomes and FRET was studied following the fluorescence of the donor, C(12)-NBD-FA. Working with a molar ratio of acceptor to donor (A/D) of 1, we have found that FRET efficiency (E) decreases during DPPC hydrolysis. After 60min, the decrease is equivalent to a reduction of more than five times in the effective A/D ratio, as estimated by interpolation in an efficiency vs. A/D reference curve. Using a more complete, empirical approach, the efficiency data, calculated from experiments at variable A/D proportions and constant donor concentration, were fitted by a rectangular hyperbolic function. The parameter K of this function, representing the A/D ratio at half-maximum transfer efficiency, increases more that five times after 60min hydrolysis. This agrees with the reduction of the effective acceptor density sensed by the donor after hydrolysis, detected by the interpolation procedure. The heterogeneous distribution of acceptor and donor induced by hydrolysis can be attributed to the formation of product domains in the phospholipid membranes and is consistent with the preferential segregation of the donor, which is an analog of the fatty acid reaction product, in those domains. In conclusion, FRET between non-substrate probes detects the heterogeneities generated in phospholipid membranes by PLA(2) action.
Subject(s)
Lipid Bilayers/metabolism , Phospholipases A2, Secretory/metabolism , 1,2-Dipalmitoylphosphatidylcholine/chemistry , 1,2-Dipalmitoylphosphatidylcholine/metabolism , 4-Chloro-7-nitrobenzofurazan/analogs & derivatives , Animals , Biophysical Phenomena , Fluorescence Resonance Energy Transfer , Fluorescent Dyes , Hydrogen-Ion Concentration , Hydrolysis , In Vitro Techniques , Kinetics , Lauric Acids , Lipid Bilayers/chemistry , Liposomes , Pancreas/enzymology , Rhodamines , Substrate Specificity , SwineABSTRACT
Different subunit aggregates of the Na,K-ATPase may be formed depending on the method used to solubilize and purify the enzyme. We have studied the thermal unfolding of detergent-solubilized and dipalmitoylphosphatidylcholine/ dipalmitoylphosphatidylethanolamine liposome-reconstituted forms of the Na,K-ATPase by circular dichroism (CD) spectroscopy and p-nitrophenylphosphatase activity. The ellipticity at 222 nm of the solubilized and reconstituted forms showed a sigmoid decrease in the absolute value of the signal of 36 and 31% with T(50%) of 44 and 42 degrees C, respectively. The catalytic activity was reduced in two steps with T(50%) of 32 and 52 degrees C in the detergent-solubilized enzyme and T(50%) of 25 and 53 degrees C in the reconstituted enzyme. The reduction in catalytic activity of the detergent-solubilized enzyme was bi-exponential with t(1/2) of 8.3 and 67.9 min, resulting in the total loss of activity after 120 min. However, under the same conditions, the ATPase activity of the reconstituted enzyme was reduced by approx 35% with a t(1/2) of 145 min. The results suggest that the alpha- and beta-subunits present different thermal stability that may be modulated by the nature of the co-solvent (detergent or lipid) used in the preparations of the Na,K-ATPase. In addition, distinct processes of beta-subunit displacement and alpha-alpha-subunit aggregate formation may also contribute to the changes in both the CD spectra and the enzyme activity. Furthermore, we have demonstrated the protective role of the phospholipid bilayer in the reconstituted enzyme compared with the detergent-solubilized enzyme.
Subject(s)
1,2-Dipalmitoylphosphatidylcholine/metabolism , Liposomes/chemistry , Phosphatidylethanolamines/metabolism , Proteolipids/chemistry , Sodium-Potassium-Exchanging ATPase/metabolism , 1,2-Dipalmitoylphosphatidylcholine/chemistry , Animals , Ca(2+) Mg(2+)-ATPase , Catalysis , Cell Membrane/metabolism , Circular Dichroism/methods , Detergents/chemistry , Enzyme Activation/physiology , Enzyme Stability/drug effects , Hot Temperature , Kidney Medulla/metabolism , Lipid Metabolism/physiology , Membrane Lipids/chemistry , Ouabain/pharmacology , Phosphatidylcholines , Phosphatidylethanolamines/chemistry , Protein Denaturation/drug effects , Rabbits , Sodium-Potassium-Exchanging ATPase/chemistry , Sodium-Potassium-Exchanging ATPase/isolation & purificationABSTRACT
In this work the interaction of Hydroxyzine, Promethazine and Thioridazine with Langmuir films of dipalmitoylphosphatidylcholine (dpPC) and dipalmitoylphosphatidic acid (dpPA), is studied. Temporal variations in lateral surface pressure (pi) were measured at different initial pi (pi(i)), subphase pH and drug-concentration. Drugs with the smallest (PRO) and largest (HYD) molecular size exhibited the lowest adsorption (k(a)) and the highest desorption (k(d)) rate constant values, respectively. The affinity binding constants (K(b)) obtained in monolayers followed the same profile (K(b,PRO) < K(b,HYD) < K(b,THI)) of the egg-PC/water partition coefficients (P) determined in bilayers. The drug concentration required to reach the half-maximal Deltapi at pi(i) = 14 mN/m (K(0.5)), was very sensitive to pH. The maximal increment in pi upon drug incorporation into the monolayer (deltapi(max)) will depend on the phospholipid collapse pressure (pi(c)), the monolayers's compressibility and drug's size, shape and charge. The higher pi(c) of dpPC lead to higher pi(cut-off) values (maximal pi allowing drug penetration), if compared with dpPA. In dpPC and dpPA pi(cut-off) decreased as a function of the molecular size of the uncharged drugs. In dpPA, protonated drugs became electrostatically trapped at the monolayer surface hence drug penetration, monolayer deformation and pi increase were impaired and the correlation between pi(cut-off) and drug molecular size was lost.
Subject(s)
1,2-Dipalmitoylphosphatidylcholine/metabolism , Hydroxyzine/metabolism , Phosphatidic Acids/metabolism , Promethazine/metabolism , Thioridazine/metabolism , 1,2-Dipalmitoylphosphatidylcholine/chemistry , Air , Hydroxyzine/chemistry , Lipid Bilayers , Phosphatidic Acids/chemistry , Promethazine/chemistry , Surface Properties , Thioridazine/chemistry , WaterABSTRACT
The study of interactions between biological molecules and model membranes is essential for the understanding of a number of physiological mechanisms involved in viral infections and dissemination. In this paper, the analysis of the interaction between a peptide from the p24 protein of Human Immunodeficiency Virus type 1 (HIV-1) and a phospholipid monolayer has pointed to a cooperative response in which very small amounts of peptide p24-1 (e.g. 0.05 mol%) can lead to measurable effects. Monolayer surface pressure and surface potential isotherms were affected for peptide concentrations as low as 0.05 mol%, with saturation at 0.5 mol%. The expansion effect from p24-1 is confirmed by changes in morphology of the monolayers using Brewster angle microscopy. Even though p24-1 is disordered in aqueous solutions, the interaction with dipalmitoyl phosphatidylcholine (DPPC) causes it to adopt an alpha-helix structure, as shown by circular dichroism (CD) data for multilamellar vesicles (MLV). The expansion of the phospholipid monolayer in a cooperative way may imply that p24-1 has potential antiviral activity, by participating in the cell rupture, with no need of specific receptors in the membrane.