Oleate Binding Research Articles

Reverse flotation separation of calcite/dolomite from hemimorphite based on their surface sulphophile and oxyphile affinity differences

This study attempts to selectively inhibit hemimorphite flotation using sodium sulphide and uncover the mechanisms behind it. With the use of a fatty acid collector, the flotation test results showed that sodium sulphide had a strong inhibition performance on hemimorphite flotation while having no adverse impact on calcite and dolomite flotation, which were consistent with the contact angle measurement results. According to the results of attenuated total reflectance-Fourier transform infrared spectroscopy and adsorption proportion measurement of sodium oleate, sodium sulphide can inhibit sodium oleate binding on hemimorphite surfaces while having no effect on calcite and dolomite surfaces. The sulphide species exhibited an extremely high binding affinity for the hemimorphite surface but not for the surfaces of the calcium/magnesium-bearing gangue minerals, as demonstrated by the X-ray photoelectron spectroscopy measurements. Because of the strong sulphophile affinity of zinc, when the sodium sulphide concentration was sufficiently high, an interface boundary layer of a liquid solution with the remaining sulphide species formed on the hemimorphite surface, preventing sodium oleate from binding on the hemimorphite surfaces and thus selectively inhibiting the hemimorphite flotation.

Impact of lipid binding on the tertiary structure and allergenic potential of Jug r 3, the non-specific lipid transfer protein from walnut

Plant non-specific lipid transfer proteins type 1 (nsLTP1) are small basic proteins with a hydrophobic cavity able to host a number of different ligands: i.e. fatty acids, fatty acyl-CoA, phospholipids, glycolipids, and hydroxylated fatty acids. However, ligand binding specificity differs among nsLTPs. Within this protein family, Jug r 3 from walnut has been identified as a major allergen. So far, data on the structural characterization of Jug r 3 and its lipid binding capacity are lacking. We report the results from a fluorescence-based ligand-binding assay and ligand-based NMR experiments, to study the binding interactions between Jug r 3 and the 18-carbon monounsaturated oleic acid. Furthermore, protein-based NMR experiments were employed to detect the oleate binding site of Jug r 3. The NMR data were used to dock the oleate molecule into the structural model of Jug r 3. Finally, the impact of the interaction on the allergenic potential of Jug r 3 was investigated by IgE ELISA with 6 sera from walnut allergic patients. Our data corroborate the hypothesis of direct impact of food-derived matrix on the IgE reactivity of nsLTPs.

Long chain fatty acids alter the interactive binding of ligands to the two principal drug binding sites of human serum albumin.

A wide variety of drugs bind to human serum albumin (HSA) at its two principal sites, namely site I and site II. A number of reports indicate that drug binding to these two binding sites are not completely independent, and that interactions between ligands of these two discrete sites can play a role. In this study, the effect of the binding of long-chain fatty acids on the interactive binding between dansyl-L-asparagine (DNSA; site I ligand) and ibuprofen (site II ligand) at pH6.5 was examined. Binding experiments showed that the binding of sodium oleate (Ole) to HSA induces conformational changes in the molecule, which, in turn, changes the individual binding of DNSA and ibuprofen, as well as the mode of interaction between these two ligands from a ‘competitive-like’ allosteric interaction in the case of the defatted HSA conformer to a ‘nearly independent’ binding in the case of non-defatted HSA conformer. Circular dichroism measurements indicated that ibuprofen and Ole are likely to modify the spatial orientation of DNSA at its binding site. Docking simulations suggest that the long-distance electric repulsion between DNSA and ibuprofen on defatted HSA contributes to a ‘competitive-like’ allosteric interaction, whereas extending the distance between ligands and/or increasing the flexibility or size of the DNSA binding site in fatted HSA evokes a change in the interaction mode to ‘nearly independent’ binding. The present findings provide further insights into the structural dynamics of HSA upon the binding of fatty acids, and its effects on drug binding and drug-drug interactions that occur on HSA.

Effects of pH and protein conformation on in-solution complexation between bovine α-lactalbumin and oleic acid: Binding trend analysis by using SPR and ITC

To analyze the trends in molecular interaction between bovine α-LA (BLA) and oleic acid, we investigated the effects of pH and protein conformation on oleate binding to BLA and compared the order-of-magnitude differences in binding behavior. Both isothermal titration calorimetry (ITC) and surface plasmon resonance (SPR) were used. For the ITC experiments, a solution of holo- or apo-BLA was titrated with sodium oleate at pH 4.0 and 10.0 to look for protein surface charge effects. In the SPR experiments, BLA was immobilized on the chip surface and sodium oleate solutions at various pHs (4.0, 5.5, 7.0, 8.5 and 10.0) were injected. Binding stoichiometry of ca. 5.2 molecules of oleate per unit molecule of BLA was observed. apo-BLA at a lower pH (lower than the pI of α-LA) yielded stronger binding avidity and affinity, which indicated that electrostatic interactions between the basic residues and the negatively charged carboxyl groups may play a major role in the complexation, in addition to hydrophobic interactions. The complexes formed at pH 4.0 and 5.5 were relatively unstable and rapidly dissociated when exposed to pH 7.0. In summary, we propose that the complexation can be regarded as a very weak (or, transient) and multivalent binding, requiring both electrostatic and hydrophobic interactions, probably in series and also in a dynamic equilibrium.

Formation of cytotoxic α-lactalbumin / sodium oleate complexes: Concentration and temperature effects

Cytotoxic complexes may be formed by mixing whey proteins and oleic acid or sodium oleate. The present study investigated the kinetics of complex formation when the whey protein α-lactalbumin (α-LA) is mixed with sodium oleate. Intrinsic fluorescence showed that the complex formed at room temperature during the mixing of α-LA and sodium oleate. However, some of the structural changes brought about by the binding of the sodium oleate can be reversed by dialysis, when loosely bound oleate is removed. During dialysis, solutions with lower protein concentrations had reduced buffering capacity, and so the pH decreased more; this decrease in pH was correlated with a loss of oleate from the complex. The cytotoxicity of all the samples produced in this study was strongly correlated with the amount of oleate bound in the complex, confirming previous studies that suggested that oleic acid is the cytotoxic agent in the complex.

Liver Fatty Acid-binding Protein Binds Monoacylglycerol in Vitro and in Mouse Liver Cytosol

Liver fatty acid-binding protein (LFABP; FABP1) is expressed both in liver and intestinal mucosa. Mice null for LFABP were recently shown to have altered metabolism of not only fatty acids but also monoacylglycerol, the two major products of dietary triacylglycerol hydrolysis (Lagakos, W. S., Gajda, A. M., Agellon, L., Binas, B., Choi, V., Mandap, B., Russnak, T., Zhou, Y. X., and Storch, J. (2011) Am. J. Physiol. Gastrointest. Liver Physiol. 300, G803-G814). Nevertheless, the binding and transport of monoacylglycerol (MG) by LFABP are uncertain, with conflicting reports in the literature as to whether this single chain amphiphile is in fact bound by LFABP. In the present studies, gel filtration chromatography of liver cytosol from LFABP(-/-) mice shows the absence of the low molecular weight peak of radiolabeled monoolein present in the fractions that contain LFABP in cytosol from wild type mice, indicating that LFABP binds sn-2 MG in vivo. Furthermore, solution-state NMR spectroscopy demonstrates two molecules of sn-2 monoolein bound in the LFABP binding pocket in positions similar to those found for oleate binding. Equilibrium binding affinities are ∼2-fold lower for MG compared with fatty acid. Finally, kinetic studies examining the transfer of a fluorescent MG analog show that the rate of transfer of MG is 7-fold faster from LFABP to phospholipid membranes than from membranes to membranes and occurs by an aqueous diffusion mechanism. These results provide strong support for monoacylglycerol as a physiological ligand for LFABP and further suggest that LFABP functions in the efficient intracellular transport of MG.

Molecular Modeling and Functional Confirmation of a Predicted Fatty Acid Binding Site of Mitochondrial Aspartate Aminotransferase

Molecular interactions are necessary for proteins to perform their functions. The identification of a putative plasma membrane fatty acid transporter as mitochondrial aspartate aminotransferase (mAsp-AT) indicated that the protein must have a fatty acid binding site. Molecular modeling suggests that such a site exists in the form of a 500-Å3 hydrophobic cleft on the surface of the molecule and identifies specific amino acid residues that are likely to be important for binding. The modeling and comparison with the cytosolic isoform indicated that two residues (Arg201 and Ala219) were likely to be important to the structure and function of the binding site. These residues were mutated to determine if they were essential to that function. Expression constructs with wild-type or mutated cDNAs were produced for bacteria and eukaryotic cells. Proteins expressed in Escherichia coli were tested for oleate binding affinity, which was decreased in the mutant proteins. 3T3 fibroblasts were transfected with expression constructs for both normal and mutated forms. Plasma membrane expression was documented by indirect immunofluorescence before [3H]oleic acid uptake kinetics were assayed. The Vmax for uptake was significantly increased by overexpression of the wild-type protein but changed little after transfection with mutated proteins, despite their presence on the plasma membrane. The hydrophobic cleft in mAsp-AT can serve as a fatty acid binding site. Specific residues are essential for normal fatty acid binding, without which fatty acid uptake is compromised. These results confirm the function of this protein as a fatty acid binding protein.

Lipid binding to cytoglobin leads to a change in haem co-ordination: a role for cytoglobin in lipid signalling of oxidative stress

Cytoglobin is a recently discovered hexa-co-ordinate haemoglobin that does not appear to function as a classical oxygen-binding protein. Its function is unknown and studies on the effects of changes in its expression have not decisively determined its role within the cell. In the present paper, we report that the protein is transformed from hexa-co-ordinate to penta-co-ordinate on binding a lipid molecule. This transformation occurs with the ferric oxidation state of the protein, but not the ferrous state, indicating that this process only occurs under an oxidative environment and may thus be related to redox-linked cell signalling mechanisms. Oleate binds to the protein in a 1:1 stoichiometry and with high affinity (K(d)=0.7 μM); however, stopped-flow kinetic measurements yield a K(d) value of 110 μM. The discrepancy between these K(d) values may be rationalized by recognizing that cytoglobin is a disulfide-linked dimer and invoking co-operativity in oleate binding. The lipid-induced transformation of cytoglobin from hexa-co-ordinate to penta-co-ordinate does not occur with similar hexa-co-ordinate haemoglobins such as neuroglobin, and therefore appears to be a unique property of cytoglobin among the haemoglobin superfamily. The lipid-derived transformation may explain why cytoglobin has enhanced peroxidatic activity, converting lipids into various oxidized products, a property virtually absent from neuroglobin and much decreased in myoglobin. We propose that the binding of ferric cytoglobin to lipids and their subsequent transformation may be integral to the physiological function of cytoglobin, generating cell signalling lipid molecules under an oxidative environment.

S-Nitrosylated Human Serum Albumin-mediated Cytoprotective Activity Is Enhanced by Fatty Acid Binding

Binding of oleate to S-nitrosylated human serum albumin (SNO-HSA) enhances its cytoprotective effect on liver cells in a rat ischemia/reperfusion model. It enhances the antiapoptotic effect of SNO-HSA on HepG2 cells exposed to anti-Fas antibody. To identify some of the reasons for the increased cytoprotective effects, additional experiments were performed with glutathione and HepG2 cells. As indicated by 5,5′-dithiobis-2-nitrobenzoic acid binding, the addition of oleate increased the accessibility of the single thiol group of albumin. Binding of increasing amounts of oleate resulted in increasing and more rapid S-transnitrosation of glutathione. Likewise, binding of oleate, or of a mixture of endogenous fatty acids, improved S-denitrosation of SNO-HSA by HepG2 cells. Oleate also enhanced S-transnitrosation by HepG2 cells, as detected by intracellular fluorescence of diaminofluorescein-FM. All of the S-transnitrosation caused by oleate binding was blocked by filipin III. Oleate also increased, in a dose-dependent manner, the binding of SNO-HSA labeled with fluorescein isothiocyanate to the surface of the hepatocytes. A model in two parts was worked out for S-transnitrosation, which does not involve low molecular weight thiols. Fatty acid binding facilitates S-denitrosation of SNO-HSA, increases its binding to HepG2 cells and greatly increases S-transnitrosation by hepatocytes in a way that is sensitive to filipin III. A small nitric oxide transfer takes place in a slow system, which is unaffected by fatty acid binding to SNO-HSA and not influenced by filipin III. Thus, fatty acids could be a novel type of mediator for S-transnitrosation.

Native UCP1 Displays Simple Competitive Kinetics between the Regulators Purine Nucleotides and Fatty Acids

Elucidation of the regulation of uncoupling protein 1 (UCP1) activity in its native environment, i.e. the inner membrane of brown-fat mitochondria, has been hampered by the presence of UCP1-independent, quantitatively unresolved effects of investigated regulators on the brown-fat mitochondria themselves. Here we have utilized the availability of UCP1-ablated mice to dissect UCP1-dependent and UCP1-independent effects of regulators. Using a complex-I-linked substrate (pyruvate), we found that UCP1 can mediate a 4-fold increase in thermogenesis when stimulated with the classical positive regulator fatty acids (oleate). After demonstrating that the fatty acids act in their free form, we found that UCP1 increased fatty acid sensitivity approximately 30-fold (as compared with the 1.5-fold increase reported earlier based on nominal fatty acid values). By identifying the UCP1-mediated fraction of the response, we could conclude that the interaction between purine nucleotides (GDP) and fatty acids (oleate) unexpectedly displayed simple competitive kinetics. In GDP-inhibited mitochondria, oleate apparently acted as an activator. However, only a model in which UCP1 is inherently active (i.e."activating" fatty acids cannot be included in the model), where GDP functions as an inhibitor with a K(m) of 0.05 mm, and where oleate functions as a competitive antagonist for the GDP effect (with a K(i) of 5 nm) can fit all of the experimental data. We conclude that, when examined in its native environment, UCP1 functions as a proton (equivalent) carrier in the absence of exogenous or endogenous fatty acids.

A Novel Phospholipase D of Arabidopsis That Is Activated by Oleic Acid and Associated with the Plasma Membrane

Oleate-dependent phospholipase D (PLD; EC 3.1.4.4) has been reported in animal systems, but its molecular nature is unkown. Multiple PLDs have been characterized in plants, but none of the previously cloned PLDs exhibits the oleate-activated activity. Here, we describe the biochemical and molecular identification and characterization of an oleate-activated PLD in Arabidopsis. This PLD, designated PLDdelta, was associated tightly with the plasma membrane, and its level of expression was higher in old leaves, stems, flowers, and roots than in young leaves and siliques. A cDNA encoding the oleate-activated PLD was identified, and catalytically active PLDdelta was expressed from its cDNA in Escherichia coli. PLDdelta was activated by free oleic acid in a dose-dependent manner, with the optimal concentration being 0.5 mM. Other unsaturated fatty acids, linoleic and linolenic acids, were less effective than oleic acid, whereas the saturated fatty acids, stearic and palmitic acids, were totally ineffective. Phosphatidylinositol 4,5-bisphosphate stimulated PLDdelta to a lesser extent than oleate. Mutation at arginine (Arg)-611 led to a differential loss of the phosphatidylinositol 4,5-bisphosphate-stimulated activity of PLDdelta, indicating that separate sites mediate the oleate regulation of PLDdelta. Oleate stimulated PLDdelta's binding to phosphatidylcholine. Mutation at Arg-399 resulted in a decrease in oleate binding by PLDdelta and a loss of PLDdelta activity. However, this mutation bound similar levels of phosphatidylcholine as wild type, suggesting that Arg-399 is not required for PC binding. These results provide the molecular information on oleate-activated PLD and also suggest a mechanism for the oleate stimulation of this enzyme.

A Novel Phospholipase D of Arabidopsis That Is Activated by Oleic Acid and Associated with the Plasma Membrane

Oleate-dependent phospholipase D (PLD; EC 3.1.4.4) has been reported in animal systems, but its molecular nature is unkown. Multiple PLDs have been characterized in plants, but none of the previously cloned PLDs exhibits the oleate-activated activity. Here, we describe the biochemical and molecular identification and characterization of an oleate-activated PLD in Arabidopsis. This PLD, designated PLDδ, was associated tightly with the plasma membrane, and its level of expression was higher in old leaves, stems, flowers, and roots than in young leaves and siliques. A cDNA encoding the oleate-activated PLD was identified, and catalytically active PLDδ was expressed from its cDNA inEscherichia coli. PLDδ was activated by free oleic acid in a dose-dependent manner, with the optimal concentration being 0.5 mm. Other unsaturated fatty acids, linoleic and linolenic acids, were less effective than oleic acid, whereas the saturated fatty acids, stearic and palmitic acids, were totally ineffective. Phosphatidylinositol 4,5-bisphosphate stimulated PLDδ to a lesser extent than oleate. Mutation at arginine (Arg)-611 led to a differential loss of the phosphatidylinositol 4,5-bisphosphate-stimulated activity of PLDδ, indicating that separate sites mediate the oleate regulation of PLDδ. Oleate stimulated PLDδ's binding to phosphatidylcholine. Mutation at Arg-399 resulted in a decrease in oleate binding by PLDδ and a loss of PLDδ activity. However, this mutation bound similar levels of phosphatidylcholine as wild type, suggesting that Arg-399 is not required for PC binding. These results provide the molecular information on oleate-activated PLD and also suggest a mechanism for the oleate stimulation of this enzyme.

Ligand-Dependent Conformational Equilibria of Serum Albumin Revealed by Tryptophan Fluorescence Quenching

Ligand-dependent structural changes in serum albumin are suggested to underlie its role in physiological solute transport and receptor-mediated cellular selection. Evidence of ligand-induced (oleic acid) structural changes in serum albumin are shown in both time-resolved and steady-state fluorescence quenching and anisotropy measurements of tryptophan 214 (Trp 214). These studies were augmented with column chromatography separations. It was found that both the steady-state and time-resolved Stern-Volmer collisional quenching studies of Trp 214 with acrylamide pointed to the existence of an oleate-dependent structural transformation. The bimolecular quenching rate constant of defatted human serum albumin, 1.96 × 10 9 M −1 s −1, decreased to 0.94 × 10 9 M −1 s −1 after incubation with oleic acid (9:1). Furthermore, Stern-Volmer quenching studies following fractionation of the structural forms by hydrophobic interaction chromatography were in accordance with this interpretation. Time-resolved fluorescence anisotropy measurements of the Trp 214 residue yielded information of motion within the protein together with the whole protein molecule. Characteristic changes in these motions were observed after the binding of oleate to albumin. The addition of oleate was accompanied by an increase in the rotational diffusion time of the albumin molecule from ∼22 to 33.6 ns. Within the body of the protein, however, the rotational diffusion time for Trp 214 exhibited a slight decrease from 191 to 182 ps and was accompanied by a decrease in the extent of the angular motion of Trp 214, indicating a transition after oleate binding to a more spatially restricted but less viscous environment.

Effects of uremic toxins and fatty acids on serum protein binding of furosemide: possible mechanism of the binding defect in uremia

To elucidate the mechanism of impaired serum binding of furosemide observed in patients with renal dysfunction, we examined in vitro the serum protein binding of furosemide in the absence and presence of uremic toxins that are endogenously retained solutes in uremic serum and act as inhibitors of drug binding. Analysis of the binding data of furosemide at its therapeutic concentration (6.6 mg/L) indicated that, among the four uremic toxins studied, 3-carboxy-4-methyl-5-propyl-2-furanpropionate (CMPF) showed the greatest inhibitory potency for the binding of furosemide to serum; moreover, the inhibition was competitive. CMPF thus most likely represents the primary determinant for the serum binding defect of furosemide in uremia. However, CMPF and oleate appear to exert a synergistic effect on the inhibition of furosemide serum binding--perhaps through a cascade effect on furosemide-binding inhibition in the oleate-CMPF-furosemide system, in which the binding of oleate to its low-affinity sites indirectly displaces furosemide from albumin and thus increases the transiently liberated CMPF molecules. Similar cascade effects on furosemide binding in the presence of CMPF were also originated by other long-chain (C18) fatty acids, linoleate and stearate, although to a lesser extent. Because CMPF is not effectively removed by ordinary hemodialysis treatment, the combined direct and cascade effects of CMPF and fatty acids appear to contribute to the increase in the free fraction of furosemide during hemodialysis.

Mutants of rat intestinal fatty acid-binding protein illustrate the critical role played by enthalpy-entropy compensation in ligand binding.

Site-specific variants of rat intestinal fatty acid-binding protein were constructed to identify the molecular interactions that are important for binding to fatty acids (FAs). Several variants displayed affinities that appeared incompatible with the crystal structure of the protein-FA complex. Thermodynamic measurements provided an explanation for these apparent inconsistencies and revealed that binding affinities often inaccurately reported changes in protein-FA interactions because changes in the binding entropy and enthalpy were usually compensatory. These results demonstrate that understanding the effects of amino acid replacements on ligand binding requires measurements of enthalpy and entropy, in addition to affinity.

Probing the cysteine 34 residue in human serum albumin using fluorescence techniques

The microenvironment surrounding Cys-34 in human serum albumin (HSA) has been studied using acrylodan, a cysteine-specific fluorescence probe. Fluorescence emission maximum (Em max) of acrylodan covalently attached to Cys-34 was observed at 476 nm, which was nearly the same as for acetonitrile. The Em max exhibited a shift toward longer wavelength with oleate binding. The acrylodan fluorescence of HSA exhibited heterogeneous decay kinetics, which adequately fit a double-exponential decay model. When three or more oleate molecules were bound to one albumin molecule, the fractional intensity was shifted in favor of the shorter lived component. These results suggest that oleate binding induces a conformational or dynamic change which is localized in the Cys-34 region. Unfolding studies with guanidine-HCl indicate that Cys-34 appears to be located on the surface of HSA molecule and that it is protected by adjacent amino acid residues. Solvent accessibility of acrylodan with HSA in the absence and presence of oleate was determined from acrylamide quenching, and suggests that oleate binding enhances the solvent exposure of the acrylodan fluorophore. In order to determine the nature of the electrostatic potential near Cys-34, the quenching rate constants for anionic (iodide) and cationic (thallium) quenchers were determined as a function of ionic strength of solvent. The ionic strength dependence of quenching indicated that there was an electrostatic attractive force between the fluorophore and both ionic quenchers. These results are consistent with a model in which amphoteric charges which arise from charged amino acid residues were surrounding Cys-34. Interestingly, oleate binding resulted in changes in the spatial relationships between acrylodan and these charged residues. Thus, the experiments described herein provide the information concerning an oleate-induced alteration in the nature of the local environment surrounding Cys-34 and suggests that long chain fatty acid binding provides a method for regulating the radical-trapping antioxidant activity of Cys-34 in HSA in vivo.

The Crystal Structure of the Liver Fatty Acid-binding Protein: A COMPLEX WITH TWO BOUND OLEATES

The crystal structure of the recombinant form of rat liver fatty acid-binding protein was completed to 2.3 A and refined to an R factor of 19.0%. The structural solution was obtained by molecular replacement using superimposed polyalanine coordinates of six intracellular lipid-binding proteins as a search probe. The entire amino acid sequence of rat liver fatty acid-binding protein along with an amino-terminal formyl-methionine was modeled in the crystal structure. In addition, the crystal was obtained in the presence of oleic acid, and the initial electron density clearly showed two fatty acid molecules bound within a central cavity. The carboxylate of one fatty acid molecule interacts with arginine 122 and is shielded from free solvent. It has an overall bent conformation. The more solvent-exposed carboxylate of the other oleate is located near the helix-turn-helix that caps one end of the beta-barrel, while the acyl chain lies in the interior. The cavity contains both polar and nonpolar residues but also shows extensive hydrophobic character around the nonpolar atoms of the ligands. The primary and secondary oleate binding sites appear to be totally interdependent, mainly because favorable hydrophobic interactions form between both aliphatic chains.

Myocardial cell damage by fatty acid ethyl esters.

Fatty acid ethyl ester (FAEE), a myocardial metabolite of ethanol, causes mitochondrial dysfunction in vitro in rabbits. We investigated the effect of these esters on rat heart mitochondria in vitro and in vivo. In vitro studies were conducted to investigate the binding of ethyl oleate (FAEE) to mitochondria and their capacity to hydrolyze these FAEE. In vivo effects of ethyl esters were studied by the direct transfer of [3H]oleate into the myocardium. Mitochondria were prepared from the myocardium of injected rats, and the amount of [3H]oleate bound to them was determined. In another in vivo study, 50 microliters of 50 microM cold oleic acid ethyl ester was injected into the rat myocardium and the histopathological changes induced by oleic acid ethyl ester were examined by light microscopy. Our results show that fatty acid ethyl ester can bind to myocardial mitochondria in vitro as well as in vivo and the mitochondria can hydrolyze FAEE to fatty acid, which is a known uncoupler of oxidative phosphorylation. Of the total ethyl [3H] oleate injected, 8 microM [3H]oleate and 1 microM ethyl [3H]oleate was bound to the mitochondria. Significant myocardial cell damage was first observed on day 4 and markedly increased on day 30 after ethyl ester injection, with cells showing gross deformation and enlargement. However, no significant histopathological changes were observed in the myocardial tissue on day 2 after injection. Our results suggest that the FAEE may damage the myocardial cells as well as the mitochondria and may provide a metabolic link between ethanol abuse and myocardial dysfunction.

Changes in the Iron Coordination Sphere of Fe(II) Lipoxygenase-1 from Soybeans upon Binding of Linoleate or Oleate

Fe K-edge X-ray absorption spectra of the non-heme iron constituent of lipoxygenase-1 from soybeans were obtained. The spectrum of 2.5 mM Fe(II) lipoxygenase, mixed with 1.2 M linoleate in the absence of O2, was compared to the spectrum of the native (i.e. untreated) enzyme. In the lipoxygenase-linoleate complex, an edge shift to lower energy was observed. This indicated that the iron-ligand distances in this complex are slightly longer than those in the untreated enzyme species. The extended X-ray absorption fine structure spectrum of Fe(II) lipoxygenase, prepared by anaerobic reduction of 2.5 mM Fe(II) lipoxygenase with 1.2 M linoleate, was very similar to the spectrum of the anaerobic lipoxygenase-linoleate complex. We conclude that the conformational differences between the iron coordination spheres of native and cycled Fe(II) lipoxygenase must be ascribed to the presence of linoleate, and not to changes in the enzyme that occur only after one cycle of oxidation and reduction. Furthermore, spectra of 2.5 mM Fe(II) lipoxygenase mixed with 1.2 M oleate, either in the absence or in the presence of O2, were also identical to the spectrum of the Fe(II) lipoxygenase-linoleate complex. This finding is in agreement with our observation that oleate is a competitive inhibitor of the lipoxygenase reaction. Moreover, the similarity of the lipoxygenase-oleate complexes in the presence and absence of O2 excludes the possibility that O2 binding to the iron cofactor is induced upon binding of a fatty acid to lipoxygenase.

Changes in the iron coordination sphere of Fe(II) lipoxygenase-1 from soybeans upon binding of linoleate or oleate.

Fe K-edge X-ray absorption spectra of the non-heme iron constituent of lipoxygenase-1 from soybeans were obtained. The spectrum of 2.5 mM Fe(II) lipoxygenase, mixed with 1.2 M linoleate in the absence of O2, was compared to the spectrum of the native (i.e. untreated) enzyme. In the lipoxygenase-linoleate complex, an edge shift to lower energy was observed. This indicated that the iron-ligand distances in this complex are slightly longer than those in the untreated enzyme species. The extended X-ray absorption fine structure spectrum of Fe(II) lipoxygenase, prepared by anaerobic reduction of 2.5 mM Fe(II) lipoxygenase with 1.2 M linoleate, was very similar to the spectrum of the anaerobic lipoxygenase-linoleate complex. We conclude that the conformational differences between the iron coordination spheres of native and cycled Fe(II) lipoxygenase must be ascribed to the presence of linoleate, and not to changes in the enzyme that occur only after one cycle of oxidation and reduction. Furthermore, spectra of 2.5 mM Fe(II) lipoxygenase mixed with 1.2 M oleate, either in the absence or in the presence of O2, were also identical to the spectrum of the Fe(II) lipoxygenase-linoleate complex. This finding is in agreement with our observation that oleate is a competitive inhibitor of the lipoxygenase reaction. Moreover, the similarity of the lipoxygenase-oleate complexes in the presence and absence of O2 excludes the possibility that O2 binding to the iron cofactor is induced upon binding of a fatty acid to lipoxygenase.