Fluorescent Fatty Acid Analogue Research Articles

Identification and characterization of small compound inhibitors of human FATP2

Fatty acid transport proteins (FATPs) are bifunctional proteins, which transport long chain fatty acids into cells and activate very long chain fatty acids by esterification with coenzyme A. In an effort to understand the linkage between cellular fatty acid transport and the pathology associated with excessive accumulation of exogenous fatty acids, we targeted FATP-mediated fatty acid transport in a high throughput screen of more than 100,000 small diverse chemical compounds in yeast expressing human FATP2 (hsFATP2). Compounds were selected for their ability to depress the transport of the fluorescent long chain fatty acid analogue, C 1-BODIPY-C 12. Among 234 hits identified in the primary screen, 5 compounds, each representative of a structural class, were further characterized in the human Caco-2 and HepG2 cell lines, each of which normally expresses FATP2, and in 3T3-L1 adipocytes, which do not. These compounds were effective in inhibiting uptake with IC 50s in the low micromolar range in both Caco-2 and HepG2 cells. Inhibition of transport was highly specific for fatty acids and there were no effects of these compounds on cell viability, trans-epithelial electrical resistance, glucose transport, or long chain acyl-CoA synthetase activity. The compounds were less effective when tested in 3T3-L1 adipocytes suggesting selectivity of inhibition. These results suggest fatty acid transport can be inhibited in a FATP-specific manner without causing cellular toxicity.

In vitro assay for drug‐induced hepatosteatosis using rat primary hepatocytes, a fluorescent lipid analog and gene expression analysis

To evaluate new drugs' potential for hepatosteatosis, we developed a cell-based assay using a fluorescent fatty acid analog: BODIPY558/568 C12. Rat primary hepatocytes were exposed to positive reference compounds [cyclosporine A (CsA), clofibrate (CFR), tetracycline (TC), valproic acid (VPA), carbon tetrachloride (CCl4), tamoxifen (TMX)] in the presence of BODIPY558/568 C12. The formation of fluorscecent particles or lipid droplets in the cytoplasm was confirmed by confocal laser scanning microscopy and electron microscopy respectively. The accumulation of BODIPY558/568 C12 was measured by fluorometry and high content imaging method. All positive reference compounds increased fluorescent particles in number and fluorescence intensity. High content imaging was more sensitive and selective method than fluorometry to detect fluorescent particles. Gene expression analysis of the hepatocytes showed two patterns: genes related to lipid metabolism/synthesis were down-regulated by oxidative stress inducing compounds: CsA, TC and TMX, and up-regulated by peroxisome proliferator-activated receptor-alpha agonists: CFR and VPA. From these findings, we concluded that the cell-based assay developed in this study is an appropriate method to predict drugs' potential for hepatosteatosis, and gene expression analysis is useful to profile the mechanism of the hepatosteatosis.

A novel function of bamboo extract in relieving lipotoxicity

Lipotoxicity is closely related to the etiology and complications of type 2 diabetes mellitus. This study investigated the protective effect of an extract from bamboo Phyllostachys edulis against palmitic acid (PA)-induced lipoapoptosis. The lipo-detoxification function of the bamboo extract (BEX) was evaluated using cell culture models. Cell viability was measured by MTT assay and cell apoptosis was monitored by Annexin V staining. Cellular uptake of fluorescent free fatty acid (FFA) analog was measured by flow cytometry. Protein levels of total protein kinase B (Akt) and phosphorylated Akt (p-Akt) were measured by western blotting. The results show that co-incubating BEX with mouse myoblast C2C12 cells had no effect on the cellular uptake of FFA, but dramatically decreased PA-induced cell apoptosis and protected cell viability. A similar antilipotoxicity effect of BEX was observed in other mammalian cells. BEX significantly decreased the protein levels of both Akt and p-Akt in C2C12 cells under normal cell culture conditions but not under lipotoxic conditions, indicating the regulatory effect of BEX on cell signaling pathways and its response to a high FFA environment. This study demonstrated a novel function of bamboo extract in preventing lipotoxicity in mammalian cells, implicating a promising phytotherapeutic approach for lipo-detoxification.

The use of fluorescent fatty acid analogs as labels in trematode experimental infections

We examined the utility of fluorescent fatty acid analog dyes for labeling larval trematodes to use in experimental infections. Our goals were to identify two dyes that label larval trematodes belonging to the species Maritrema novaezealandensis and Coitocaecum parvum, determine if the dyes influence survival and infectivity of larval trematodes and/or host mortality, and if larval trematodes labeled with alternative dyes could be distinguished post-infection. The two dyes tested, BODIPY FL C12 and BODIPY 558/568 C12, successfully labeled all treated larval trematodes, did not influence cercariae survival or infectivity, and did not influence host mortality in either host–parasite system. All larval parasites were fluorescent and distinguishable after 5 days in amphipod intermediate hosts. In addition, larval Acanthoparyphium sp. were strongly fluorescent with both dyes after 5 weeks within cockle hosts. This method should be extremely useful for experimental studies using trematode–host systems as models for addressing a range of ecological and evolutionary questions.

Comparative studies of human fatty acid transport proteins 1, 4 and 6 in the vectorial acylation of exogenous long chain fatty acids

The fatty acid transport protein (FATP) family consists of six members that are involved in the transport of exogenous long chain fatty acids (LCFAs) and the activation of very long chain fatty acids. Genetic and biochemical studies support the idea that several of the FATPs function in transport with a long chain acyl CoA synthetase by a process named as vectorial acylation. Two lines of experiments are being completed to define the role of selected FATP isoforms in vectorial acylation. In the first, we have expressed FATP1, FATP4 and FATP6 in a yeast strain devoid of both transport and activation activity. Our initial studies demonstrate FATP1 and 6 fail to function in fatty acid transport; FATP4 confers very low levels of it. Additional studies are evaluating complementation patterns relying on exogenous fatty acid transport and defining acyl CoA synthetase profiles using different fatty acid substrates. Second, we have cloned FATP1, 4 and 6 into pcDNA4/TO/myc‐His‐A and a tetracycline‐inducible promoter drives their regulated expression when transfected into T‐Rex 293 cells. We have generated stable lines expressing FATP1, 4 and 6 and the kinetic parameters of fatty acid transport are being determined using a live cell assay and the fluorescent fatty acid analog C1‐BODIPY‐C12. Additional studies are addressing localization of each of these proteins by immunofluorescence. Supported by a grant from the NIH (GM56850).

High-throughput screening for fatty acid uptake inhibitors in humanized yeast identifies atypical antipsychotic drugs that cause dyslipidemias

Fatty acids are implicated in the development of dyslipidemias, leading to type 2 diabetes and cardiovascular disease. We used a standardized small compound library to screen humanized yeast to identify compounds that inhibit fatty acid transport protein (FATP)-mediated fatty acid uptake into cells. This screening procedure used live yeast cells expressing human FATP2 to identify small compounds that reduced the import of a fluorescent fatty acid analog, 4,4-difluoro-5-methyl-4-bora-3a,4a-diaza-s-indacene-3-dodecanoic acid (C(1)-BODIPY-C(12)). The library used consisted of 2,080 compounds with known biological activities. Of these, approximately 1.8% reduced cell-associated C(1)-BODIPY-C(12) fluorescence and were selected as potential inhibitors of human FATP2-mediated fatty acid uptake. Based on secondary screens, 28 compounds were selected as potential fatty acid uptake inhibitors. Some compounds fell into four groups with similar structural features. The largest group was structurally related to a family of tricyclic, phenothiazine-derived drugs used to treat schizophrenia and related psychiatric disorders, which are also known to cause metabolic side effects, including hypertriglyceridemia. Potential hit compounds were studied for specificity of interaction with human FATP and efficacy in human Caco-2 cells. This study validates this screening system as useful to assess the impact of drugs in preclinical screening for fatty acid uptake.

Charge modification at conserved positively charged residues of fatty acid binding protein (FABP) from the giant liver fluke Fasciola gigantica: its effect on oligomerization and binding properties

Fatty acid binding proteins (FABPs) are capable of binding hydrophobic ligands with high affinity; thereby facilitating the cellular uptake and intracellular trafficking of fatty acids. In this study, functional characteristics of a cytoplasmic FABP from the giant liver fluke Fasciola gigantica (FgFABP) were determined. Binding of a fluorescent fatty acid analogue 11-[[5-dimethy aminonaphtalene-1-sulphonyl] amino] undecanoic acid (DAUDA) to FgFABP resulted in changes in the emission spectrum. The optimal excitation wavelength and maximum emission of fluorescence for binding activities with DAUDA were 350 nm and 550 nm, respectively. The binding activity for DAUDA was determined from titration experiments and revealed a Kd value of 2.95+/-0.54 microM. Furthermore, we found that cross-linking profile of FgFABP with dithiobis-(succinimidylpropionate) (DSP) in the presence of DAUDA resulted in increased formation of higher-ordered oligomers compared to that in the absence of DAUDA. We also replaced five highly conserved positively charged residues (K9, K58, K91, R107 and K131) with alanine and studied their oligomerization and binding properties of the modified FgFABPs. The obtained data demonstrate that these residues do not appear to be involved in oligomerization. However, the K58A and R107A substitutions exhibited a reduction in binding affinities. K91A and R107A revealed an increase in maximal specific binding.

Abstracts for Oral Presentation

Abstracts for Oral Presentation

Triacylglycerol Is Synthesized in a Specific Subclass of Caveolae in Primary Adipocytes

A principal metabolic function of adipocytes is to synthesize triacylglycerol (TG) from exogenous fatty acids. The level of fatty acids has to be tightly controlled in the adipocyte, as they can act as detergents that rapidly dissolve the plasma membrane, causing cell lysis if allowed to accumulate. Fatty acids therefore have to be efficiently converted to TG and stored in the central lipid droplet. We report that in intact primary adipocytes exogenous oleic acid was taken up and directly converted to TG in the plasma membrane, in a novel subclass of caveolae that specifically contains the protein perilipin. Isolated caveolae catalyzed de novo TG synthesis from oleic acid and glycerol 3-phosphate. Electron microscopy revealed the presence of caveolin and perilipin in caveolae and in lipid-laden bulbs in the plasma membrane, and fluorescence microscopy demonstrated colocalization of fatty acids/TG with caveolin and perilipin at the plasma membrane. A second caveolae fraction was isolated, which lacked perilipin and the triacylglycerol synthesizing enzymes. Both caveolae fractions contained caveolin-1 and the insulin receptor. The findings demonstrate that specific subclasses of caveolae carry out specific functions in cell metabolism. In particular, triacylglycerol is synthesized at the site of fatty acid entry in one of these caveolae classes.

Conformational and functional analysis of the lipid binding protein Ag‐NPA‐1 from the parasitic nematode Ascaridia galli

Ag-NPA-1 (AgFABP), a 15 kDa lipid binding protein (LBP) from Ascaridia galli, is a member of the nematode polyprotein allergen/antigen (NPA) family. Spectroscopic analysis shows that Ag-NPA-1 is a highly ordered, alpha-helical protein and that ligand binding slightly increases the ordered secondary structure content. The conserved, single Trp residue (Trp17) and three Tyr residues determine the fluorescence properties of Ag-NPA-1. Analysis of the efficiency of the energy transfer between these chromophores shows a high degree of Tyr-Trp dipole-dipole coupling. Binding of fatty acids and retinol was accompanied by enhancement of the Trp emission, which allowed calculation of the affinity constants of the binary complexes. The distance between the single Trp of Ag-NPA-1 and the fluorescent fatty acid analogue 11-[(5-dimethylaminonaphthalene-1- sulfonyl)amino]undecanoic acid (DAUDA) from the protein binding site is 1.41 nm as estimated by fluorescence resonance energy transfer. A chemical modification of the Cys residues of Ag-NPA-1 (Cys66 and Cys122) with the thiol reactive probes 5-({[(2-iodoacetyl)amino]ethyl}amino) naphthalene-1-sulfonic acid (IAEDANS) and N,N'-dimethyl-N-(iodoacetyl)-N'-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)ethylenediamine (IANBD), followed by MALDI-TOF analysis showed that only Cys66 was labeled. The observed similar affinities for fatty acids of the modified and native Ag-NPA-1 suggest that Cys66 is not a part of the protein binding pocket but is located close to it. Ag-NPA-1 is one of the most abundant proteins in A. galli and it is distributed extracellularly mainly as shown by immunohistology and immunogold electron microscopy. This suggests that Ag-NPA-1 plays an important role in the transport of fatty acids and retinoids.

A live-cell high-throughput screening assay for identification of fatty acid uptake inhibitors

We developed a live-cell high-throughput assay system using the baker’s yeast Saccharomyces cerevisiae to screen for chemical compounds that will inhibit fatty acid uptake. The target for the inhibitors is a mammalian fatty acid transport protein (mmFATP2), which is involved in the fatty acid transport and activation pathway. The mmFATP2 was expressed in a S. cerevisiae mutant strain deficient in Fat1p-dependent fatty acid uptake and reduced in long-chain fatty acid activation, fat1Δ faa1Δ. To detect fatty acid import, a fluorescent fatty acid analog, 4,4-difluoro-5-methyl-4-bora-3 a,4 a-diaza- s-indacene-3-dodecanoic acid (C 1-BODIPY-C 12), was incubated with cells expressing FATP2 in a 96-well plate. The mmFATP2-dependent C 1-BODIPY-C 12 uptake was monitored by measuring intracellular C 1-BODIPY-C 12 fluorescence on a microtiter plate reader, whereas extracellular fluorescence was quenched by a cell viability dye, trypan blue. Using this high-throughput screening method, we demonstrate that the uptake of the fluorescent fatty acid ligand was effectively competed by the natural fatty acid oleate. Inhibition of uptake was also demonstrated to occur when cells were pretreated with sodium azide or Triacsin C. This yeast live-cell-based assay is rapid to execute, inexpensive to implement, and has adequate sensitivity for high-throughput screening. The assay basis and limitations are discussed.

A native 13-kDa fatty acid binding protein from the liver fluke Fasciola hepatica.

A 13-kDa fatty acid binding protein (FABP) (Fh13) has been isolated from the cytosol of adult Fasciola hepatica and its physicochemical and binding characteristics determined. Fh13 appears to exist as a dimer in native solution. Binding of the fluorescent fatty acid analogue 11-((5-dimethyl aminonaphthalene-1-sulfonyl) amino) undecanoic acid (DAUDA) to Fh13 results in changes in the emission spectrum, which are reversed by oleic acid. The binding activity for DAUDA determined from titration experiments revealed a single binding site per monomeric unit with Kd of 1.5 microM. The displacement of DAUDA by competitive nonfluorescent ligands allowed Kd values for oleic (2.5 microM), retinoic (2.8 microM), palmitic (4.1 microM) and arachidonic acid (6.1 microM) to be calculated. Ten commonly used anthelmintics were evaluated for binding to Fh13, but only bithionol showed binding activity commensurate with those of the putative natural ligands (Kd 6.8 microM).

Regulation of sterol carrier protein gene expression by the Forkhead transcription factor FOXO3a

The SCP gene encodes two proteins, sterol carrier protein X (SCPx) and SCP2, that are independently regulated by separate promoters. SCPx has been shown to be the thiolase involved in the breakdown of branched-chain fatty acids and in the biosynthesis of bile acids. The in vivo function of SCP2 however remains to be established. The transcriptional regulation of SCPx and SCP2 is unclear, but their promoter regions contain several putative regulatory domains. We show here that both SCPx and SCP2 are upregulated by the daf-16-like Forkhead transcription factor FOXO3a (also known as FKHRL1) on the level of promoter activity. It was recently described that Forkheads regulate protection against (oxidative) stress in both Caenorhabditis elegans and mammalian cells. We looked into a role for SCP2 in the cellular defense against oxidative damage and found that a fluorescent fatty acid analog bound to SCP2 is protected against H2O2/Cu2+-induced oxidative damage. We propose a model for the way in which SCP2 could protect fatty acids from peroxidation.

Effect of Charge Reversal Mutations on the Ligand- and Membrane-binding Properties of Liver Fatty Acid-binding Protein

Liver fatty acid-binding protein (FABP) is able to bind to anionic phospholipid vesicles under conditions of low ionic strength. This binding results in the release of ligand, the fluorescent fatty acid analogue 11-dansylaminoundecanoic acid (DAUDA), with loss of fluorescence intensity (Davies, J. K., Thumser, A. E. A., and Wilton, D. C. (1999) Biochemistry 38, 16932-16940). Using a strategy of charge reversal mutagenesis, the potential role of specific cationic residues in promoting interfacial binding of FABP to anionic phospholipid vesicles has been investigated. Cationic residues chosen included those within the alpha-helical region (Lys-20, Lys-31, and Lys-33) and those that make a significant contribution to the positive surface potential of the protein (Lys-31, Lys-36, Lys-47, Lys-57, and Arg-126). Only three cationic residues make a significant contribution to interfacial binding, and these residues (Lys-31, Lys-36, and Lys-57) are all located within the ligand portal region, where the protein may be predicted to exhibit maximum disorder. The binding of tryptophan mutants, F3W, F18W, and C69W, to dioleoylphosphatidylglycerol vesicles, containing 5 mol% of the fluorescent phospholipid dansyldihexadecanoylphosphatidylethanolamine, was monitored by fluorescence resonance energy transfer (FRET). All three mutants showed enhanced dansyl fluorescence due to FRET on addition of phospholipid to protein; however, this fluorescence was considerably greater with the F3W mutant, consistent with the N-terminal region of the protein coming in close proximity to the phospholipid interface. These results were confirmed by succinimide quenching studies. Overall, the results indicate that the portal region of liver FABP and specifically Lys-31, Lys-36, and Lys-57 are involved in the interaction with the interface of anionic vesicles and that the N-terminal region of the protein undergoes a conformational change, resulting in DAUDA release.

The Acyl-CoA Synthetases Encoded within FAA1 andFAA4 in Saccharomyces cerevisiaeFunction as Components of the Fatty Acid Transport System Linking Import, Activation, and Intracellular Utilization

Exogenous long-chain fatty acids are activated to coenzyme A derivatives prior to metabolic utilization. In the yeast Saccharomyces cerevisiae, the activation of these compounds prior to metabolic utilization proceeds through the fatty acyl-CoA synthetases Faa1p and Faa4p. Faa1p or Faa4p are essential for long-chain fatty acid import, suggesting that one or both of these enzymes are components of the fatty acid transport system, which also includes Fat1p. By monitoring the intracellular accumulation of the fluorescent long-chain fatty acid analogue 4,4-difluoro-5-methyl-4-bora-3a,4a-diaza-s-indacene-3-dodecanoic acid, long-chain fatty acid transport was shown to be severely restricted in a faa1 Delta faa4 Delta strain. These data established for the first time a mechanistic linkage between the import and activation of exogenous fatty acids in yeast. To investigate this linkage further, oleoyl CoA levels were defined following incubation of wild type and mutant cells with limiting concentrations of exogenous oleate. These studies demonstrated oleoyl CoA levels were reduced to less than 10% wild-type levels in faa1 Delta and faa1 Delta faa4 Delta strains. Defects in metabolic utilization and intracellular trafficking were also found in the fatty acyl-CoA synthetase-deficient strains. The faa1 Delta faa4 Delta strain had a marked reduction in endogenous acyl-CoA pools, suggesting these enzymes play a role in maintenance of endogenous acyl-CoA pools, metabolism and trafficking. In addition, this strain had levels of in vivo beta-oxidation of exogenous oleate reduced 3-fold when compared with the isogenic parent. Northern analyses demonstrated an additional defect in fatty acid trafficking as FAA1 or FAA4 were required for the transcriptional regulation of the genes encoding the peroxisomal enzymes acyl-CoA oxidase (POX1) and medium-chain acyl-CoA synthetase (FAA2). These data support the hypothesis that fatty acyl-CoA synthetase (Faa1p or Faa4p) functions as a component of the fatty acid import system by linking import and activation of exogenous fatty acids to intracellular utilization and signaling.

Kinetic model of protein-mediated ligand transport: influence of soluble binding proteins on the intermembrane diffusion of a fluorescent fatty acid.

The mechanism (or mechanisms) whereby fatty acids and other amphipathic compounds are transported from the plasma membrane to intracellular sites of biotransformation remains poorly defined. In an attempt to better characterize the role of cytosolic binding proteins in this process, a kinetic model of intermembrane ligand transport was developed in which diffusional transfer of ligand between membrane and protein is assumed. The model was tested by utilizing stopped-flow techniques to monitor the transfer of the fluorescent fatty acid analogue, 12-anthroyloxy stearate (12-AS), between model membrane vesicles. Studies were conducted in the presence or absence of bovine serum albumin (BSA), liver fatty acid-binding protein (L-FABP), and intestinal fatty acid-binding protein (I-FABP) in order to determine the effect of soluble proteins on the rate of intermembrane ligand transfer. As predicted by the model, the initial velocity of 12-AS arrival at the acceptor membrane increases in an asymptotic manner with the acceptor concentration. Furthermore, probe transfer velocity was found to decline asymptotically with increasing concentrations of BSA or L-FABP, proteins that exhibit diffusional transfer kinetics. This observation was found to hold true independent of whether donor or acceptor vesicles were preequilibrated with the protein. In contrast, 12-AS transfer velocity exhibited a linear correlation with the concentration of I-FABP, a protein that is thought to transport fatty acids, at least in part, via a collisional mechanism. Taken together, these findings validate the derived kinetic model of protein-mediated ligand transport and further suggest that the mechanism of ligand-protein interaction is a key determinant of the effect of cytosolic proteins on intracellular ligand diffusion.

Binding of recombinant rat liver fatty acid-binding protein to small anionic phospholipid vesicles results in ligand release: a model for interfacial binding and fatty acid targeting.

A number of intracellular proteins bind to negatively charged phospholipid membranes, and this interfacial binding results in a conformational change that modulates the activity of the protein. Using a fluorescent fatty acid analogue, 11-[5-(dimethylamino)naphthalenesulfonyl]undecanoic acid (DAUDA), it is possible to demonstrate the release of this ligand from recombinant rat liver FABP in the presence of phospholipid vesicles that contain a significant proportion of anionic phospholipids. The ligand release that is observed with anionic phospholipids is sensitive to the ionic strength of the assay conditions and the anionic charge density of the phospholipid at the interface, indicating that nonspecific electrostatic interactions play an important role in the process. The stoichiometric relationship between anionic phospholipid and liver FABP suggests that the liver FABP coats the surface of the phospholipid vesicle. The most likely explanation for ligand release is that interaction of FABP with an anionic membrane interface induces a rapid conformational change, resulting in a reduced affinity of DAUDA for the protein. The nature of this interaction involves both electrostatic and nonpolar interactions as maximal release of liver FABP from phospholipid vesicles with recovery of ligand binding cannot be achieved with high salt and requires the presence of a nonionic detergent. The precise interfacial mechanism that results in the rapid release of ligand from L-FABP remains to be determined, but studies with two mutants, F3W and F18W, suggest the possible involvement of the amino-terminal region of the protein in the process. The conformational change linked to interfacial binding of this protein could provide a mechanism for fatty acid targeting within the cell.

Binding studies of tear lipocalin: the role of the conserved tryptophan in maintaining structure, stability and ligand affinity

The principal lipid binding protein in tears, tear lipocalin (TL), binds the spin-labeled analog of lauric acid and the fluorescent fatty acid analogs, DAUDA and 16-AP at one site. The native ligands of TL compete for this binding site. A fluorescent competitive binding assay revealed that apo-TL has a high affinity for phospholipids and stearic acid ( K i) of 1.2 μM and 1.3 μM, respectively, and much less affinity for cholesterol ( K i) of 15.9 μM. For fatty acids, binding affinity correlated with the length of the hydrocarbon chain. TL binds most strongly the least soluble lipids permitting these lipids to exceed their maximum solubility in aqueous solution. These data implicate TL in solubilizing and transporting lipids in the tear film. Phenylalanine, tyrosine and cysteine were substituted for Trp 17, the only invariant residue throughout the lipocalin superfamily. Cysteine substitution resulted in some loss of secondary structure, relaxation of aromatic side chain rigidity, decreased binding affinity for DAUDA and destabilization of structure. Mutants of TL, W17Y, and W17F showed a higher binding affinity for DAUDA than wild-type TL. Comparison of the results of tryptophan 17 substitution in lipocalin with those of tryptophan 19 substitution in β-lactoglobulin revealed important differences in binding characteristics that reflect the functional heterogeneity within the lipocalin family.

Ascaridia galli fatty acid-binding protein, a member of the nematode polyprotein allergens family.

A fatty acid-binding protein from the nematode Ascaridia galli was characterized. The gene was isolated and recombinantly expressed in Escherichia coli. According to the deduced amino acid sequence A. galli fatty acid-binding protein (AgFABP) belongs to the family of nematode polyprotein allergens, as shown by Western blotting and PCR analysis with genomic DNA and cDNA. Both native and recombinant proteins bind fatty acids and retinoids with high affinity. The fluorescent fatty acid analogue 11-[(5-dimethylaminonaphthalene-1-sulfonyl)amino] undecanoic acid (DAUDA) shows substantial changes in its emission spectrum when bound to AgFABP; this binding is reversed by fatty acids such as oleate. Moreover, changes of the intrinsic fluorescence of retinol and retinoic acid confirm retinoid binding activity of AgFABP. Fluorescence titration experiments with DAUDA indicate stoichiometric binding to a single binding site per monomer unit with affinities (Kd) of 1.6 and 1.8 x 10(-7) m for native and the recombinant protein, respectively. The apparent binding affinities of the nonfluorescent ligands were calculated in displacement experiments with DAUDA and values in the same range were obtained for myristic, palmitic, oleic, linoleic, arachidonic and retinoic acid. Additionally, the binding affinity of AgFABP for oleate and palmitate was determined by direct and indirect radiochemical analysis and the values obtained were similar to those from the fluorescent experiments. Both proteins show a preference for the binding of long-chain saturated and unsaturated fatty acids, but not for short chain (C3-C12) and branched fatty acids, cholesterol and tryptophan.

Phenotypic Adaptation of Membrane Fluidity in the Tonoplast and Plasmalemma the C 3 Plant Hordeum vulgare cv. Alexis

Summary The influence of growth temperature and salinity stress on the fluidity of the tonoplast and plasmalemma in Hordeum vulgare cv. Alexis was studied. Membrane fluidity was monitored by measurements of fluorescence polarisation in membrane vesicles labeled with the fluorescent fatty acid analogue 1,6-diphenyl- 1,3,5-hexatrien (DPH) as a probe. It was found that, both in the tonoplast and plasmalemma, an increase in growth temperature lowered membrane fluidity, whereas lowering of growth temperature did the opposite. There is evidence that the rigidization of the membranes due to increase in growth temperature is mainly caused by membrane proteins. Accumulation of salt in the vacuoles of plants did not significantly shift the fluidity of the membranes, neither in the tonoplast nor in the plasmalemma. The results suggest that phenotypic adaptation of membrane fluidity also occurs in the tonoplast and plasmalemma of C3 plants, and not only in the tonoplast of CAM plants as previously reported.