Rat Liver Fatty Acid-binding Protein Research Articles

Mapping Hydration Dynamics around a β-Barrel Protein.

Protein surface hydration is fundamental to its structure, flexibility, dynamics, and function, but it has been challenging to disentangle their ultimate relationships. Here, we report our systematic characterization of hydration dynamics around a β-barrel protein, rat liver fatty acid-binding protein (rLFABP), to reveal the effect of different protein secondary structures on hydration water. We employed a tryptophan scan to the protein surface one at a time and examined a total of 17 different sites. We observed three types of hydration water relaxation with distinct time scales, from hundreds of femtoseconds to a hundred picoseconds. We also examined the anisotropy dynamics of the corresponding tryptophan side chains and observed two distinct relaxations from tens to hundreds of picoseconds. Integrating our previous findings on α-helical proteins, we conclude the following: (1) The hydration dynamics is highly heterogeneous around the protein surface of both α-helical and β-sheet proteins. The outer-layer water of the hydration shell behave like a bulk and relaxes in hundreds of femtoseconds. The inner-layer water collectively relaxes in two time scales, reorientation motions in a few picoseconds and network restructuring in tens to a hundred picoseconds. (2) The hydration dynamics are always faster than local protein relaxations and in fact drive the protein fluctuations on the picosecond time scale. (3) The hydration dynamics in general are more retarded around β-sheet structures than α-helical motifs. A thicker hydration shell and a more rigid interfacial hydration network are observed in the β-sheet protein. Overall, these findings elucidate the intimate relationship between water-protein interactions and dynamics on the ultrafast time scale.

Solution Structure and Backbone Dynamics of Human Liver Fatty Acid Binding Protein: Fatty Acid Binding Revisited

Liver fatty acid binding protein (L-FABP), a cytosolic protein most abundant in liver, is associated with intracellular transport of fatty acids, nuclear signaling, and regulation of intracellular lipolysis. Among the members of the intracellular lipid binding protein family, L-FABP is of particular interest as it can i), bind two fatty acid molecules simultaneously and ii), accommodate a variety of bulkier physiological ligands such as bilirubin and fatty acyl CoA. To better understand the promiscuous binding and transport properties of L-FABP, we investigated structure and dynamics of human L-FABP with and without bound ligands by means of heteronuclear NMR. The overall conformation of human L-FABP shows the typical β-clam motif. Binding of two oleic acid (OA) molecules does not alter the protein conformation substantially, but perturbs the chemical shift of certain backbone and side-chain protons that are involved in OA binding according to the structure of the human L-FABP/OA complex. Comparison of the human apo and holo L-FABP structures revealed no evidence for an “open-cap” conformation or a “swivel-back” mechanism of the K90 side chain upon ligand binding, as proposed for rat L-FABP. Instead, we postulate that the lipid binding process in L-FABP is associated with backbone dynamics.

Experimental characterization of the mechanism of perfluorocarboxylic acids' liver protein bioaccumulation: The key role of the neutral species

Perfluorocarboxylic acids (PFCAs) of chain length greater than seven carbon atoms bioconcentrate in the livers of fish. However, a mechanistic cause for the empirically observed increase in the bioconcentration potential of PFCAs as a function of chain length has yet to be determined. To this end, recombinant rat liver fatty acid-binding protein (L-FABP) was purified, and its interaction with PFCAs was characterized in an aqueous system at pH 7.4. Relative binding affinities of L-FABP with PFCAs of carbon chain lengths of five to nine were established fluorimetrically. The energetics, mechanism, and stoichiometry of the interaction of perfluorooctanoic acid (PFOA) with L-FABP were examined further by isothermal titration calorimetry (ITC) and electrospray ionization combined with tandem mass spectrometry (ESI-MS/MS). Perfluorooctanoic acid was shown to bind to L-FABP with an affinity approximately an order of magnitude less than the natural ligand, oleic acid, and to have at least 3:1 PFOA:L-FABP stoichiometry. Two distinct modes of PFOA binding to L-FABP were observed by ESI-MS/MS analysis; in both cases, PFOA binds solely as the neutral species under typical physiological pH and aqueous concentrations of the anion. A comparison of their chemical and physical properties with other well-studied biologically relevant chemicals showed that accumulation of PFCAs in proteins as the neutral species is predictable. For example, the interaction of PFOA with L-FABP is almost identical to that of the acidic ionizing drugs ketolac, ibuprofen, and warfarin that show specificity to protein partitioning with a magnitude that is proportional to the K(OW) (octanol-water partitioning) of the neutral species. The experimental results suggest that routine pharmacochemical models may be applicable to predicting the protein-based bioaccumulation of long-chain PFCAs.

Molecular mechanism of recombinant liver fatty acid binding protein's antioxidant activity

Hepatocytes expressing liver fatty acid binding protein (L-FABP) are known to be more resistant to oxidative stress than those devoid of this protein. The mechanism for the observed antioxidant activity is not known. We examined the antioxidant mechanism of a recombinant rat L-FABP in the presence of a hydrophilic (AAPH) or lipophilic (AMVN) free radical generator. Recombinant L-FABP amino acid sequence and its amino acid oxidative products following oxidation were identified by MALDI quadrupole time-of-flight MS after being digested by endoproteinase Glu-C. L-FABP was observed to have better antioxidative activity when free radicals were generated by the hydrophilic generator than by the lipophilic generator. Oxidative modification of L-FABP included up to five methionine oxidative peptide products with a total of approximately 80 Da mass shift compared with native L-FABP. Protection against lipid peroxidation of L-FABP after binding with palmitate or alpha-bromo-palmitate by the AAPH or AMVN free radical generators indicated that ligand binding can partially block antioxidant activity. We conclude that the mechanism of L-FABP's antioxidant activity is through inactivation of the free radicals by L-FABP's methionine and cysteine amino acids. Moreover, exposure of the L-FABP binding site further promotes its antioxidant activity. In this manner, L-FABP serves as a hepatocellular antioxidant.

Crystal Structure of Soluble Domain of Malaria Sporozoite Protein UIS3 in Complex with Lipid

Malaria parasite UIS3 (up-regulated in infective sporozoites gene 3) is essential for sporozoite development in infected hepatocytes. UIS3 encodes for a membrane protein that is localized to the parasite parasitophorous vacuolar membrane in infected hepatocytes. We describe here 2.5-Å resolution crystal structure of Plasmodium falciparum UIS3 soluble domain (PfUIS3130-229) in complex with the lipid phosphatidylethanolamine (PE). PfUIS3130-229 is a novel, compact, and all α-helical structure bound to one molecule of PE. The PfUIS3130-229-PE complex structure reveals a novel binding site with specific interactions between PfUIS3130-229 and the PE head group. One acyl chain of PE wraps around part of PfUIS3130-229 and docks onto a hydrophobic channel. We additionally provide new structural and biochemical evidence of PfUIS3130-229 interactions with lipids (phosphatidylethanolamine), with phospholipid liposomes, and with the human liver fatty acid-binding protein. The direct interaction of PfUIS3130-229 with liver fatty acid-binding protein most likely provides the parasite with a conduit for importing essential fatty acids/lipids. Therefore, our analyses have implications for lipid transport into the parasite during the rapid growth phases of sporozoites. Given that PfUIS3 is essential for establishment of liver stage infection by P. falciparum, our data provide a new target for abrogating parasite development within liver cells before typical symptoms of malaria can manifest.

Hepatic Fatty Acid Transporter Cd36 Is a Common Target of LXR, PXR, and PPARγ in Promoting Steatosis

Liver X receptor (LXR) is known to promote hepatic lipogenesis by activating the lipogenic transcriptional factor sterol regulatory element-binding protein (Srebp). Pregnane X receptor (PXR), a previously known "xenobiotic receptor," could mediate a Srebp-independent lipogenic pathway by activating the free fatty acid uptake transporter Cd36. The goal of this study is to investigate further the role of Cd36 in hepatic steatosis. Wild-type, LXR transgenic, PXR transgenic, and Cd36 null mice were used to study the regulation of Cd36 and other hepatic lipogenic genes and the implication of this regulation in hepatic steatosis. Promoter sequences of Cd36 and peroxisome proliferator-activated receptor (PPAR) gamma were cloned, and their respective regulation by LXR and PXR was investigated by combinations of receptor-DNA binding and reporter gene assays. We showed that genetic (transgene) or pharmacologic (ligands) activation of LXR induced Cd36. Promoter analysis established Cd36 as a novel transcription target of LXRalpha. Moreover, the hepatic steatosis induced by LXR agonists was largely abolished in Cd36 null mice. We also showed that PPARgamma, a positive regulator of Cd36, is a transcriptional target of PXR, suggesting that PXR can regulate Cd36 directly or through its activation of PPARgamma. Interestingly, both LXR-mediated Cd36 regulation and PXR-mediated PPARgamma regulation are liver specific. We conclude that Cd36 is a shared target of LXR, PXR, and PPARgamma. The network of CD36 regulation by LXR, PXR, and PPARgamma establishes this free fatty acid transporter as a common target of orphan nuclear receptors in their mediation of lipid homeostasis.

Solution-State Molecular Structure of Apo and Oleate-Liganded Liver Fatty Acid-Binding Protein

Rat liver fatty acid-binding protein (LFABP) is distinctive among intracellular lipid-binding proteins (iLBPs): more than one molecule of long-chain fatty acid and a variety of diverse ligands can be bound within its large cavity, and in vitro lipid transfer to model membranes follows a mechanism that is diffusion-controlled rather than mediated by protein-membrane collisions. Because the apoprotein has proven resistant to crystallization, nuclear magnetic resonance spectroscopy offers a unique route to functionally informative comparisons of molecular structure and dynamics for LFABP in free (apo) and liganded (holo) forms. We report herein the solution-state structures determined for apo-LFABP at pH 6.0 and for holoprotein liganded to two oleates at pH 7.0, as well as the structure of the complex including locations of the ligands. 1H, 13C, and 15N resonance assignments revealed very similar types and locations of secondary structural elements for apo- and holo-LFABP as judged from chemical shift indices. The solution-state tertiary structures of the proteins were derived with the CNS/ARIA computational protocol, using distance and angular restraints based on 1H-1H nuclear Overhauser effects (NOEs), hydrogen-bonding networks, 3J(HNHA) coupling constants, intermolecular NOEs, and residual dipolar (NH) couplings. The holo-LFABP solution-state conformation is in substantial agreement with a previously reported X-ray structure [Thompson, J., Winter, N., Terwey, D., Bratt, J., and Banaszak, L. (1997) The crystal structure of the liver fatty acid-binding protein. A complex with two bound oleates, J. Biol. Chem. 272, 7140-7150], including the typical beta-barrel capped by a helix-turn-helix portal. In the solution state, the internally bound oleate has the expected U-shaped conformation and is tethered electrostatically, but the extended portal ligand can adopt a range of conformations based on the computationally refined structures, in contrast to the single conformation observed in the crystal structure. The apo-LFABP also has a well-defined beta-barrel structural motif typical of other members of the iLBP protein family, but the portal region that is thought to facilitate ligand entry and exit exhibits conformational variability and an unusual "open cap" orientation with respect to the barrel. These structural results allow us to propose a model in which ligand binding to LFABP occurs through conformational fluctuations that adjust the helix-turn-helix motif to open or close the top of the beta-barrel, and solvent accessibility to the protein cavity favors diffusion-controlled ligand transport.

Liver Fatty Acid-binding Protein Initiates Budding of Pre-chylomicron Transport Vesicles from Intestinal Endoplasmic Reticulum

The rate-limiting step in the transit of absorbed dietary fat across the enterocyte is the generation of the pre-chylomicron transport vesicle (PCTV) from the endoplasmic reticulum (ER). This vesicle does not require coatomer-II (COPII) proteins for budding from the ER membrane and contains vesicle-associated membrane protein 7, found in intestinal ER, which is a unique intracellular location for this SNARE protein. We wished to identify the protein(s) responsible for budding this vesicle from ER membranes in the absence of the requirement for COPII proteins. We chromatographed rat intestinal cytosol on Sephacryl S-100 and found that PCTV budding activity appeared in the low molecular weight fractions. Additional chromatographic steps produced a single major and several minor bands on SDS-PAGE. By tandem mass spectroscopy, the bands contained both liver and intestinal fatty acid-binding proteins (L- and I-FABP) as well as four other proteins. Recombinant proteins for each of the six proteins identified were tested for PCTV budding activity; only L-FABP and I-FABP (23% the activity of L-FABP) were active. The vesicles generated by L-FABP were sealed, contained apolipoproteins B48 and AIV, were of the same size as PCTV on Sepharose CL-6B, and by electron microscopy, excluded calnexin and calreticulin but did not fuse with cis-Golgi nor did L-FABP generate COPII-dependent vesicles. Gene-disrupted L-FABP mouse cytosol had 60% the activity of wild type mouse cytosol. We conclude that L-FABP can select cargo for and bud PCTV from intestinal ER membranes.

Targeted Intestinal Overexpression of the Immediate Early Gene tis7 in Transgenic Mice Increases Triglyceride Absorption and Adiposity

Following loss of functional small bowel surface area due to surgical resection, the remnant gut undergoes an adaptive response characterized by increased crypt cell proliferation and enhanced villus height and crypt depth, resulting in augmented intestinal nutrient absorptive capacity. Previous studies showed that expression of the immediate early gene tis7 is markedly up-regulated in intestinal enterocytes during the adaptive response. To study its role in the enterocyte, transgenic mice were generated that specifically overexpress TIS7 in the gut. Nucleotides -596 to +21 of the rat liver fatty acid-binding protein promoter were used to direct abundant overexpression of TIS7 into small intestinal upper crypt and villus enterocytes. TIS7 transgenic mice had increased total body adiposity and decreased lean muscle mass compared with normal littermates. Oxygen consumption levels, body weight, surface area, and small bowel weight were decreased. On a high fat diet, transgenic mice exhibited a more rapid and proportionately greater gain in body weight with persistently elevated total body adiposity and increased hepatic fat accumulation. Bolus fat feeding resulted in a greater increase in serum triglyceride levels and an accelerated appearance of enterocytic, lamina propria, and hepatic fat. Changes in fat homeostasis were linked to increased expression of genes involved in enterocytic triglyceride metabolism and changes in growth with decreased insulin-like growth factor-1 expression. Thus, TIS7 overexpression in the intestine altered growth, metabolic rate, adiposity, and intestinal triglyceride absorption. These results suggest that TIS7 is a unique mediator of nutrient absorptive and metabolic adaptation following gut resection.

An improved method for the purification of rat liver-type fatty acid binding protein from Escherichia coli

Rat liver fatty acid binding protein (L-FABP) was efficiently expressed in Escherichia coli and purified to homogeneity. The cDNA encoding L-FABP was ligated into the pTrc99A expression vector and expressed by induction with isopropyl-β- d-thiogalactopyranoside under the control of the P trc promoter. Following an 18 h induction period, L-FABP constituted approximately 3% of the cytosolic protein. The protein could be purified to electrophoretic homogeneity (silver-stained polyacrylamide gel detection) by ammonium sulfate fractionation (65% saturation) of the soluble bacterial lysate followed by the chromatographic sequence of anion-exchange → hydrophobic interaction → anion-exchange chromatography. The recombinant protein displayed an isoelectric point of 7.0 and cross-reactivity with rabbit anti-(human L-FABP) polyclonal antibody. The ligand binding properties of the delipidated L-FABP were examined by titration with the fluorescent probe 1-anilino-8-naphthalene sulfonic acid and isothermal titration calorimetric analysis of oleic acid binding. The purified rat L-FABP displayed a binding stoichiometry of 2:1 (ANS:L-FABP) with dissociation constants ( K d) of 1.7 and 15.5 μM for the high and low affinity binding sites, respectively. The K d values determined by ITC for oleic acid binding were 0.155 and 4.04 μM with a binding stoichiometry of approximately 2 mol of fatty acid/mol of protein. These physicochemical and binding properties are in agreement with those of L-FABP isolated from rat liver tissue.

Statin Induction of Liver Fatty Acid-Binding Protein (L-FABP) Gene Expression Is Peroxisome Proliferator-activated Receptor-α-dependent

Statins are drugs widely used in humans to treat hypercholesterolemia. Statins act by inhibiting cholesterol synthesis resulting in the activation of the transcription factor sterol-responsive element-binding protein-2 that controls the expression of genes involved in cholesterol homeostasis. Statin therapy also decreases plasma triglyceride and non-esterified fatty acid levels, but the mechanism behind this effect remains more elusive. Liver fatty acid-binding protein (L-FABP) plays a role in the influx of long-chain fatty acids into hepatocytes. Here we show that L-FABP is a target for statins. In rat hepatocytes, simvastatin treatment induced L-FABP mRNA levels in a dose-dependent manner. Moreover, L-FABP promoter activity was induced by statin treatment. Progressive 5'-deletion analysis revealed that the peroxisome proliferator-activated receptor (PPAR)-responsive element located at position -67/-55 was responsible for the statin-mediated transactivation of the rat L-FABP promoter. Moreover, treatment with simvastatin and the PPARalpha agonist Wy14,649 resulted in a synergistic induction of L-FABP expression (mRNA and protein) in rat Fao hepatoma cells. This effect was also observed in vivo in wild-type mice but not in PPARalpha-null animals demonstrating the direct implication of PPARalpha in L-FABP regulation by statin treatment. Statin treatment resulted in a rise in PPARalpha mRNA levels both in vitro and in vivo and activated the mouse PPARalpha promoter in a reporter assay. Altogether, these data demonstrate that L-FABP expression is up-regulated by statins through a mechanism involving PPARalpha. Moreover, PPARalpha might be a statin target gene. These effects might contribute to the triglyceride/non-esterified fatty acid-lowering properties of statins.

Fatty Acid-Binding Proteins Inhibit Hydration of Epoxyeicosatrienoic Acids by Soluble Epoxide Hydrolase

Epoxyeicosatrienoic acids (EETs) are potent regulators of vascular homeostasis and are bound by cytosolic fatty acid-binding proteins (FABPs) with K(d) values of approximately 0.4 microM. To determine whether FABP binding modulates EET metabolism, we examined the effect of FABPs on the soluble epoxide hydrolase (sEH)-mediated conversion of EETs to dihydroxyeicosatrienoic acids (DHETs). Kinetic analysis of sEH conversion of racemic [(3)H]11,12-EET yielded K(m) = 0.45 +/- 0.08 microM and V(max) = 9.2 +/- 1.4 micromol min(-1) mg(-)(1). Rat heart FABP (H-FABP) and rat liver FABP were potent inhibitors of 11,12-EET and 14,15-EET conversion to DHET. The resultant inhibition curves were best described by a substrate depletion model, with K(d) = 0.17 +/- 0.01 microM for H-FABP binding to 11,12-EET, suggesting that FABP acts by reducing EET availability to sEH. The EET depletion by FABP was antagonized by the co-addition of arachidonic acid, oleic acid, linoleic acid, or 20-hydroxyeicosatetraenoic acid, presumably due to competitive displacement of FABP-bound EET. Collectively, these findings imply that FABP might potentiate the actions of EETs by limiting their conversion to DHET. However, the effectiveness of this process may depend on metabolic conditions that regulate the levels of competing FABP ligands.

Structural and biochemical characterization of toad liver fatty acid-binding protein.

Two paralogous groups of fatty acid-binding proteins (FABPs) have been described in vertebrate liver: liver FABP (L-FABP) type, extensively characterized in mammals, and liver basic FABP (Lb-FABP) found in fish, amphibians, reptiles, and birds. We describe here the toad Lb-FABP complete amino acid sequence, its X-ray structure to 2.5 A resolution, ligand-binding properties, and mechanism of fatty acid transfer to phospholipid membranes. Alignment of the amino acid sequence of toad Lb-FABP with known L-FABPs and Lb-FABPs shows that it is more closely related to the other Lb-FABPs. Toad Lb-FABP conserves the 12 characteristic residues present in all Lb-FABPs and absent in L-FABPs and presents the canonical fold characteristic of all the members of this protein family. Eight out of the 12 conserved residues point to the lipid-binding cavity of the molecule. In contrast, most of the 25 L-FABP conserved residues are in clusters on the surface of the molecule. The helix-turn-helix motif shows both a negative and positive electrostatic potential surface as in rat L-FABP, and in contrast with the other FABP types. The mechanism of anthroyloxy-labeled fatty acids transfer from Lb-FABP to phospholipid membranes occurs by a diffusion-mediated process, as previously shown for L-FABP, but the rate of transfer is 1 order of magnitude faster. Toad Lb-FABP can bind two cis-parinaric acid molecules but only one trans-parinaric acid molecule while L-FABP binds two molecules of both parinaric acid isomers. Although toad Lb-FABP shares with L-FABP a broad ligand-binding specificity, the relative affinity is different.

Interactions of flurochemicals with rat liver fatty acid-binding protein

Liver-fatty acid binding protein (L-FABP) is an abundant intracellular lipid-carrier protein. The hypothesis that perfluorooctanesulfonate (PFOS), perfluorooctanoate (PFOA), and certain related perfluorooctanesulfonamide-based fluorochemicals (PFOSAs) can interfere with the binding affinity of L-FABP for fatty acids was tested. The relative effectiveness of PFOA, PFOS, N-ethylperfluorooctanesulfonamide (N-EtFOSA), N-ethylperfluorooctanesulfonamido ethanol (N-EtFOSE), and of the strong peroxisome proliferator Wyeth-14 643 (WY) to inhibit 11-(5-dimethylaminonapthalenesulphonyl)-undecanoic acid (DAUDA) binding to-L-FABP was determined. The dissociation constant (Kd) of the DAUDA-L-FABP complex was 0.47 nM. PFOS exhibited the highest level of inhibition of DAUDA-L-FABP binding in the competitive binding assays, followed by N-EtFOSA, WY, and, with equal IC 50s, N-EtFOSE and PFOA. The in vitro data presented in this study support the hypothesis that these fluorochemicals may interfere with the binding of fatty acids or other endogenous ligands to L-FABP. Furthermore, this work provides evidence to support the hypothesis that displacement of endogenous ligands from L-FABP may contribute to toxicity in rodents fed these fluorochemicals.

Titration and exchange studies of liver fatty acid-binding protein with 13C-labeled long-chain fatty acids.

Uniformly (13)C-labeled long-chain fatty acids were used to probe ligand binding to rat liver fatty acid-binding protein (LFABP), an atypical member of the fatty acid-binding protein (FABP) family that binds more than one molecule of long-chain fatty acid, accommodates a variety of diverse ligands, and exhibits diffusion-mediated lipid transport to membranes. Two sets of (1)H-(13)C resonances were found in a titration series of NMR spectra for oleate-LFABP complexes, indicating that two molecules of the fatty acid are situated in the protein cavity. However, no distinct resonances were observed for the excess fatty acid in solution, suggesting that at least one ligand undergoes rapid exchange with oleate in the bulk solution. An exchange rate of 54 +/- 6 s(-1) between the two sets of resonances was measured directly using (13)C z,z-exchange spectroscopy. In light of these NMR measurements, possible molecular mechanisms for the ligand-exchange process are evaluated and implications for the anomalous fatty acid transport mechanism of LFABP are discussed.

A fatty acid-binding protein and a protein disulphide isomerase-related protein expressed in urochordate gonad cytosol

Despite the evolutionary-tree data suggesting that gene duplication leading to the divergence of the three branches which heart, liver and intestinal fatty acid-binding proteins belong to must have occurred before the vertebrate/invertebrate split, only the heart fatty acid-binding protein has been reported for invertebrates. In an attempt to shed light on this apparent inconsistency the presence of the other two branch members was investigated in the Urochordata Molgula pedunculata, an ascidian species close to vertebrates. The mantle-, gonad- and digestive tube-cytosolic fractions, obtained by centrifugation at 106,000 g , were incubated separately with [1- 14C]palmitic acid and then fractionated on a Sephadex G-75 column. In the case of gonads and digestive tube, radioactive peaks corresponding to a molecular mass of 14–16 kDa, characteristic of fatty acid-binding proteins, were detected. When the experiment was performed on the mantle, this peak showing fatty acid binding capacity was absent. Western Blot of the radioactive 14–16 kDa Sephadex fraction from the urochordate gonad cross-reacted with rat liver fatty acid-binding protein anti-serum but did not do so with anti-rat intestinal, adipocyte or heart fatty acid-binding protein antisera. The material from the digestive tube was not recognized by any of the antisera. The most abundant protein in said 14–16 kDa fraction was a protein disulphide isomerase-related protein. Its partial amino acid sequence was determined.

Isolation, characterization and binding properties of two rat liver fatty acid-binding protein isoforms

Mammalian liver has only one fatty acid-binding protein (L-FABP) while the liver of non-mammalian vertebrates expresses a liver basic FABP (Lb-FABP) in addition to other members of the FABP family. We explore the possibility that L-FABP isoforms accomplish, in the liver of mammals, the metabolic functions corresponding to the different FABPs present in the liver of non-mammalian vertebrates. We have isolated isoforms I and II which have a different residue 105, Asn in the former and Asp in the latter. We made a conformational comparison of the apo-isoforms by intrinsic fluorescence emission and fourth-derivative spectroscopy, native-state proteolysis and unfolding curves. Ligand affinity was studied by measuring cis-parinaric acid displacement by different ligands. They have differences in their molecular conformation, including the environment of the binding site. Isoform II has probably a more open conformation than isoform I, thus allowing the binding of a greater variety of ligands. The affinity of isoform II for lysophospholipids, prostaglandins, retinoids, bilirubin and bile salts is greater than that of isoform I. These characteristics of rat L-FABP isoforms I and II suggest that they may accomplish different functions as happens with those of the different FABP types in non-mammalian species.

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

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

Isolation and characterization of two distinct forms of liver fatty acid binding protein from the rat

Liver fatty acid binding protein (L-FABP) appears to contain several different forms that may result from post-translational modification or bound ligand. To further assess this possibility, L-FABP was purified from rat liver homogenate and two putative isoforms separated using a sulfonyl column, a strong cation exchange resin. Fraction I eluted at 0.2 M NaCl, had a p I of 7.59, and following a final size exclusion step contained >98% L-FABP. Fraction II eluted at 1.0 M NaCl, had a p I of 7.59, and following a final size exclusion step contained >99% L-FABP. Both fractions contained approx. 0.15 moles of endogenous bound fatty acid per mole of protein, while L-FABP not subjected to the cation exchange step contained 0.75 moles of fatty acid per mole of protein. Fractions I and II had a greater proportion of saturated and monounsaturated fatty acids with a large reduction in polyunsaturated fatty acids compared to L-FABP not fractionated by cation exchange. Mass spectral analysis indicated the molecular mass of Fraction I was 14 315.02±0.35 Da and Fraction II was 14 315.86±0.34 Da. The peptide map for each fraction was determined by limited digestion of each fraction with either trypsin, Asp-N, or chymotrypsin to yield overlapping peptide fragments. Mass spectral analysis of these digests indicated the two proteins had identical amino acid fragments and that Cys 69 was reduced and there were no Asn to Asp exchanges. Hence, these two forms of L-FABP were not isoforms and were not the result of differences in bound fatty acid. It is proposed that these two distinct forms of rat L-FABP were structural conformers based on two alternative folding pathways.

Fatty acid binding to rat liver fatty acid-binding protein is modulated by early glycolytic intermediates

Fatty acid binding to rat liver fatty acid binding protein in the presence of glycolytic metabolites and at different pH (optimal 7.2) and ionic strength was studied. Binding decreased logarithmically with ionic strength. Glucose and glucose-6-phosphate increased fatty acid binding significantly with K 0.5 within physiological ranges while glucose-1-phosphate and phosphate ion caused no effect.