Studies on the mechanism of inhibition of nuclear triiodothyronine binding by fatty acids

Studies were designed to elucidate the mechanism by which unsaturated fatty acids inhibit the binding of triiodothyronine (T3) ro rat liver nuclei. The possibility of a direct interaction between oleic acid and T3 was excluded by dialysis experiments. Oleic acid inhibits nuclear T3 binding in a strictly competitive manner. The Ki value of oleic acid was approx. 10(4) times greater than that of T3. The inhibitory effect of oleic acid could be reversed by bovine serum albumin.     

Fatty acyl-CoAs are potent inhibitors of the nuclear thyroid hormone receptor in vitro

We report that long-chain fatty acyl-CoAs are potent inhibitors of the thyroid hormone (T3) receptor isolated from rat liver nuclei. Both saturated and unsaturated fatty acyl-CoAs were similarly potent. Fifty per cent inhibition of T3 binding by the receptor was observed at an oleoyl-CoA concentration as low as 1.3 microM, and the affinity of oleoyl-CoA for the receptor (Ki) was estimated to be 0.45 microM. Fatty acyl-CoAs also promoted dissociation of the hormone bound to the receptor. The action of fatty acyl-CoAs was competitive for the hormone binding site, resulting in a reduction in the receptor's affinity for T3. These observations suggest that fatty acyl-CoAs modulate the binding of the thyroid hormone to its nuclear receptor, in vitro. Whether or not such events occur in vivo remains to be determined.     

Fatty acyl-CoA binding activity of the nuclear thyroid hormone receptor

Long-chain fatty acids and their acyl-CoA esters are potent inhibitors of nuclear thyroid hormone (T₃) receptor in vitro. In the present study, we obtained evidence for acyl-CoA binding activity in the nuclear extract from rat liver. The activity sedimented at a position (3.5 S) identical with that of the T₃ receptor, and the two activities sedimented together. Similarly, they coeluted on DEAE-Sephadex. After partial purification of the receptor, it was again inhibited strongly by acyl-CoAs. Heat stability and a partial trypsin digestion of the receptor both suggested that the action site of oleoyl-CoA overlapped the T₃-binding domain of the receptor. In addition, thyroid hormone receptor β1, synthesized in vitro, bound oleoyl-CoA specifically and its T₃-binding activity was inhibited. The dissociation constant for oleoyl-CoA binding to the partially purified receptor was 1.2 × 10^?7 M. This value as well as its molecular size distinguished the nuclear binding sites from the cytoplasmic fatty acid/acyl-CoA binding proteins. Oleoyl-CoA had no effect on the glucocorticoid receptor, another member of the nuclear hormone-receptor superfamily. From these results, we propose that thyroid hormone receptor is a specific acyl-CoA binding protein of the cell nucleus.     

Inhibitory effect of fatty acids on the binding of androgen receptor and R1881

Unsaturated long chain fatty acids are known to inhibit the binding between estrogen and estrogen receptor, or progesterone and progesterone receptor in rat uterus. The effects of long chain fatty acids on the binding between androgen receptor of castrated rat prostate and 3H-R1881 were studied. The binding was not affected by saturated fatty acids such as palmitic acid (16:0) or stearic acid (18:0). But unsaturated fatty acids such as oleic acid (18:1), arachidonic acid (20:4) and docosahexaenoic acid (22:6) inhibited the binding between androgen receptor and 3H-R1881. The inhibitory effect of arachidonic acid was dose dependent. Scatchard analysis showed that the addition of arachidonic acid markedly decrease the number of binding sites of androgen receptor. But the dissociation constant was not affected. The inhibitory effect of arachidonic was not a competitive one.     

Unsaturated fatty acids as endogenous inhibitors of tamoxifen binding to anti-oestrogen-binding sites.

It is known that triphenylethylene anti-oestrogens such as tamoxifen bind to specific high-affinity anti-oestrogen-binding sites, which are distinct from oestrogen receptors. These binding sites are widely distributed in human and animal tissues, but their function and endogenous ligands are unknown. By using [3H]tamoxifen and a rat liver microsomal fraction, a radio-ligand-binding assay was developed in an attempt to identify endogenous ligands for the anti-oestrogen-binding sites in the rat. An ether extract of rat serum inhibited [3H]tamoxifen binding to rat liver binding sites in a dose-dependent manner. Identification of the active serum constituents that inhibited [3H]tamoxifen binding was achieved by g.l.c.-mass spectrometry after preliminary purification of a rat serum extract by silica-gel t.l.c. Three unsaturated fatty acids (oleic, linoleic and arachidonic) accounted for about 50% of the total inhibiting activity of the serum extract. The concentrations of these fatty acids required to inhibit [3H]tamoxifen binding were in the range of 10-100 microM, comparable with those found in the rat circulation under physiological conditions. Saturated fatty acids present in rat serum (palmitic and stearic) did not inhibit [3H]tamoxifen binding. A survey of other fatty acids revealed that, in general, unsaturated fatty acids were far more potent than saturated fatty acids in inhibiting [3H]tamoxifen binding. These studies demonstrate that unsaturated fatty acids are quantitatively the most important circulating inhibitors of [3H]tamoxifen binding to the anti-oestrogen-binding sites. The biological significance of their interaction with these sites, however, remains to be clarified.     

Polyunsaturated fatty acids inhibit mouse hepatic glucocorticoid receptor activation in vitro

The effect of saturated fatty acids (SFAs) stearic and palmitic acids and polyunsaturated fatty acids (PUFAs) oleic, linoleic and arachidonic acids was studied on in vitro heat activation of mouse hepatic glucocorticoid receptor (GR) complex, as assessed by binding to DNA-cellulose and purified nuclei. Significant dose-dependent inhibition of heat activation of hormone-receptor complex by the PUFAs was observed. Linoleic and arachidonic acids were found to be more potent (caused approximately 70% inhibition maximally at 160 microM) inhibitors of GR heat activation, compared to oleic acid (approximately 38% inhibition at 40 microM). However, stearic and palmitic acids were unable to modulate GR heat activation, suggesting that the unsaturated moieties in PUFAs are possibly the important determinants of receptor activation. Thus, our study shows an inhibitory effect of PUFAs on in vitro hepatic GR activation.     

Glutathione-protein mixed disulfide decreases the affinity of rat liver fatty acid-binding protein for unsaturated fatty acid

.16 +/- 0.062% of the fatty acid-binding protein purified from 50 mM N-ethylmaleimide-treated rat liver (L-FABP) was determined as a form S-thiolated by glutathione (L-FABP-SSG). L-FABP-SSG, which was prepared in vitro through thiol-disulfide exchange reaction, showed more acidic pI (approximately 5.0) than the pI (approximately 7.0) of reduced L-FABP. S-thiolation of L-FABP by glutathione decreased the affinity of the protein for unsaturated fatty acids without changing the equimolar maximum binding. The changes in Kd were from 0.63 +/- 0.054 microM to 1.03 +/- 0.14 microM for oleic acid, from 0.63 +/- 0.028 microM to 0.97 +/- 0.12 microM for linoleic acid and from 0.85 +/- 0.050 microM to 1.45 +/- 0.024 microM for arachidonic acid. This modification did not alter the affinity nor the maximum binding for saturated fatty acids, which were determined to be Kd of approximately 1.0 microM for palmitic acid and approximately 0.9 microM for stearic acids, and equimolar maximum binding for both fatty acids. The binding affinity of L-FABP for unsaturated fatty acid may be regulated by redox state of the liver.     

Cis-unsaturated fatty acids modulate the function of the cell-surface cAMP receptor of Dictyostelium discoideum

The binding of cAMP to the chemotactic cAMP receptor in intact Dictyostelium discoideum cells and isolated membranes is strongly inhibited by unsaturated fatty acids. In isolated membranes, cis-unsaturated fatty acids decreased the number of accessible cAMP binding sites, without significantly altering their affinity. Most potent were C₁₈ and C₂₀ cis-poly unsaturated fatty acids, like arachidonic acid, linoleic acid and linolenic acid. Trans-unsaturated fatty acid was less potent than its cis isomer, while saturated fatty acids did not affect the binding of cAMP to receptors at all. Oxidation reactions were not important for the effect of unsaturated fatty acids. When membranes were preincubated with millimolar concentrations of Ca²⁺, the effect of unsaturated fatty acids was strongly diminished. Mg²⁺ was ineffective. Ca²⁺, if presented after the incubation of membranes with unsaturated fatty acids, did not reverse the inhibitory effect. The specificity of the fatty acid effect, and the interference with Ca²⁺, but not Mg²⁺, suggest that the properties of the cAMP receptor are changed as a result of alterations in the lipid bilayer structure of the membrane.     

Altered Microviscosity at Brain Membrane Surface Induces Distinct and Reversible Inhibition of Opioid Receptor Binding

In synaptosomal membranes from rat and monkey brain cortex, the addition of petroselenic (18:1, cis-delta 6) acid, oleic (18:1, cis-delta 9) acid, and vaccenic (18:1, cis-delta 11) acid or their corresponding methyl esters at 0.5 mumol/mg of membrane protein caused a similar 7-10% decrease in the microviscosity of the membrane core, whereas at the membrane surface the microviscosity was reduced 5-7% by the fatty acids but only 1% by their methyl esters. Concomitantly, the fatty acids, but not the methyl esters, inhibited the specific binding of the tritiated mu-, delta-, and kappa-opioids Tyr-D-Ala-Gly-(Me)Phe-Gly-ol (DAMGO), [D-Pen2,D-Pen5]enkephalin (DPDPE), and U69,593, respectively. As shown with oleic acid, the sensitivity of opioid receptor binding toward inhibition by fatty acids was in the order delta greater than mu much greater than kappa, whereby the binding of [3H]DPDPE was abolished, but significant inhibition of [3H]U69,593 binding, determined in membranes from monkey brain, required membrane modification with a twofold higher fatty acid concentration. Except for the unchanged KD of [3H]U69,593, the inhibition by oleic acid involved both the Bmax and affinity of opioid binding. Cholesteryl hemisuccinate (0.5-3 mumol/mg of protein), added to membranes previously modified by fatty acids, reversed the fluidization caused by the latter compounds and restored inhibited mu-, delta-, and kappa-opioid binding toward control values. In particular, the Bmax of [3H]-DPDPE binding completely recovered after being undetectable.(ABSTRACT TRUNCATED AT 250 WORDS)     

Action of long-chain fatty acids in vitro on Ca2+-stimulatable, Mg2+-dependent ATPase activity in human red cell membranes.

Human red cell membrane Ca2+-stimulatable, Mg2+-dependent adenosine triphosphatase (Ca2+-ATPase) activity and its response to thyroid hormone have been studied following exposure of membranes in vitro to specific long-chain fatty acids. Basal enzyme activity (no added thyroid hormone) was significantly decreased by additions of 10(-9)-10(-4) M-stearic (18:0) and oleic (18:1 cis-9) acids. Methyl oleate and elaidic (18:1 trans-9), palmitic (16:0) and lauric (12:0) acids at 10(-6) and 10(-4) M were not inhibitory, nor were arachidonic (20:4) and linolenic (18:3) acids. Myristic acid (14:0) was inhibitory only at 10(-4) M. Thus, chain length of 18 carbon atoms and anionic charge were the principal determinants of inhibitory activity. Introduction of a cis-9 double bond (oleic acid) did not alter the inhibitory activity of the 18-carbon moiety (stearic acid), but the trans-9 elaidic acid did not cause enzyme inhibition. While the predominant effect of fatty acids on erythrocyte Ca2+-ATPase in situ is inhibition of basal activity, elaidic, linoleic (18:2) and palmitoleic (16:1) acids at 10(-6) and 10(-4) M stimulated the enzyme. Methyl elaidate was not stimulatory. These structure-activity relationships differ from those described for fatty acids and purified red cell Ca2+-ATPase reconstituted in liposomes. Thyroid hormone stimulation of Ca2+-ATPase was significantly decreased by stearic and oleic acids (10(-9)-10(-4) M), but also by elaidic, linoleic, palmitoleic and myristic acids. Arachidonic, palmitic and lauric acids were ineffective, as were the methyl esters of oleic and elaidic acids. Thus, inhibition of the iodothyronine effect on Ca2+-ATPase by fatty acids has similar, but not identical, structure-activity relationships to those for basal enzyme activity. To examine mechanisms for these fatty acid effects, we studied the action of oleic and stearic acids on responsiveness of the enzyme to purified calmodulin, the Ca2+-binding activator protein for Ca2+-ATPase. Oleic and stearic acids (10(-9)-10(-4) M) progressively inhibited, but did not abolish, enzyme stimulation by calmodulin (10(-9) M). Double-reciprocal analysis of the effect of oleic acid on calmodulin stimulation indicated noncompetitive inhibition. Addition of calmodulin to membranes in the presence of equimolar oleic acid restored basal enzyme activity. Oleic acid also reduced 125I-calmodulin binding to membranes, but had no effect on the binding of [125I]T4 by ghosts. The mechanism of the decrease by long chain fatty acids of Ca2+-ATPase activity in situ in human red cell ghosts thus is calmodulin-dependent and involves reduction in membrane binding of calmodulin.     

Ligand-dependent interaction of hepatic fatty acid-binding protein with the nucleus

Our studies were conducted to explore the role of hepatic fatty acid-binding protein (L-FABP) in fatty acid transport to the nucleus. Purified rat L-FABP facilitated the specific interaction of [(3)H]oleic acid with the nuclei. L-FABP complexed with unlabeled oleic acid decreased the nuclear association of [(3)H]oleic acid:L-FABP; however, oleic acid-saturated bovine serum albumin (BSA) or fatty acid-free L-FABP did not. The peroxisome-proliferating agents LY171883, bezafibrate, and WY-14,643 were also effective competitors when complexed to L-FABP. Nuclease treatment did not affect the nuclear association of [(3)H]oleic acid:L-FABP; however, proteinase treatment of the nuclei abolished the binding. Nuclei incubated with fluorescein-conjugated L-FABP in the presence of oleic acid were highly fluorescent whereas no fluorescence was observed in reactions lacking oleic acid, suggesting that L-FABP itself was binding to the nuclei. The nuclear binding of FABP was concentration dependent, saturable, and competitive. LY189585, a ligand for L-FABP, also facilitated the nuclear binding of fluorescein-conjugated L-FABP, although it was less potent than oleic acid. A structural analog that does not bind L-FABP, LY163443, was relatively inactive in stimulating the nuclear binding. Potential interactions between L-FABP and nuclear proteins were analyzed by Far-Western blotting and identified a 33-kDa protein in the 500 mm NaCl extract of rat hepatocyte nuclei that bound strongly to biotinylated L-FABP. Oleic acid enhanced the interaction of L-FABP with the 33-kDa protein as well as other nuclear proteins.We propose that L-FABP is involved in communicating the state of fatty acid metabolism from the cytosol to the nucleus through an interaction with lipid mediators that are involved in nuclear signal transduction.     

Platelet desensitization by arachidonic acid is associated with the suppression of endoperoxide/thromboxane A2 binding to the membrane receptor

The inhibitory mechanism of high levels of exogenously added arachidonic acid on activation of washed human platelets was investigated. While low levels of arachidonic acid (5-10 microM) induced aggregation, ATP secretion and increase in cytoplasmic free Ca2+ concentration (first phase of activation), these platelet responses did not occur significantly at high concentrations (30-50 microM). However, much higher concentrations than 80 microM again elicited these responses (second phase). The first phase of platelet activation was inhibited by cyclooxygenase inhibitor, indomethacin, whereas the second one was independent of such treatment. Thromboxane B2 was produced dose-dependently until reaching a plateau at arachidonic acid concentrations higher than 20 microM, irrespective of the lack of aggregation and secretion at high concentrations. After that the amount of free arachidonic acid which remained unmetabolized in platelets gradually increased. High concentrations of arachidonic acid as well as other polyunsaturated fatty acids caused desensitization of platelets in response to U46619, and also depressed the specific [3H]U46619-binding to the receptor as well as other polyunsaturated fatty acids. The amount free arachidonic acid needed in platelets to suppress [3H]U46619 binding corresponded to that needed to inhibit platelet aggregation. Furthermore, arachidonic acid dose-dependently induced fluidization of lipid phase of platelet membranes as detected by 1,6-diphenyl-1,3,5-hexatriene. These results suggest that the inhibition of platelet response by high levels of arachidonic acid can be attributed to interference with endoperoxide/thromboxane A2 binding to the receptor, probably due to perturbation of the membrane lipid phase due to excess amounts of free arachidonic acid remaining in the membranes.     

Eicosapentaenoic acid reduces hepatic synthesis and secretion of triacylglycerol by decreasing the activity of acyl-coenzyme A:1,2-diacylglycerol acyltransferase

The mechanism for the reduced hepatic production of triacylglycerol in the presence of eicosapentaenoic acid was explored in short-term experiments using cultured parenchymal cells and microsomes from rat liver. Oleic, palmitic, stearic, and linoleic acids were the most potent stimulators of triacyl[3H]glycerol synthesis and secretion by hepatocytes, whereas erucic, alpha-linolenic, gamma-linolenic, arachidonic, docosahexaenoic, and eicosapentaenoic acids (in decreasing order) were less stimulatory. There was a linear correlation (r = 0.85, P less than 0.01) between synthesis and secretion of triacyl[3H]glycerol for the fatty acids examined. The extreme and opposite effects of eicosapentaenoic and oleic acids on triacylglycerol metabolism were studied in more detail. With increasing number of free fatty acid molecules bound per molecule of albumin, the rate of synthesis and secretion of triacyl[3H]glycerol increased, most markedly for oleic acid. Cellular uptake of the two fatty acids was similar, but more free eicosapentaenoic acid accumulated intracellularly. Eicosapentaenoic acid caused higher incorporation of [3H]water into phospholipid and lower incorporation into triacylglycerol and cholesteryl ester as compared to oleic acid. No difference was observed between the fatty acids on incorporation into cellular free fatty acids, monoacylglycerol and diacylglycerol. The amount of some 16- and 18-carbon fatty acids in triacylglycerol was significantly higher in the presence of oleic acid compared with eicosapentaenoic acid. Rat liver microsomes in the presence of added 1,2-dioleoyl-glycerol incorporated eicosapentaenoic acid and eicosapentaenoyl-CoA into triacylglycerol to a lesser extent than oleic acid and its CoA derivative. Decreased formation of triacylglycerol was also observed when eicosapentaenoyl-CoA was given together with oleoyl-CoA, whereas palmitoyl-CoA, stearoyl-CoA, linoleoyl-CoA, linolenoyl-CoA, and arachi-donoyl-CoA had no inhibitory effect. In conclusion, inhibition of acyl-CoA:1,2-diacylglycerol O-acyltransferase (EC 2.3.1.20) by eicosapentaenoic acid may be important for reduced synthesis and secretion of triacylglycerol from the liver.     

Modulation of thyroid hormone nuclear receptors by short-chain fatty acids in glial C6 cells. Role of histone acetylation

We have studied the effect of butyrate and other short-chain fatty acids on thyroid hormone nuclear receptors in C6 cells, a rat glioma cell line. Exposure of C6 cells to butyrate leads to increased levels of L-triiodothyronine (T3) in the nuclear and extranuclear compartments. The rise in nuclear binding is not merely a reflection of the higher cellular hormone content, and Scatchard analysis of T3 binding to isolated nuclei reveals that butyrate increases receptor number without changing affinity. The effect on the receptor is quantitatively important: a 48-h incubation with 2 mM butyrate increases nuclear binding by 2-3-fold, and 5 mM butyrate by 3-5-fold. Other short-chain fatty acids were found to similarly influence both nuclear receptor and extranuclear T3 levels with the following potency: butyrate greater than valerate greater than propionate greater than acetate. On the contrary, ketone bodies were ineffective. Butyrate increases receptor levels by decreasing receptor degradation, since the apparent t1/2 of receptor disappearance increased by approximately 3-fold in cells incubated with 2 mM butyrate for 48 h. The regulation of receptor number might be secondary to an action of butyrate on regions of the chromatin to which the receptor associates. We then examined the effect of butyrate on histone acetylation. The fatty acid had little effect in increasing the level of multiacetylated forms of H3 and H4 histone when studied in acid-urea gels, but it markedly inhibited the turnover of [3H] acetate from the histone fraction. There was a striking similarity in the dose-response of butyrate for increasing receptor levels and inhibiting histone deacetylation. Furthermore, a very close correlation between receptor levels and [3H]acetate release was also found when different short-chain fatty acids were used. We thus conclude that the effect of butyrate on the receptor could be explained by a modification of the chromatin structure of C6 cells secondary to acetylation.     

Interactions of the nuclear thyroid hormone receptor with core histones

These studies concern the interactions of the rat liver thyroid hormone nuclear receptor with histones and factors influencing the receptor's assay and stability. Heating certain crude receptor preparations at 50 degrees C produces a selective loss of triiodothyronine (T3) but not thyroxine (T4) binding activity, whereas, with more purified preparations, such heating decreases both T3 and T4 binding. The selective T3-binding loss in crude preparations was found to be due to the simultaneous denaturation of the receptor's high-affinity hormone-binding activity for both T3 and T4 and generation of new low-affinity T4-binding sites. The fraction in which T4 binding can be activated could be separated from the receptors by Sephadex G-100 chromatography. Core histones stimulated both T3- and T4-binding activity of 6-fold-purified receptor preparations, and data from several different experimental approaches suggest that this stimulation is due to the capability of the core histones to prevent the receptor from binding to or being denatured by Sephadex G-25 assay columns. The core histones were also found to stabilize 500-fold-purified but not 6-fold-purified or crude receptor preparations. A number of other acidic or basic proteins had little or none of these stimulatory effects, whereas a few proteins (such as the insulin B chain and histone H1) did have activity, although it was less than that of the core histones. There were no significant differences between the purified core histone subfractions (H2A, H2B, H3, and H4). That core histones can interact with the thyroid hormone receptors was demonstrated more directly by the finding that the receptors bind to histone-Sepharose but not Sepharose or insulin- or ovalbumin-Sepharose columns and that this binding was blocked by core histones at concentrations suggestive of an affinity for the receptor-core histone interaction of around 3 microM at 0.15 M salt concentration. The results demonstrate the utility of the histones in the assay and stabilization of purified thyroid hormone receptors, but they fail to support our previous hypothesis of a receptor subunit where T3- but not T4-binding activity is regulated selectively by histones. However, the results indicate that histones may interact with the receptors with some degree of specificity, and they raise the possibility that the histones participate in the nuclear localization of the receptors.     

Modulation of Opioid Receptor Binding by Cis and Trans Fatty Acids

In synaptosomal brain membranes, the addition of oleic acid (cis), elaidic acid (trans), and the cis and trans isomers of vaccenic acid, at a concentration of 0.87 mumol of lipid/mg of protein, strongly reduced the Bmax and, to a lesser degree, the binding affinity of the mu-selective opioid [3H]Tyr-D-Ala-Gly-(Me)Phe-Gly-ol ([3H]DAMGO). At comparable membrane content, the cis isomers of the fatty acids were more potent than their trans counterparts in inhibiting ligand binding and in decreasing membrane microviscosity, both at the membrane surface and in the core. However, trans-vacenic acid affected opioid receptor binding in spite of just marginally altering membrane microviscosity. If the receptors were uncoupled from guanine nucleotide regulatory protein, an altered inhibition profile was obtained: the impairment of KD by the fatty acids was enhanced and that of Bmax reduced. Receptor interaction of the delta-opioid [3H](D-Pen2,D-Pen5)enkephalin was modulated by lipids to a greater extent than that of [3H]DAMGO: saturable binding was abolished by both oleic and elaidic acids. The binding of [3H]naltrexone was less susceptible to inhibition by the fatty acids, particularly in the presence of sodium. In the absence of this cation, however, cis-vaccenic acid abolished the low-affinity binding component of [3H]naltrexone. These findings support the membrane model of opioid receptor sequestration depicting different ionic environments for the mu- and delta-binding sites. The results of this work show distinct modulation of different types and molecular states of opioid receptor by fatty acids through mechanisms involving membrane fluidity and specific interactions with membrane constituents.     

Interaction of fatty acids with recombinant rat intestinal and liver fatty acid-binding proteins

Intestinal enterocytes contain two homologous fatty acid-binding proteins, intestinal fatty acid-binding protein (I-FABP)2 and liver fatty acid-binding protein (L-FABP). Since the functional basis for this multiplicity is not known, the fatty acid-binding specificity of recombinant forms of both rat I-FABP and rat L-FABP was examined. A systematic comparative analysis of the 18 carbon chain length fatty acid binding parameters, using both radiolabeled (stearic, oleic, and linoleic) and fluorescent (trans-parinaric and cis-parinaric) fatty acids, was undertaken. Results obtained with a classical Lipidex-1000 binding assay, which requires separation of bound from free fatty acid, were confirmed with a fluorescent fatty acid-binding assay not requiring separation of bound and unbound ligand. Depending on the nature of the fatty acid ligand, I-FABP bound fatty acid had dissociation constants between 0.2 and 3.1 microM and a consistent 1:1 molar ratio. The dissociation constants for L-FABP bound fatty acids ranged between 0.9 and 2.6 microM and the protein bound up to 2 mol fatty acid per mole of protein. Both fatty acid-binding proteins exhibited relatively higher affinity for unsaturated fatty acids as compared to saturated fatty acids of the same chain length. cis-Parinaric acid or trans-parinaric acid (each containing four double bonds) bound to L-FABP and I-FABP were displaced in a competitive manner by non-fluorescent fatty acid. Hill plots of the binding of cis- and trans- parinaric acid to L-FABP showed that the binding affinities of the two sites were very similar and did not exhibit cooperativity. The lack of fluorescence self-quenching upon binding 2 mol of either trans- or cis-parinaric acid/mol L-FABP is consistent with the presence of two binding sites with dissimilar orientation in the L-FABP. Thus, the difference in binding capacity between I-FABP and L-FABP predicts a structurally different binding site or sites.     

Competitive inhibition of T3 binding to alpha 1 and beta 1 thyroid hormone receptors by fatty acids.

This study was undertaken to investigate whether fatty acids inhibit the binding of T3 to the alpha 1 and beta 1 form of the thyroid hormone receptor. Fatty acids inhibited the binding of T3 to both receptor proteins isolated from a bacterial expression system. The effectiveness of inhibition depends on the chain length and degree of saturation of the fatty acids. The inhibition of T3 binding to the alpha 1 and beta 1 receptor by oleic acid is competitive in nature; the Ki value was 5.4 10(-6) M for the c-erbA alpha 1 protein and 3.3 10(-6) M for the c-erb beta 1 protein. The findings indicate a direct interaction of fatty acids with T3 receptor proteins.     

Fatty acids and cholesterol: effect on the interaction of theophylline with bovine serum albumin

The binding of theophylline (Th, 11-840 microM) to bovine serum albumin (BSA, 10 microM) using microdialysis technique in the presence of fatty acids (2.5-50.0 microM) and cholesterol (20-500 nM) indicates that fatty acids and cholesterol inhibit the binding of Th to BSA. The maximum inhibition (90.5%) occurs in presence of acetic acid (AA) followed by lauric acid (LA, 83.3%), palmitic acid (PA, 72.2%), oleic acid (OA, 44.4%) and cholesterol (22.2%). Fatty acids and cholesterol also decrease the number of binding sites and the affinity for the binding of Th to BSA. Such a decrease is maximum in the presence of AA followed by LA, PA, OA and cholesterol. Complete abolition of the low affinity binding site in the presence of AA indicates that the low affinity binding is predominantly ionic in nature while the high affinity binding involves ionic and other type(s) of unidentified force(s). This makes high affinity binding stronger than low affinity binding.