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Biophysical Journal Jan 2019Cholesterol is an essential molecule in the membranes of mammalian cells. It is known to be distributed heterogeneously within the cells, between the bilayer leaflets,...
Cholesterol is an essential molecule in the membranes of mammalian cells. It is known to be distributed heterogeneously within the cells, between the bilayer leaflets, as well as between lateral domains within the bilayer. However, we do not know exactly how cholesterol is distributed and what forces drive this sorting process because it extremely difficult to study using currently available methods. To further elucidate this distribution, we measured how cholesterol partitions between different phospholipid (PL) environments using different methods based on cholesterol, TopFluor-cholesterol, and cholesta-5,7,9(11)-triene-3-β-ol. Based on the obtained relative partition coefficients, we made predictions regarding how cholesterol would be distributed between lateral domains and between the inner and outer leaflets of the plasma membrane. In addition, using a trans-parinaric acid fluorescence-based method, we tested how cholesterol could influence lateral segregation through its interaction with unsaturated PLs with different headgroups. The results showed that the lower the affinity of cholesterol was for the different unsaturated PLs, the more cholesterol stimulated lateral segregation in a ternary bilayer of unsaturated PL/N-palmitoyl-D-erythro-sphingomyelin and cholesterol. Overall, the results indicate that both the distribution of cholesterol between different lipid environments and the impact of cholesterol on lateral segregation can be predicted relatively accurately from determined relative partition coefficients.
Topics: Animals; Cell Membrane; Cholesterol; Cyclodextrins; Humans; Lipid Bilayers
PubMed: 30583790
DOI: 10.1016/j.bpj.2018.11.3135 -
Biophysical Journal Nov 1993The effects of hydrostatic pressure on the physical properties of large unilamellar vesicles of single lipids dipalmitoyl phosphatidylcholine (DPPC) and dimyristoyl...
The effects of hydrostatic pressure on the physical properties of large unilamellar vesicles of single lipids dipalmitoyl phosphatidylcholine (DPPC) and dimyristoyl phosphatidylcholine (DMPC) and lipid mixtures of DMPC/DPPC have been studied from time-resolved fluorescence of trans-parinaric acid. Additional experiments were carried out using diphenylhexatriene to compare the results extracted from both probes. Fluorescence decays were analyzed by the maximum entropy method. Pressure does not influence the fluorescence lifetime distribution of trans-parinaric acid in isotropic solvents. However, in pressurized lipid bilayers an abrupt change was observed in the lifetime distribution which was associated with the isothermal pressure-induced phase transition. The pressure to temperature equivalence values, dT/dP, determined from the midpoint of the phase transitions, were 24 and 14.5 degrees C kbar-1 for DMPC and POPC, respectively. Relatively moderate pressures of about 500 bar shifted the DMPC/DPPC phase diagram 11.5 degrees C to higher temperatures. The effects of pressure on the structural properties of these lipid vesicles were investigated from the anisotropy decays of both probes. Order parameters for all systems increased with pressure. In the gel phase of POPC the order parameter was smaller than that obtained in the same phase of saturated phospholipids, suggesting that an efficient packing of the POPC hydrocarbon chains is hindered.
Topics: 1,2-Dipalmitoylphosphatidylcholine; Biophysical Phenomena; Biophysics; Dimyristoylphosphatidylcholine; Diphenylhexatriene; Fatty Acids, Unsaturated; Fluorescence Polarization; Fluorescent Dyes; Hydrostatic Pressure; Lipid Bilayers; Models, Chemical; Molecular Structure; Phosphatidylcholines; Viscosity
PubMed: 8298048
DOI: 10.1016/S0006-3495(93)81258-X -
Journal of Andrology 1998To determine how the individual components of extenders affected boar sperm function and membrane structure and to test a new surfactant's cryoprotective ability, boar...
To determine how the individual components of extenders affected boar sperm function and membrane structure and to test a new surfactant's cryoprotective ability, boar sperm were cryopreserved in straws in BF5 extender plus or minus egg yolk plus or minus glycerol plus or minus a surfactant (Orvus ES Paste [OEP] or various concentrations of Pluronic F-127). After thawing, sperm function and fluidity of the isolated head plasma membrane (HPM) were determined. Total motility and adenosine triphosphate content (a measure of viability) were superior postthaw in sperm extended in egg yolk plus glycerol (P < 0.05); neither surfactant improved function. Egg yolk plus any other ingredients improved normal acrosome morphology, whereas a combined measure of motility and normal acrosome morphology was better in the presence of 0.33% OEP or 0.1% Pluronic F-127 (P < 0.05 vs. controls). Head plasma membrane was isolated from freshly collected spermatozoa and spermatozoa cryopreserved in the various extenders. Membrane fluidity was monitored with the probes cis-parinaric acid (cPNA), transparinaric acid (tPNA), and 1,6-diphenyl-1 ,3,5-hexatriene (DPH). The cPNA and the DPH monitor the fluidity of gel and liquid-crystalline areas of the membrane, whereas the tPNA preferentially monitors the gel-phase domains of the membrane. Additionally, DPH monitors the hydrophobic core of the bilayer. In the HPM from fresh sperm, the fluidity of each domain changed over time in a manner unique to that domain, and the behavior of the DPH domain varied among boars. The fluidity dynamics of each domain responded uniquely to cryopreservation. The cPNA domain was unaffected, the tPNA domain was altered by four of the eight extenders, and all extenders affected the fluidity of the DPH domain. Membrane structure was significantly correlated with cell function for sperm cryopreserved in extenders that preserved viability and motility. Sperm cryopreserved in egg yolk plus glycerol plus either OEP or 0.1% Pluronic F-127 functioned best when the bulk domains were less fluid initially and the gel domain solidified more slowly. Therefore, the behavior of domains in the HPM of boar spermatozoa is affected by cryopreservation and is related to the postthaw function of boar sperm cryopreserved in different extenders.
Topics: Adenosine Triphosphate; Animals; Cell Membrane; Cryopreservation; Fluorescence Polarization; Male; Membrane Fluidity; Spermatozoa; Surface-Active Agents; Swine
PubMed: 9876025
DOI: No ID Found -
European Journal of Biochemistry Jul 1984Human skin fibroblasts were taken from age-matched male and female subjects. The cells were then cultured under identical conditions and passage-number matched. Plasma...
Human skin fibroblasts were taken from age-matched male and female subjects. The cells were then cultured under identical conditions and passage-number matched. Plasma membranes were isolated and membrane enzyme activities, lipid composition, and structure of isolated plasma membranes were measured in order to determine the presence of significant sex differences in human fibroblast membrane properties. The results indicated that plasma membranes from normal female subjects had a 1.6-fold and 3.6-fold higher cholesterol/phospholipid ratio and oleic acid (18:2) content than normal male subjects. The limiting anisotropy and the rotational relaxation time of fluorescence probe molecules such as trans-parinaric acid and 1,6-diphenyl-1,3,5-hexatriene in the plasma membranes was not significantly different from fibroblasts of male versus female normal subjects. The total activity of plasma membrane (Na+, K+)-ATPase was significantly higher in female than male normal subjects. A potential 'membrane structural disorder', Huntington's disease, was confirmed in fibroblast membranes from male but not from female Huntington's disease subjects. The possibility that Huntington's disease was a 'premature membrane aging' phenomenon was considered. A comparison of plasma membrane enzymes, lipids, and structure from old and young Huntington's disease subjects did not show differences consistent with accelerated membrane aging as explaining the molecular basis for the disease. The age-dependent differences noted in aged Huntington's disease subjects: increased phosphatidylcholine/phosphatidylethanolamine ratio and sphingomyelin + lysophosphatidylcholine content of fibroblast plasma membranes were not significantly altered when compared to normal age-matched controls. However, (Na+, K+)-ATPase activity was significantly enhanced in fibroblast plasma membranes of older Huntington's disease subjects unlike those of control subjects. In conclusion, sex and age differences in membrane properties of cultured cells represent important potential variables in the elucidation of human genetic disorders that may be membrane-related.
Topics: Adult; Age Factors; Cell Membrane; Cells, Cultured; Female; Fibroblasts; Fluorescent Dyes; Humans; Huntington Disease; Kinetics; Male; Membrane Lipids; Middle Aged; Sex Factors; Skin; Sodium-Potassium-Exchanging ATPase
PubMed: 6086340
DOI: 10.1111/j.1432-1033.1984.tb08268.x -
The Journal of Biological Chemistry Apr 2005The F-domain of rat HNF-4alpha1 has a crucial impact on the ligand binding affinity, ligand specificity and secondary structure of HNF-4alpha. (i) Fluorescent binding...
The F-domain of rat HNF-4alpha1 has a crucial impact on the ligand binding affinity, ligand specificity and secondary structure of HNF-4alpha. (i) Fluorescent binding assays indicate that wild-type, full-length HNF-4alpha (amino acids 1-455) has high affinity (Kd=0.06-12 nm) for long chain fatty acyl-CoAs (LCFA-CoA) and low affinity (Kd=58-296 nm) for unesterified long chain fatty acids (LCFAs). LCFA-CoA binding was due to close molecular interaction as shown by fluorescence resonance energy transfer (FRET) from full-length HNF-4alpha tryptophan (FRET donor) to bound cis-parinaroyl-CoA (FRET acceptor), which yielded an intermolecular distance of 33 A, although no FRET to cis-parinaric acid was detected. (ii) Deleting the N-terminal A-D-domains, comprising the AF1 and DNA binding functions, only slightly affected affinities for LCFA-CoAs (Kd=0.9-4 nm) and LCFAs (Kd=93-581 nm). (iii) Further deletion of the F-domain robustly reduced affinities for LCFA-CoA and reversed ligand specificity (i.e. high affinity for LCFAs (Kd=1.5-32 nm) and low affinity for LCFA-CoAs (Kd=54-302 nm)). No FRET from HNF-4alpha-E (amino acids 132-370) tryptophan (FRET donor) to bound cis-parinaroyl-CoA (FRET acceptor) was detected, whereas an intermolecular distance of 28 A was calculated from FRET between HNF-4alpha-E and cis-parinaric acid. (iv) Circular dichroism showed that LCFA-CoA, but not LCFA, altered the secondary structure of HNF-4alpha only when the F-domain was present. (v) cis-Parinaric acid bound to HNF-4alpha with intact F-domain was readily displaceable by S-hexadecyl-CoA, a nonhydrolyzable thioether analogue of LCFA-CoAs. Truncation of the F-domain significantly decreased cis-parinaric acid displacement. Hence, the C-terminal F-domain of HNF-4alpha regulated ligand affinity, ligand specificity, and ligand-induced conformational change of HNF-4alpha. Thus, characteristics of F-domain-truncated mutants may not reflect the properties of full-length HNF-4alpha.
Topics: Acyl Coenzyme A; Animals; Arachidonic Acid; Circular Dichroism; DNA, Complementary; DNA-Binding Proteins; Dose-Response Relationship, Drug; Fatty Acids, Unsaturated; Fluorescence Resonance Energy Transfer; Gene Deletion; Hepatocyte Nuclear Factor 4; Kinetics; Ligands; Mutation; Myristic Acid; Palmitic Acid; Phosphoproteins; Protein Binding; Protein Conformation; Protein Structure, Secondary; Protein Structure, Tertiary; Rats; Spectrometry, Fluorescence; Stearic Acids; Transcription Factors; Tryptophan
PubMed: 15741159
DOI: 10.1074/jbc.M405906200 -
FEBS Letters Jul 1998Peroxisome proliferator activated receptor gamma (PPARgamma) is the subject of intense investigation as a target for drugs against diabetes, atherosclerosis and cancer....
cis-parinaric acid is a ligand for the human peroxisome proliferator activated receptor gamma: development of a novel spectrophotometric assay for the discovery of PPARgamma ligands.
Peroxisome proliferator activated receptor gamma (PPARgamma) is the subject of intense investigation as a target for drugs against diabetes, atherosclerosis and cancer. For this reason there is considerable interest in the spectrum of compounds that bind this receptor. In this paper we have identified cis-parinaric acid (CPA) as a novel hPPARgamma ligand. The binding of this fatty acid to the receptor increases its fluorescence and causes a shift in the UV spectrum. This spectral shift is reversible by competition with other known ligands for PPARgamma. This report represents the first direct demonstration of a fatty acid binding to PPARgamma.
Topics: Base Sequence; Binding, Competitive; DNA, Complementary; Fatty Acids, Unsaturated; Humans; Ligands; Protein Binding; Receptors, Cytoplasmic and Nuclear; Recombinant Proteins; Spectrometry, Fluorescence; Spectrophotometry, Ultraviolet; Transcription Factors
PubMed: 9714568
DOI: 10.1016/s0014-5793(98)00818-7 -
Biophysical Journal Mar 2017We examined how the length of the long-chain base or the N-linked acyl chain of ceramides affected their lateral segregation in...
We examined how the length of the long-chain base or the N-linked acyl chain of ceramides affected their lateral segregation in 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) bilayers. Lateral segregation and ceramide-rich phase formation was ascertained by a lifetime analysis of trans-parinaric acid (tPA) fluorescence. The longer the length of the long-chain base (d16:1, d17:1, d18:1, d19:1, and d20:1 in N-palmitoyl ceramide), the less ceramide was needed for the onset of lateral segregation and ceramide-rich phase formation. A similar but much weaker trend was observed when sphingosine (d18:1)-based ceramide had N-linked acyl chains of increasing length (14:0 and 16:0-20:0 in one-carbon increments). The apparent lateral packing of the ceramide-rich phase, as determined from the longest-lifetime component of tPA fluorescence, also correlated strongly with the long-chain base length, but not as strongly with the N-acyl chain length. Finally, we compared two ceramide analogs with equal carbon numbers (d16:1/17:0 or d20:1/13:0) and observed that the analog with a longer sphingoid base segregated at lower bilayer concentrations to a ceramide-rich phase compared with the shorter sphingoid base analog. The gel phase formed by d20:1/13:0 ceramide also was more thermostable than the gel phase formed by d16:1/17:0 ceramide. H NMR data for 10 mol % stearoyl ceramide in POPC also showed that the long-chain base was more ordered than the acyl chain at comparable chain positions and temperatures. We conclude that the long-chain base length of ceramide is more important than the acyl chain length in determining the lateral segregation of the ceramide-rich gel phase and intermolecular interactions therein.
Topics: Ceramides; Phosphatidylcholines
PubMed: 28297656
DOI: 10.1016/j.bpj.2017.01.016 -
Biophysical Journal Jan 1982Nanosecond fluorescence polarization anisotropy decay is used to determine the effect of the bacteriophage M13 coat protein on lipid bilayer acyl chain dynamics and...
Nanosecond fluorescence polarization anisotropy decay is used to determine the effect of the bacteriophage M13 coat protein on lipid bilayer acyl chain dynamics and order. The fluorescent acyl chain analogues cis- and trans-parinaric acid were used to determine the rate and extent of the angular motion of acyl chains in liquid crystalline (39 degrees C) dimyristoylphosphatidylcholine bilayers free of coat protein or containing the coat protein at a protein:lipid ratio of 1:30. Subnanosecond time resolution was obtained by using synchrotron radiation as the excitation source for single photon counting detection. Previous measurements of Förster energy transfer from coat protein tryptophan to cis- or trans-parinaric acid have shown that these probes are randomly distributed in the bilayer with respect to the protein. The anisotropy decay observed for pure bilayers has the form of a rapid drop, followed by a nonzero constant region extending from roughly 3 ns to at least 12 ns. The magnitude of the anisotropy in the plateau region is simply related to the acyl chain order parameter. The effect of the M13 coat protein is to increase the acyl chain order parameter significantly while having only a small effect on the rate of angular relaxation. This behavior is rationalized in terms of a simple microscopic model. The order parameters for pure lipid and coat protein containing bilayers are compared to 2H-NMR values.
Topics: Coliphages; Dimyristoylphosphatidylcholine; Fatty Acids, Unsaturated; Fluorescence Polarization; In Vitro Techniques; Isomerism; Lipid Bilayers; Membranes, Artificial; Models, Biological; Phosphatidylcholines; Viral Proteins
PubMed: 7055623
DOI: 10.1016/S0006-3495(82)84674-2 -
European Journal of Biochemistry Oct 1997The complete amino acid sequence of a basic liver fatty acid-binding protein (L-FABP) from catfish (Rhamdia sapo) was determined. Alignment of sequences shows that it...
The complete amino acid sequence of a basic liver fatty acid-binding protein (L-FABP) from catfish (Rhamdia sapo) was determined. Alignment of sequences shows that it has more similarity to chicken basic L-FABP than to mammalian L-FABP. The phylogenetic analysis suggests that basic L-FABP from catfish, chicken and iguana diverged from the mammalian protein before the fish-tetrapod divergence, thus implying that the two types are encoded by different genes. Supporting this conclusion, a 14-kDa protein, structurally closely related to mammalian L-FABP, was isolated from catfish intestine, indicating the presence of the two genes in the same species. The catfish basic L-FABP binds only one fatty acid/molecule, while mammalian L-FABP bind two. The former has more affinity for trans-parinaric acid than for cis-parinaric acid, in constrast to the latter proteins.
Topics: Amino Acid Sequence; Animals; Carrier Proteins; Catfishes; Chickens; Evolution, Molecular; Fatty Acid-Binding Protein 7; Fatty Acid-Binding Proteins; Fatty Acids; Humans; Iguanas; Intestinal Mucosa; Kinetics; Liver; Mice; Molecular Sequence Data; Myelin P2 Protein; Neoplasm Proteins; Nerve Tissue Proteins; Peptide Fragments; Phylogeny; Rats; Sequence Alignment; Sequence Homology, Amino Acid; Sharks; Tumor Suppressor Proteins
PubMed: 9370361
DOI: 10.1111/j.1432-1033.1997.00510.x -
Molecular and Biochemical Parasitology Nov 2014Nematodes are unable to synthesize fatty acids de novo and must acquire them from the environment or host. It is hypothesized that two unique classes of fatty acid and...
Nematodes are unable to synthesize fatty acids de novo and must acquire them from the environment or host. It is hypothesized that two unique classes of fatty acid and retinol binding proteins that nematodes produce (fatty acid and retinol binding (FAR) and nematode polyprotein antigen/allergen (NPA)) are used to meet this need. A partial cDNA has been cloned corresponding to four subunits of a putative Ancylostoma ceylanicum NPA (AceNPA). The translated amino acid sequence of AceNPA shares sequence identity with similar proteins from Dictyocaulus viviparus, Ascaris suum, and Ostertagia ostertagi. Immunoblot experiments using a polyclonal anti-AceNPA IgG revealed proteins corresponding to the expected sizes of single, as well as two or three un-cleaved NPA subunits in adult excretory/secretory proteins and soluble adult worm extracts. Immunohistochemistry experiments localize AceNPA to the cuticle, pseudocoelomic space and testes suggesting a role in hookworm biology that is distinct from what has previously been defined for other hookworm lipid binding proteins. A single recombinant subunit of AceNPA (rAceNPAb) demonstrated binding in vitro to fluorescent fatty acids DAUDA, cis-parinaric acid, as well as retinol, at equilibrium dissociation constants in the low micromolar range. Further, in vitro data reveal that rAceNPAb binds fatty acids with chain lengths of C12-C22, with the greatest affinities for arachidonic, linoleic (C18), and eicosapentaenoic (C20) acids.
Topics: Amino Acid Sequence; Ancylostoma; Ancylostomiasis; Animals; Antigens, Helminth; Cloning, Molecular; Cricetinae; DNA, Complementary; Fatty Acids; Female; Helminth Proteins; Humans; Male; Molecular Sequence Data; Sequence Alignment
PubMed: 25481749
DOI: 10.1016/j.molbiopara.2014.11.005