Interaction of prostaglandins and fatty acids with native and acetaldehyde-alkylated proteins

Using intrinsic and probe fluorescence, microcalorimetry and isotopic methods, the interactions of prostaglandins (PG) E2 and F2 alpha and some fatty acids with native and alkylated proteins (human serum albumin (HSA) and rat liver plasma membrane PG receptors), were studied. The fatty acid and PG interactions with human serum albumin (HSA) resulted in effective quenching of fluorescence of the probe, 1.8-anilinonaphthalene sulfonate (ANS), bound to the protein. Fatty acids competed with ANS for the binding sites; the efficiency of this process increased with an increase in the number of double bonds in the fatty acid molecule. PG induced a weaker fluorescence quenching of HSA-bound ANS and stabilized the protein molecule in a lesser degree compared to fatty acids. The sites of PG E2 and F2 alpha binding did not overlap with the sites of fatty acid binding on the HSA molecule. Nonenzymatic alkylation of HSA by acetaldehyde resulted in the abnormalities of binding sites for fatty acids and PG. Modification of the plasma membrane proteins with acetaldehyde sharply diminished the density of PG E2 binding sites without changing the association constants. Alkylation did not interfere with the parameters of PG F2 alpha binding to liver membrane proteins.     

Competitive binding of fatty acids and the fluorescent probe 1-8-anilinonaphthalene sulfonate to bovine beta-lactoglobulin

The use of spectroscopy in the study of fatty acids binding to bovine beta-lactoglobulin (BLG) appears to be a difficult task, as these acid compounds, assumed as the protein natural ligands, do not exhibit favorable optical response such as, for example, absorption or fluorescence. Therefore, the BLG fatty-acid equilibrium has been tackled by exploiting the competition between fatty acids and ANS, a widely used fluorescent hydrophobic probe, whose binding sites on the protein have been characterized recently. Two lifetime decays of the ANS-BLG complex have been found; the longer one has been attributed to the internal binding site and the shorter one to the external site. At increasing fatty acids concentration, the fractional weight associated with ANS bound to the internal site drops, in agreement with a model describing the competition of the dye with fatty acids, whereas the external site occupancy appears to be unaffected by the fatty acids binding to BLG. This model is supported by docking studies. An estimate of the acid-binding affinities for BLG has been obtained by implementing the fitting of the bound ANS intensities with a competitive binding model. A relevant dependence has been found upon the solution pH, in the range from 6 to 8, which correlates with the calyx accessibility modulated by the conformation of the EF loop. Fatty acids with longer aliphatic chains (palmitate and laurate) are found to display larger affinities for the protein and the interaction free energy nicely correlates with the number of contacts inside the protein calyx, in agreement with docking simulations.     

Characterization of the sources of protein-ligand affinity: 1-sulfonato-8-(1'')anilinonaphthalene binding to intestinal fatty acid binding protein.

1-Sulfonato-8-(1')anilinonaphthalene (1,8-ANS) was employed as a fluorescent probe of the fatty acid binding site of recombinant rat intestinal fatty acid binding protein (1-FABP). The enhancement of fluorescence upon binding allowed direct determination of binding affinity by fluorescence titration experiments, and measurement of the effects on that affinity of temperature, pH, and ionic strength. Solvent isotope effects were also determined. These data were compared to results from isothermal titration calorimetry. We obtained values for the enthalpy and entropy of this interaction at a variety of temperatures, and hence determined the change in heat capacity of the system consequent upon binding. The ANS-1-FABP is enthalpically driven; above approximately 14 degrees C it is entropically opposed, but below this temperature the entropy makes a positive contribution to the binding. The changes we observe in both enthalpy and entropy of binding with temperature can be derived from the change in heat capacity upon binding by integration, which demonstrates the internal consistency of our results. Bound ANS is displaced by fatty acids and can itself displace fatty acids bound to I-FABP. The binding site for ANS appears to be inside the solvent-containing cavity observed in the x-ray crystal structure, the same cavity occupied by fatty acid. From the fluorescence spectrum and from an inversion of the Debye-Hueckel formula for the activity coefficients as a function of added salt, we inferred that this cavity is fairly polar in character, which is in keeping with inferences drawn from the x-ray structure. The binding affinity of ANS is considered to be a consequence of both electrostatic and conditional hydrophobic effects. We speculate that the observed change in heat capacity is produced mainly by the displacement of strongly hydrogen-bonded waters from the protein cavity.     

Identification of fatty acid molecules in a Fasciola hepatica immunoprophylactic fatty acid-binding protein

Fasciola hepatica adult flukes have a native protein complex denoted nFh12 and consisting of fatty acid binding proteins that comprise at least 8 isoforms. It is a potent immunogen because in several animal hosts it induces an early antibody response to F. hepatica infection. It is also a potent cross-protective immunogen because it induces a protective immune response in mice to challenge infection with Schistosoma mansoni cercariae. The gene encoding this protein has been cloned and sequenced. It produces a polypeptide of 132 amino acids with a predicted molecular mass of 14.7 kDa and is denoted rFh15. It also has a significant homology to a 14-kDa S. mansoni fatty acid binding protein (Sm14). In the present study, nFh12 was delipidated with charcoal treatment and then studied by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Additionally, a lipid analysis of nFh12 was undertaken using gas chromatography-mass spectrometry to demonstrate that the nFh12 protein complex is, in fact, a complex of fatty acid binding proteins. Five long-chain saturated and unsaturated fatty acids were detected. The most abundant were palmitic acid (38%), stearic acid (24%), and oleic acid (13%). These fatty acid molecules do not have covalent bonds attached to the protein molecule. Because both nFh12 and Sm14 protect mice against challenge infection with F. hepatica and S. mansoni, it is possible that they have common protective epitopes in which fatty acids could be involved. Further studies are in progress to determine the chemical nature of these potential common epitopes.     

Metabolism of 15-hydroxyeicosatetraenoic acid by Caco-2 cells

Monolayers of Caco-2 cells, a human enterocyte cell line, were incubated with [1-14C]15-hydroxyeicosatetraenoic acid (15-HETE), a lipid mediator of inflammation, and [1-14C]arachidonic acid. Both fatty acids were taken up readily and metabolized by Caco-2 cells. [1-14C]Arachidonic acid was directly esterified in cellular phospholipids and, to a lesser extent, in triglycerides. When [1-14C]15-hydroxyeicosatetraenoic acid was incubated with Caco-2 cells, about 10% was directly esterified into cellular lipids but most (55%) was beta-oxidized to ketone bodies, CO2, and acetate, with very little accumulation of shorter carbon chain products of partial beta-oxidation. The radiolabeled acetate generated from beta-oxidation of [1-14C]15-hydroxyeicosatetraenoic acid was incorporated into the synthesis of new fatty acids, primarily [14C]palmitate, which in turn was esterified into cellular phospholipids, with lesser amounts in triglycerides. Caco-2 cells were also incubated with [5,6,8,9,11,12,14,15-3H]15-hydroxyeicosatetraenoic acid; most of the radiolabel was recovered either in ketone bodies or in [3H]palmitate esterified in phospholipids and triglycerides, demonstrating that most of the [3H]15-hydroxyeicosatetraenoic acid underwent several cycles of beta-oxidation. The binding of both 15-hydroxyeicosatetraenoic acid and arachidonic acid to hepatic fatty acid binding protein, the only fatty acid binding protein in Caco-2 cells, was measured. The Kd (6.0 microM) for 15-HETE was three-fold higher than that for arachidonate (2.1 microM).     

The two calcium-binding proteins, S100A8 and S100A9, are involved in the metabolism of arachidonic acid in human neutrophils.

Recently, we identified the two myeloid related protein-8 (MRP8) (S100A8) and MRP14 (S100A9) as fatty acid-binding proteins (Klempt, M., Melkonyan, H., Nacken, W., Wiesmann, D., Holtkemper, U., and Sorg, C. (1997) FEBS Lett. 408, 81-84). Here we present data that the S100A8/A9 protein complex represents the exclusive arachidonic acid-binding proteins in human neutrophils. Binding and competition studies revealed evidence that (i) fatty acid binding was dependent on the calcium concentration; (ii) fatty acid binding was specific for the protein complex formed by S100A8 and S100A9, whereas the individual components were unable to bind fatty acids; (iii) exclusively polyunsaturated fatty acids were bound by S100A8/A9, whereas saturated (palmitic acid, stearic acid) and monounsaturated fatty acids (oleic acid) as well as arachidonic acid-derived eicosanoids (15-hydroxyeicosatetraenoic acid, prostaglandin E(2), thromboxane B(2), leukotriene B(4)) were poor competitors. Stimulation of neutrophil-like HL-60 cells with phorbol 12-myristate 13-acetate led to the secretion of S100A8/A9 protein complex, which carried the released arachidonic acid. When elevation of intracellular calcium level was induced by A23187, release of arachidonic acid occurred without secretion of S100A8/A9. In view of the unusual abundance in neutrophilic cytosol (approximately 40% of cytosolic protein) our findings assign an important role for S100A8/A9 as mediator between calcium signaling and arachidonic acid effects. Further investigations have to explore the exact function of the S100A8/A9-arachidonic acid complex both inside and outside of neutrophils.     

Brugia malayi: microfilarial polyunsaturated fatty acid composition and synthesis

The polyunsaturated fatty acid composition of Brugia malayi microfilariae was analyzed by gas chromatography and compared to that of sera from B. malayi-infected jirds. The essential fatty acid, linoleic acid (18:2 omega 6), was the most abundant fatty acid present in both microfilarial total lipids and phospholipids as well as in jird sera. In contrast, arachidonic acid (20:4 omega 6), as well as the 18:3 omega 6, 20:2 omega 6, and 20:3 omega 6 intermediates that are formed in the enzymatic conversion of linoleic acid to arachidonic acid, were proportionally more abundant in microfilariae than in jird sera. To assess the capacity of microfilariae to transform linoleic acid into arachidonic acid, B. malayi microfilariae were incubated with [14C]linoleic acid. Microfilarial lipids were extracted and resolved by high-pressure liquid chromatography and thin-layer chromatography. A portion of [14C]linoleic acid incorporated by microfilariae was converted to [14C]arachidonic acid. Thus, microfilariae can not only incorporate exogenous arachidonic acid, as previously demonstrated, but can also synthesize arachidonic acid from exogenous linoleic acid. The capacity of microfilariae to utilize specific host polyunsaturated fatty acids raises the possibility that intravascular filarial parasites may synthesize eicosanoid metabolites of arachidonic acid that could mediate filarial-host cell interactions.     

Alpha-fetoprotein-mediated uptake of fatty acids by human T lymphocytes.

The binding to resting and activated T lymphocytes of two radiolabelled fatty acids (oleic and arachidonic) was studied in the presence or in the absence of alpha-fetoprotein (AFP) as carrier protein. Fatty acid binding by resting and activated T lymphocytes was determined at 4 degrees C as a function of the concentration of fatty acid and AFP. Under the conditions employed, the following observations were made: (1) in the presence of AFP, fatty acids (oleic and arachidonic acid) are bound to cells by a two-component pathway; one is a saturable process, evidenced when the fatty acid to AFP (FA/AFP) molar ratio was fixed at 1 and the concentration of the fatty acid and the protein varied from 0.1 to 3.2 microM, and the second is a nonsaturable function of FA/AFP molar ratio and was linearly related to the unbound fatty acid concentration in the medium over the entire range studied; (2) in the absence of AFP, the nonsaturable process appears to be the only component of fatty acid binding; 3) at all tested concentrations of free (unbound) fatty acid in the medium, net fatty acid binding by either resting or activated T cells was considerably greater in the presence than in the absence of AFP; (4) in the presence of AFP, fatty acid binding was much higher in activated T cells than in resting T cells, whereas in the absence of AFP, nonsignificant differences were observed between activated and resting T cells; and (5) the time course of fatty acid and AFP binding at 4 degrees C revealed that, at equilibrium, the number of fatty acid molecules bound to the cell was much greater than that of AFP suggesting an accelerated dissociation of the fatty acid upon interaction of the AFP-fatty acid complex with putative cell receptors. It is concluded to the existence of an AFP/AFP-receptor pathway that facilitates the binding of fatty acids to T lymphocytes, particularly upon their blast transformation. This pathway may fulfill the increased requirement for fatty acids characteristic of proliferating cells and may serve to regulate the endocytosis of fatty acids with modulatory effects on lymphocyte function and to protect cells from their cytotoxic potential when internalized in excess.     

beta-Oxidation of polyunsaturated fatty acids by rat liver peroxisomes. A role for 2,4-dienoyl-coenzyme A reductase in peroxisomal beta-oxidation

beta-Oxidation of unsaturated fatty acids was studied with isolated solubilized or nonsolubilized peroxisomes or with perfused liver isolated from rats treated with clofibrate. gamma-Linolenic acid gave the higher rate of beta-oxidation, while arachidonic acid gave the slower rate of beta-oxidation. Other polyunsaturated fatty acids (including docosahexaenoic acid) were oxidized at rates which were similar to, or higher than, that observed with oleic acid. Experiments with 1-14C-labeled polyunsaturated fatty acids demonstrated that these are chain-shortened when incubated with nonsolubilized peroxisomes. Spectrophotometric investigation of solubilized peroxisomal incubations showed that 2,4-dienoyl-CoA esters accumulated during peroxisomal beta-oxidation of fatty acids possessing double bond(s) at even-numbered carbon atoms. beta-Oxidation of [1-14C]docosahexaenoic acid by isolated peroxisomes was markedly stimulated by added NADPH or isocitrate. This fatty acid also failed to cause acyl-CoA-dependent NADH generation with conditions of assay which facilitate this using other acyl-CoA esters. These findings suggest that 2,4-dienoyl-CoA reductase participation is essential during peroxisomal beta-oxidation if chain shortening is to proceed beyond a delta 4 double bond. Evidence obtained using arachidionoyl-CoA, [1-14C]arachidonic acid, and [5,6,8,9,11,12,14,15-3H]arachidonic acid suggests that peroxisomal beta-oxidation also can proceed beyond a double bond positioned at an odd-numbered carbon atom. Experiments with isolated perfused livers showed that polyunsaturated fatty acids also in the intact liver are substrates for peroxisomal beta-oxidation, as judged by increased levels of the catalase-H2O2 complex on infusion of polyunsaturated fatty acids.     

Evidence for the involvement of the unique C-tail of S100A9 in the binding of arachidonic acid to the heterocomplex S100A8/A9

Protein complexes formed by S100A8 and S100A9 represent the only AA-binding capacity in the human neutrophilic cytosol and are involved in the intracellular arachidonic acid metabolism. The formation of S100A8/A9 protein complexes and the binding of calcium to the complexes are prerequisites for the specific binding of polyunsaturated fatty acids. The present study was undertaken to characterize the fatty acid binding site within the protein complex. Deletions at both termini and point mutations of different basic amino acids especially within the extended C-terminal tail of human S100A9 were introduced. The S100A9 mutant proteins were then analyzed with respect to protein-protein interaction (GST pull down-assay and yeast two-hybrid system) and functional properties (arachidonic acid and calcium binding). The data give strong evidence that the unique C-tail of S100A9 containing the three consecutive histidine residues (His103-His105) represents the region to which the fatty acid carboxy-group is bound to the protein complex. The localization of the AA-binding site within the unique C-tail of S100A9 correlates with the fact that fatty acid binding has not yet been reported for other S100 proteins.     

Relations between vitamin D and fatty acid binding properties of vitamin D-binding protein

The interaction of fatty acids with bovine vitamin D-binding protein (DBP) was studied using a partition equilibrium method. This protein has one high affinity site for binding of fatty acids with an association constant Ka = 7 x 10(5) M-1 for palmitic acid and Ka = 6 x 10(5) M-1 for arachidonic acid. Competition experiments showed that palmitic acid hardly competes with 25-hydroxycholecalciferol for binding to DBP. However, arachidonic acid showed comparatively a stronger competition for binding to this protein. The great difference in competition of palmitic and arachidonic acids with 25-hydroxycholecalciferol may be related to changes in DBP conformation promoted by the binding of different ligands.     

Ligand specificity and conformational stability of human fatty acid-binding proteins.

Fatty acid binding proteins (FABPs) are small cytosolic proteins with virtually identical backbone structures that facilitate the solubility and intracellular transport of fatty acids. At least eight different types of FABP occur, each with a specific tissue distribution and possibly with a distinct function. To define the functional characteristics of all eight human FABPs, viz. heart (H), brain (B), myelin (M), adipocyte (A), epidermal (E), intestinal (I), liver (L) and ileal lipid-binding protein (I-LBP), we studied their ligand specificity, their conformational stability and their immunological crossreactivity. Additionally, binding of bile acids to I-LBP was studied. The FABP types showed differences in fatty acid binding affinity. Generally, the affinity for palmitic acid was lower than for oleic and arachidonic acid. All FABP types, except E-FABP, I-FABP and I-LBP interacted with 1-anilinonaphtalene-8-sulphonic acid (ANS). Only L-FABP, I-FABP and M-FABP showed binding of 11-((5-dimethylaminonaphtalene-1-sulfonyl)amino)undecanoic acid (DAUDA). I-LBP showed increasing binding of bile acids in the order taurine-conjugated>glycine-conjugated>unconjugated bile acids. A hydroxylgroup of bile acids at position 7 decreased and at position 12 increased the binding affinity to I-LBP. The fatty acid-binding affinity and the conformation of FABP types were differentially affected in the presence of urea. Our results demonstrate significant differences in ligand binding, conformational stability and surface properties between different FABP types which may point to a specific function in certain cells and tissues. The preference of I-LBP (but not L-FABP) for conjugated bile acids is in accordance with a specific role in bile acid reabsorption in the ileum.     

Isolation and characterization of the three fractions (DE-I, DE-II and DE-III) of rat-liver Z-protein and the complete primary structure of DE-II

Three fractions (DE-I, DE-II and DE-III) of Z-protein (fatty acid binding protein) have been isolated from rat liver cytosol by DEAE-cellulose chromatography and characterized. They had the same molecular weight of 14000 and essentially identical amino acid composition. However, compositions of endogenous fatty acids were found to differ strikingly from one another. Long-chain fatty acids detected in DE-II were palmitic, stearic, oleic, linoleic and arachidonic acids. In contrast to DE-II, DE-III contained mainly arachidonic acid. Molar ratios of endogenous long-chain fatty acids to both DE-II and DE-III were estimated to be around 1.0. Unlike the latter two fractions, DE-I was virtually lipid-free. Analyses of the three fractions by polyacrylamide gel electrophoresis, electrofocusing and DEAE-cellulose chromatography before and after delipidation suggested that the difference between DE-I and DE-II was in part due to fatty acids bound to DE-II. In contrast, DE-III appeared to be somewhat different from these forms in its protein structure, though tryptic peptide mappings of the three fractions did not reveal clear differences among them. Analysis of the primary structure was made on the most abundant fraction, DE-II, to investigate the relationship among the three fractions and to other proteins. The protein was a single chain consisting of 127 amino acid residues and had a mostly acetylated NH2 terminus and a free sulfhydryl group. The complete sequence of Z-protein showed striking homology to cellular retinoid binding proteins and peripheral nerve myelin P2 protein, which indicated the presence of a new family of cellular lipid-binding proteins diverged from a common ancestor. A possible intragenic duplication of Z-protein was also suggested.     

Ligand binding site of tear lipocalin: contribution of a trigonal cluster of charged residues probed by 8-anilino-1-naphthalenesulfonic acid

Human tear lipocalin (TL) exhibits diverse functions, most of which are linked to ligand binding. To map the binding site of TL for some amphiphilic ligands, we capitalized on the hydrophobic and hydrophilic properties of 8-anilino-1-naphthalenesulfonic acid (ANS). In single Trp mutants, resonance energy transfer from Trp to ANS indicates that the naphthalene group of ANS is proximate to Leu105 in the cavity. Binding energies of TL to ANS and its analogues reveal contributions from electrostatic interactions. The sulfonate group of ANS interacts strongly with the nonconserved intracavitary residue Lys114 and less with neighboring residues His84 and Glu34. This trigonal cluster of residues may play a role in the ligand recognition site for some negatively charged ligands. Because many drugs possess sulfonate groups, the trigonal cluster-sulfonate interaction can also be exploited as a lipocalin-based drug delivery mechanism. The binding of lauric acid and its analogues shows that fatty acids assume heterogeneous orientations in the cavity of TL. Predominantly, the hydrocarbon tail is buried in the cavity of TL and the carboxyl group is oriented toward the mouth. However, TL can also interact, albeit relatively weakly, with fatty acids oriented in the opposite direction. As the major lipid binding protein of tears, the ability to accommodate fatty acids in two opposing orientations may have functional implications for TL. At the aqueous-lipid interface, fatty acids whose carboxyl groups are positioned toward the aqueous phase are available for interaction with TL that could augment stability of the tear film.     

Specific formation of arachidonic acid and eicosapentaenoic acid by a front-end Delta5-desaturase from Phytophthora megasperma

The biosynthesis of arachidonic acid (20:4(Delta5Z,8Z,11Z,14Z)) from linoleic acid in plants by transgenic means requires the sequential and specific action of two desaturation reactions and one elongation reaction. Here, we describe the isolation of a specific acyl-lipid-desaturase catalyzing the formation of the double bond at position 5 from a cDNA library from Phytophthora megasperma. The isolated full-length cDNA harbors a sequence of 1740 bp encoding a protein of 477 amino acids with a calculated molecular weight of 53.5 kDa. The desaturase sequence contained a predicted N-terminal cytochrome b(5)-like domain, as well as three histidine-rich domains. For functional identification, the cDNA was expressed in Saccharomyces cerevisiae, and the formation of newly formed fatty acids was analyzed. The expression of the heterologous enzyme resulted in the formation of arachidonic acid after di-homo-gamma-linolenic acid supplementation and in the formation of eicosapentaenoic acid synthesis from omega3-arachidonic acid. Results presented here on the substrate specificity identify this expressed protein as a classical Delta5-acyl-lipid-desaturase, capable of specifically introducing a double bond at the Delta5 position solely in 20-carbon-atom chain length fatty acids containing a double bond at position Delta8. Detailed analysis of the different lipid species showed a preferential occurrence of the desaturation reaction for fatty acids esterified to phosphatidylcholine.     

Structure-based ligand binding sites of protein p14.5, a member of protein family YER057c/YIL051c/YjgF

Seventeen mutants with one, two or three amino acids substitutions of human protein p14.5, homologue to well-known tumor antigen from goat liver UK114 and a member of proteins YER057c/YIL051c/YjgF family, have been used for structure-functional relation studies and ligand binding analysis using cross-linking by triacryloyl-hexahydro-s-triazine (TAT), size exclusion chromatography, free fatty acid and 8-anilino-1-naphthalenesulfonic acid (ANS) binding assays. Amino acids having the most significant impact on the ligand binding activity have been determined: R107, N93, Y21 and F89. Arginine 107 has been identified as the most accessible amino acid in the cleft. Trimeric structure of protein p14.5 has been confirmed as being essential for stoichiometric small ligand binding activity and oligomeric structure of p14. Ligand binding activity may be related with the biological functions of these proteins, which still are not understood well.     

Arachidonic acid as a second messenger. Interactions with a GTP-binding protein of human neutrophils.

Arachidonic acid (20:4) and other cis-unsaturated fatty acids exert direct effects on a variety of cells, effects that do not depend on the metabolism of fatty acids via cyclooxygenase or lipoxygenase pathways. In these studies arachidonic acid and other cis-unsaturated fatty acids (but not trans-unsaturated or saturated fatty acids) increased the specific binding of the nonhydrolyzable analog of GTP, [35S]GTP gamma S, to purified neutrophil membrane preparations and elicited superoxide anion generation from intact neutrophils. There was a positive correlation (r = 0.70) between the capacity of fatty acids to increase nucleotide binding and to elicit the respiratory burst. Scatchard plot analysis of binding at equilibrium demonstrated an increase in the number of available GTP binding sites in the presence of 50 microM arachidonic acid. Nonsteroidal antiinflammatory agents interfered with the arachidonic acid effect on [35S]GTP gamma S binding. ADP-ribosylation of the pertussis toxin substrate Gi alpha within the plasmalemma-reduced specific [35S]GTP gamma S binding and blocked arachidonate-dependent enhancement of binding. Moreover, pertussis toxin treatment of intact neutrophils inhibited arachidonic acid-induced superoxide anion generation. The data indicate that arachidonic acid directly activates a GTP binding protein in the neutrophil plasma membrane and may thereby act as a second messenger in signal transduction.     

Fatty acid binding proteins from heart

Heart contains a fatty acid binding protein (FABP) concentration comparable to liver, when it is determined with a fatty acid-binding assay. The low concentration detected with anti-liver FABP antibodies is related to the different chemical forms and physiochemical properties of liver and heart FABP. The ratio of fatty acid bound per purified protein molecule is one or lower. Rat heart mitochondria oxidize FABP-bound fatty acids. The FABP content of rat heart is dependent on sex and diurnal cycle, but is not influenced by starvation or clofibrate feeding. It is also not different in the newborn rat. FABP was obtained from human heart in a yield of 11%. It shows similar binding characteristics to palmitic, oleic and arachidonic acid. The functional significance of the specific heart FABP is discussed in relation to myocardial fatty acid metabolism in normal and pathological conditions.     

Purification and characterization of a protein capable of binding to fatty acids and bile salts in Giardia lamblia

A specific fatty acid binding protein was isolated from Giardia lamblia, using an affinity column with butyric acid acting as a ligand in place of stearic acid. This method has proved to be more efficient than the one previously described using stearic acid as ligand. The purified fraction showed 8 electrophoretic bands of proteins, with molecular weights ranging between 8 and 80 kDa. This pattern is a consequence of the aggregation of a protein with a molecular weight of 8,215 Da, corresponding to the lower molecular weight band, the only one capable of binding to fatty acids. The labeled oleic acid bound to these purified proteins was replaced by a 100-fold greater concentration of taurocholate, glycocholate, deoxycholate, palmitic acid, and arachidonic acid, having a greater displacement of the bile salts than the free fatty acids.