Showing papers on "Fatty acid-binding protein published in 1996"
TL;DR: Results indicate that aP2 is central to the pathway that links obesity to insulin resistance, possibly by linking fatty acid metabolism to expression of TNF-α.
Abstract: Fatty acid binding proteins (FABPs) are small cytoplasmic proteins that are expressed in a highly tissue-specific manner and bind to fatty acids such as oleic and retinoic acid. Mice with a null mutation in aP2 , the gene encoding the adipocyte FABP, were developmentally and metabolically normal. The aP2 -deficient mice developed dietary obesity but, unlike control mice, they did not develop insulin resistance or diabetes. Also unlike their obese wild-type counterparts, obese aP2 −/− animals failed to express in adipose tissue tumor necrosis factor-α (TNF-α), a molecule implicated in obesity-related insulin resistance. These results indicate that aP2 is central to the pathway that links obesity to insulin resistance, possibly by linking fatty acid metabolism to expression of TNF-α.
914 citations
542 citations
TL;DR: It is reported that treatment of 3T3-L1 adipocytes with TNF alpha resulted in a time- and concentration-dependent decrease in PPAR gamma mRNA expression to the level detected in preadipocytes, which supports the following conclusions: 1) PPAR Gamma expression is necessary for the maintenance of the adipocyte phenotype.
Abstract: Recent studies indicate that a peroxisome proliferator-activated receptor, PPAR gamma, functions as an important adipocyte determination factor. In contrast, tumor necrosis factor-alpha (TNF alpha) inhibits adipogenesis, causes dedifferentiation of mature adipocytes, and reduces the expression of several adipocyte-specific genes. Here, we report that treatment of 3T3-L1 adipocytes with TNF alpha resulted in a time- and concentration-dependent decrease in PPAR gamma mRNA expression to the level detected in preadipocytes. PPAR gamma mRNA levels were reduced by 95% with 3 nM TNF alpha treatment for 24 h. Half-maximal effects were seen after 3 h treatment with 3 nM TNF alpha or with 50 pM TNF alpha (24-h exposure). Parallel reductions in PPAR gamma protein levels were also observed after treatment of 3T3-L1 adipocytes with TNF alpha. Using a ribonuclease protection assay, both alternatively spliced PPAR gamma isoforms (gamma 1 and gamma 2) were shown to be negatively regulated by TNF alpha. The down-regulation of PPAR gamma by TNF-alpha preceded the diminution in expression of other adipocyte-specific genes including CCAAT/enhancer binding protein and adipocyte fatty acid-binding protein (aP2). The effect of TNF alpha was specific for the gamma-isoform of PPARs, since the expression of PPAR delta mRNA was not affected by treatment with TNF alpha. Low level constitutive expression of PPAR gamma in 3T3-L1 adipocytes (at levels approximately 2- to 3-fold higher than in preadipocytes) partially blocked the inhibitory effect of TNF alpha on aP2 and adipsin expression. These findings support the following conclusions: 1) PPAR gamma expression is necessary for the maintenance of the adipocyte phenotype. 2) PPAR gamma, but not PPAR delta, expression is sufficient to attenuate TNF alpha-mediated effects on adipocyte phenotype. 3) Reduced PPAR gamma gene expression is likely to represent an important component of the mechanism by which TNF alpha exerts its antiadipogenic effects.
333 citations
TL;DR: Comparison of the predicted secondary sequence of CD36 with that of human muscle fatty acid binding protein suggested that a potential binding site for the fatty acid on CD36 may exist in its extracellular segment between residues 127 and 279.
Abstract: Transport of long-chain fatty acids into rat adipocytes was previously shown to be inhibited by the reactive derivative sulfosuccinimidyl oleate consequent to its binding to a membrane protein FAT, which is homologous to CD36. In this report, the ability of the purified protein to bind native fatty acids was investigated. CD36 was isolated from rat adipocytes by phase partitioning into Triton X-114 followed by chromatography on DEAE and then on wheat germ agglutinin. Fatty acid binding was determined by incubating CD36, solubilized in buffer containing 0.1 Triton X-100, with fatty acids at 37°C, and then by adsorbing the unbound ligand with Lipidex 1,000 at 0°C. Bovine serum albumin was used as a positive control and gelatin, a protein that does not bind fatty acids, as a negative control. Measurements with albumin yielded reproducible binding values which were not altered by the presence of 0.1% Triton X-100. Under the same conditions, gelatin yielded reproducibly negative measurements that did not differ significantly from zero. CD36 bound various long-chain fatty acids at low ligand to protein ratios. Warming the protein-FA-Lipidex mixture to 37°C removed the FA off the protein. Thus, binding was reversible and distinct from the palmitoylation of the protein known to occur on an extracellular domain. Comparison of the predicted secondary sequence of CD36 with that of human muscle fatty acid binding protein suggested that a potential binding site for the fatty acid on CD36 may exist in its extracellular segment between residues 127 and 279.
226 citations
TL;DR: The data demonstrate that FAT and FABP are co-expressed in enterocytes, as has been shown in adipocytes, myocytes and mammary cells, and suggest that these membrane and cytosolic proteins might have complementary functions during dietary-fat absorption.
Abstract: The expression of the putative membrane fatty-acid transporter (FAT) was investigated in the small intestine. The FAT mRNA level was higher in the jejunum than in the duodenum and was lower in the ileum, as observed for cytosolic fatty-acid-binding proteins (FABP) expressed in this tissue. No FAT transcript was found in the stomach or colon. FAT mRNA was constitutively expressed in the epithelial cells located in the upper two thirds of villi, while it was undectectable in the crypt cells and submucosal cells. In jejunal mucosa, immunochemical studies showed that FAT protein was limited to the brush border of enterocytes. No fluorescence was found in the goblet cells. To determine whether FAT responded to changes in fat intake, as reported for FABP, the effect of two high-fat diets, which essentially contained either medium-chain fatty acids or long-chain fatty acids (sunflower-oil diet), was investigated. The sunflower-oil diet greatly increased FAT mRNA abundance throughout the small intestine. In contrast, a weak effect of medium-chain fatty acids was observed only in the jejunum. As found for FABP expression, treatment with the hypolipidemic drug bezafibrate affected FAT expression. These data demonstrate that FAT and FABP are co-expressed in enterocytes, as has been shown in adipocytes, myocytes and mammary cells. The data suggest that these membrane and cytosolic proteins might have complementary functions during dietary-fat absorption.
212 citations
TL;DR: The results strongly suggest that fatty acid transfer from I-FABP to membranes occurs by direct collisional interaction of the protein with the phospholipid bilayer, which is consistent with an aqueous diffusion-mediated process.
187 citations
TL;DR: The observed increase in transport of fatty acids across cells expressing Thr IFABP suggests a plausible physiologic mechanism for the prior observation that Pima Indians with a ThrIFABP genotype have increased post-absorptive lipid oxidation rates and are more insulin-resistant than Pimas with a Ala IFABp genotype.
175 citations
TL;DR: Results show in detail for the first time using an intact cell culture system that L-FABP expression not only stimulated fatty acid uptake, but also increased intracellular esterification of exogenously supplied fatty acids.
135 citations
TL;DR: The identity, nature, function, and pathobiology of these fascinating newly discovered long-chain fatty acyl-CoA binding proteins are explored.
Abstract: The physiological role of long-chain fatty acyl-CoA is thought to be primarily in intermediary metabolism of fatty acids. However, recent data show that nM to μM levels of these lipophilic molecules are potent regulators of cell functionsin vitro. Although long-chain fatty acyl-CoA are present at several hundred μM concentration in the cell, very little long-chain fatty acyl-CoA actually exists as free or unbound molecules, but rather is bound with high affinity to membrane lipids and/or proteins. Recently, there is growing awareness that cytosol contains nonenzymatic proteins also capable of binding long-chain fatty acyl-CoA with high affinity. Although the identity of the cytosolic long-chain fatty acyl-CoA binding protein(s) has been the subject of some controversy, there is growing evidence that several diverse nonenzymatic cytosolic proteins will bind long-chain fatty acyl-CoA. Not only does acyl-CoA binding protein specifically bind medium and long-chain fatty acyl-CoA (LCFA-CoA), but ubiquitous proteins with multiple ligand specificities such as the fatty acid binding proteins and sterol carrier protein-2 also bind LCFA-CoA with high affinity. The potential of these acyl-CoA binding proteins to influence the level of free LCFA-CoA and thereby the amount of LCFA-CoA bound to regulatory sites in proteins and enzymes is only now being examined in detail. The purpose of this article is to explore the identity, nature, function, and pathobiology of these fascinating newly discovered long-chain fatty acyl-CoA binding proteins. The relative contributions of these three different protein families to LCFA-CoA utilization and/or regulation of cellular activities are the focus of new directions in this field.
126 citations
TL;DR: It is suggested that the T54 variant of the intestinal fatty acid-binding protein is associated with differences in fat metabolism in this aboriginal population.
Abstract: We hypothesized that genomic variation affecting the primary amino acid sequence of the intestinal fatty acid-binding protein would be related to variation in body mass index and associated clinical phenotypes in aboriginal Canadians. We studied 507 adult native Canadians from an isolated community in Northern Ontario. We found that the frequency of the T54 variant of the intestinal fatty acid-binding protein gene was 0.14 in this sample. The presence of this variant was associated with significant increases in body mass index, percent body fat, and fasting plasma triglyceride concentration (P = 0.012, 0.019, and 0.012, respectively). However, the variant was not associated with the presence of diabetes mellitus. These findings suggest that the T54 variant of the intestinal fatty acid-binding protein is associated with differences in fat metabolism in this aboriginal population.
111 citations
TL;DR: Data support a saturable component in the transport of long chain but not short chain fatty acids by human intestinal epithelial cells, which may involve an as yet unknown plasma membrane protein.
TL;DR: A hypothesis in which the myotoxins and/or products of lipase activity are acting at a site existing on both the Na+ channel and a protein involved in Ca2+ release and probably serving a modulatory function for ion regulation is supported.
TL;DR: It is suggested that entering or leaving the FABP binding cavity involves similar mechanisms for all 3 FABPs and may involve amino acid residues located within the portal regions of these proteins.
TL;DR: Oleate and other long-chain fatty acids are strong inducers of L-FABP gene expression, and FAO cells constitute a useful tool for studying the underlying mechanism of fatty acid action.
Abstract: The role of fatty acids in the expression of the gene for liver fatty acid-binding protein (L-FABP) was investigated in the well-differentiated FAO rat hepatoma cell line. Cells were maintained in serum-free medium containing 40 microM BSA/320 microM oleate. Western blot analysis showed that oleate triggered an approx. 4-fold increase in the cytosolic L-FABP level in 16 h. Oleate specifically stimulated L-FABP mRNA in time-dependent and dose-dependent manners with a maximum 7-fold increase at 16 h in FAO cells. Preincubation of FAO cells with cycloheximide prevented the oleate-mediated induction of L-FABP mRNA, showing that protein synthesis was required for the action of fatty acids. Run-on transcription assays demonstrated that the control of L-FABP gene expression by oleate was, at least in part, transcriptional. Palmitic acid, oleic acid, linoleic acid, linolenic acid and arachidonic acid were similarly potent whereas octanoic acid was inefficient. This regulation was also found in normal hepatocytes. Therefore long-chain fatty acids are strong inducers of L-FABP gene expression. FAO cells constitute a useful tool for studying the underlying mechanism of fatty acid action.
TL;DR: It is proposed that an important function of L-FABP is to bind certain physiological amphipathic anions, thus preventing the "free' concentrations of these compounds from exceeding their critical micelle concentration, which could result in cell damage.
Abstract: The physiological role of liver fatty acid-binding protein (L-FABP) has yet to be clarified. An important feature of this member of the family of intracellular lipid-binding proteins is the wide range of compounds that have been identified as potential physiological ligands. By using recombinant L-FABP, the binding of cholesterol, bile salts and their derivatives has been investigated under conditions that allow a direct comparison of the binding affinities of these ligands for fatty acids. The results demonstrate an inability of L-FABP to bind cholesterol, although the anionic derivative, cholesteryl sulphate, will bind under similar assay conditions. Of the bile salts examined, lithocholate and taurolithocholate sulphate showed the greatest binding to L-FABP. It is proposed that an important function of L-FABP is to bind certain physiological amphipathic anions, thus preventing the "free' concentrations of these compounds from exceeding their critical micelle concentration, which could result in cell damage.
TL;DR: A short summary is provided of the latest developments in this research area of cytoplasmic fatty acid-binding proteins, thought to function in the transmembrane transport of fatty acids.
Abstract: A number of cellular fatty acid-binding proteins are being implicated in the uptake and intracellular transport of long-chain fatty acids by parenchymal cells. Having been a topic of research for more than 20 years, cytoplasmic fatty acid-binding proteins now are assigned various pivotal functions in intracellular fatty acid transport and metabolism. More recently several membrane-associated fatty acid-binding proteins have been identified and these proteins are thought to function in the transmembrane transport of fatty acids. In this review, a short summary is provided of the latest developments in this research area.
TL;DR: The tertiary structure of ILBP is similar to that of other LBPs, but appears to be unusually flexible, with a relatively weak hydrogen-bonding network, likely important in allowing bile acids to enter the central cavity of the protein.
TL;DR: It is suggested that substantial intracellular concentration gradients of fatty acids should exist, and that these gradients may help determine which metabolic pathway the fatty acid enters.
Abstract: After entering the cell, small molecules must penetrate the cytoplasm before they are metabolized, excreted or can convey information to the cell nucleus. Without efficient cytoplasmic transport, most such molecules would efflux back from the cell before they could reach their targets. Conversely, intracellular lipids generated by hydrolysis of triglycerides, phospholipids and other esters must be transported away from their site of formation to prevent toxic accumulation. Intracellular movement of all molecules is slowed by molecular crowding, tortuosity, and the greater viscosity of the cytosol relative to water. However, lipids and other amphipathic molecules are further slowed by their tendency to bind to cytoplasmic membranes. Cytoplasmic binding proteins reduce membrane binding by increasing the aqueous solubility of their ligands. These aqueous carriers catalyze the transport of lipid molecules across hydrophilic water layers just as plasma membrane carriers catalyze the transport of hydrophilic molecules across the hydrophobic membrane core. They even display the principal features of carrier-mediated transport, including saturation, mutual competition, and countertransport. Higher concentrations of cytoplasmic binding proteins are associated with more rapid cytoplasmic transport of longchain fatty acids. Available data suggest that substantial intracellular concentration gradients of fatty acids should exist, and that these gradients may help determine which metabolic pathway the fatty acid enters. Thus, cytoplasmic carrier proteins may help regulate the uptake and metabolism of fatty acids and other lipid molecules.
TL;DR: It is concluded that cytoplasmic transport of NBD-stearate is modulated by binding to soluble proteins like FABP, which enhances diffusive transport by reducing binding to immobile cytosolic membranes.
Abstract: Male livers, containing lesser amounts of fatty acid binding protein (FABP), utilize fatty acids more slowly than female livers. Conventional wisdom dictates that FABP stimulates fatty acid use by increasing cytoplasmic transport rates. Previously, we showed that the cytoplasmic diffusion of a fatty acid analogue [12-N-methyl-7-nitrobenzo-2-oxa-1,3-diazol-amino stearate (NBD-stearate)] is faster in female hepatocytes, paralleling the larger amounts of FABP. Sex differences in other cytoplasmic factors could also lead to faster diffusion, independent of FABP levels. The aim of this study was to determine the effect of inhibition of fatty acid binding to FABP on the directly measured intracellular transport rate of NBD-stearate. The binding of NBD-stearate to FABP was reduced by incubating hepatocytes isolated from male and female rats with alpha-bromo-palmitate (0-1,500 microM), a modified long-chain fatty acid that binds to FABP. The inhibition by alpha-bromo-palmitate on NBD-stearate binding to FABP was measured with the use of centrifugation to separate cytosol from cytoplasmic membranes. Laser photobleaching (fluorescence recovery after photobleaching) was used to measure the cytoplasmic diffusion of NBD-stearate in hepatocytes. Alpha-Bromo-palmitate incubation reduced NBD-stearate binding to FABP in a dose-dependent manner. The measured diffusion rate was also reduced in proportion to the degree of binding inhibition. We conclude that cytoplasmic transport of NBD-stearate is modulated by binding to soluble proteins like FABP. FABP enhances diffusive transport by reducing binding to immobile cytosolic membranes.
TL;DR: Increased gene expression for two species of cytosolic fatty acid binding proteins after systemic administration of kainic acid in the rat brain was demonstrated by Northern blotting and in situ hybridization histochemical analyses, suggesting strongly that the increased expression for B- and S-FABPs occurs in dedifferentiated and proliferated astrocytes in response to kainsic acid-induced seizure.
TL;DR: Ch21, a developmentally regulated extracellular protein expressed in chick embryos and in cultured chondrocytes, was expressed in the baculovirus system, and the recombinant protein was purified to homogeneity by gel-filtration chromatography to rename the Ch21 protein as Extracellular fatty acid-binding rotein (Ex-FABP).
TL;DR: The classical and molecular approaches to the identification of membrane transporters are examined and compared, with particular attention paid to sulfobromophthalein, bilirubin, and fatty acid transport.
Abstract: Recent efforts to identify plasma membrane transporters mediating the selective uptake of specific classes of organic anions have employed expression cloning techniques. The transporters identified by these procedures have, almost without exception, differed from putative transporters identified earlier by classical methods. In this review, the classical and molecular approaches to the identification of membrane transporters are examined and compared, with particular attention paid to the results obtained by each with respect to sulfobromophthalein, bilirubin, and fatty acid transport. The classical approach requires the initial identification and purification of a candidate transport protein, proof of its function as a transporter by antibody inhibition or liposome reconstitution studies, and following the cloning of its cDNA, genetic expression in transfected mammalian cells or in Xenopus laevis oocytes. Expression cloning affords more rapid identification of proteins which (by definition) influence uptake, avoids several potential artifacts (e.g., of conventional cDNA library screening techniques), and yields rapid access to sequence information and derived structural characteristics. However, it is ultimately necessary to express and purify the recombinant protein product, produce antibodies against synthetic peptides and/or the purified recombinant protein, use the antibodies to identify the subcellular location of the cloned protein, demonstrate that the protein binds the ligand of interest, and document that the protein mediates a facilitated process with the characteristics of the one under study. Hence, the two approaches ultimately require similar efforts. It is argued that the different putative BSP/bilirubin and fatty acid transporters identified by the two approaches may mediate different parallel transport pathways.
TL;DR: The mechanism of transfer of fluorescent anthroyloxy-labeled fatty acids (AOFA) from purified FABP's to phospholipid vesicles is examined and it is found that “cytosolic” FABPs may function in intracellular transport of fatty acids to decrease their membrane association, as well as to target fatty acid to specific subcellular sites of utilization.
Abstract: The path of a small hydrophobic molecule through the aqueous cytoplasm is not linear. Partition may favor membrane binding by several orders of magnitude; thus significant membrane association will markedly decrease the cytosolic transport rate. The presence of high concentrations of soluble binding proteins for these hydrophobic molecules would compete with membrane association and thereby increase transport rate. For long chain fatty acid molecules, a family of cytosolic binding proteins collectively known as the fatty acid-binding proteins (FABP), are thought to act as intracellular transport proteins. This paper examines the mechanism of transfer of fluorescent anthroyloxy-labeled fatty acids (AOFA) from purified FABP's to phospholipid vesicles. With the exception of the liver FABP, AOFA is transferred from FABP by collisional interaction of the protein with an acceptor membrane. The rate of transfer increased markedly when membranes contain anionic phospholipids; this suggests that positively charged residues on the surface of the FABP may interact with the membranes. Neutralization of surface lysine residues of adipocyte and heart-type FABPs decreased the AOFA transfer rate, and transfer was then found to proceed via aqueous diffusion rather than collisional interaction. Site-specific mutagenesis has further shown that the helix-turn-helix domain of the FABPs is critical for interaction with anionic acceptor membranes. In addition, direct interaction of adipocyte FABPs with anionic membranes has been demonstrated. Thus “cytosolic” FABP may function in. intracellular transport of fatty acids to decrease their membrane association, as well as to target fatty acids to specific subcellular sites of utilization.
TL;DR: Human muscle fatty acid-binding protein (M-FABP) is a 15 kDa cytosolic protein which may be involved in fatty acid transfer and modulation of non-esterified fatty acid concentration in heart, skeletal muscle, kidney and many other tissues.
Abstract: Human muscle fatty acid-binding protein (M-FABP) is a 15 kDa cytosolic protein which may be involved in fatty acid transfer and modulation of non-esterified fatty acid concentration in heart, skeletal muscle, kidney and many other tissues Crystallographic studies have suggested the importance of the amino acids Thr-40, Arg-106, Arg-126 and Tyr-128 for the hydrogen bonding network of the fatty acid carboxylate group Two phenylalanines at 16 and 57 are positioned to interact with the acyl chain of the fatty acid We prepared 13 mutant proteins by site-directed mutagenesis and tested them for fatty acid binding and stability Substitution of amino acids Phe-16, Arg-106 or Arg-126 created proteins which showed a large decrease in or complete loss of oleic acid binding Substitution of Phe-57 by Ser or Val and of Tyr-128 by Phe had no great effect The stability of the mutant proteins was tested by denaturation studies on the basis of fatty acid binding or tryptophan fluorescence and compared with that of the wild-type M-FABP There was no direct relationship between fatty acid-binding activity and stability Less stable mutants (F57S and Y128F) did not show a marked change in fatty acid-binding activity Substitution of Arg-126 by Gln or Arg-106 by Thr eliminated binding activity, but the former mutant protein showed wild-type stability, in contrast to the latter The results are in agreement with crystallographic data
TL;DR: FABP may increase flux of fatty acids through β-oxidation (in cold-acclimated fish) by an increase in the percent of FABP molecules carrying fatty acid.
Abstract: Fatty acid-binding protein was isolated from aerobic skeletal muscle of the striped bass Morone saxatilis. FABP from striped bass (MS-FABP) has a molecular mass of 14,800 Da (as estimated by SDS-PAGE) and binds long-chain fatty acids with 1:1 molar stoichiometry and with micromolar affinity. Binding dissociation constants, as estimated by liposome assay, were 0.63 ± 0.44, 0.82 ± 0.12, and 1.60 ± 0.44 μM for palmitic (16:0), oleic (18:1), and palmitoleic (16:1) acids, respectively. No significant difference was detected in MS-FABP's binding affinity among these fatty acids or those estimated by competitive displacement assay (22:6, 20:5, and 18:0). Acclimation of striped bass from 25°C to 5°C caused an increase in FABP concentration in aerobic skeletal muscle (5°C = 1.44 ± 0.12 mg/g wet weight; warm = 1.04 ± 0.11 mg/g wet weight). The magnitude of this increase matches the predicted decrease in diffusion coefficient of FABP at 5°C. Previous studies, however, report that fatty acid oxidation is not only maintained but increased in cold-acclimated fish. We speculate that FABP may increase flux of fatty acids through β-oxidation (in cold-acclimated fish) by an increase in the percent of FABP molecules carrying fatty acid. © 1996 Wiley-Liss, Inc.
TL;DR: Partial amino acid sequencing showed that the toad liver FABP is related to that of mammalian liver and is clearly different from the amphibian heart FABp as well as from the frogs' amphibian intestine FABPs.
Abstract: The fatty acid binding protein (FABP) from toad liver cytosol was purified to homogeneity by a procedure involving gel filtration and anion exchange chromatography. The protein presented a molecula...
TL;DR: It is concluded that Arg-122 plays a role in accommodating the carboxylate group of at least one fatty acid and it is proposed that physiological ligands with more bulky headgroups, such as lysophospholipids, acyl-CoA and mono-olein, bind with the headgroups in a solvent-exposed location near the portal region of the protein.
Abstract: Rat liver fatty acid-binding protein (FABP) is able to accommodate a wide range of non-polar anions in addition to long-chain fatty acids. This property means that the liver protein is functionally different from other FABPs from intestine, muscle and adipose tissue that have a more restricted ligand specificity and stoichiometry. The availability of crystal structures for the latter proteins has highlighted the importance of two arginine residues that are involved in the binding of the fatty acid carboxylate. Only one of these arginine residues, arginine-122, is conserved in liver FABP, whereas the other arginine, at position 102, is replaced by a threonine. In order to gain further insight into the nature of ligand interactions with liver FABP these key residues (102 and 122) have been changed by site-directed mutagenesis. The results with an R122Q mutant highlight the critical role of this arginine in determining ligand affinity, while similar but less dramatic effects were observed with the T102Q mutant. The double mutant T102Q/R122Q was expressed but had lost the ability to bind fluorescent ligands. It is concluded that Arg-122 plays a role in accommodating the carboxylate group of at least one fatty acid. It is proposed that physiological ligands with more bulky headgroups, such as lysophospholipids, acyl-CoA and mono-olein, bind with the headgroups in a solvent-exposed location near the portal region of the protein. The portal region is suggested to be more flexible in the mutants (R122Q and to a lesser extent T102Q). The net result is that the ligand specificity of the R122Q mutant changes to that of a protein with enhanced affinity for acyl-CoA, lysophospholipids and mono-olein.
TL;DR: It was demonstrated that in adult mice, B-FABP is predominantly expressed in the olfactory bulb and could be purified to homogeneity by anion exchange chromatography and gel filtration in a functional conformation as they bound oleic acid with high affinity.
Abstract: A novel brain-type member of the fatty acid binding protein family (B-FABP) was heterologously expressed in Escherichia coli, either as inclusion bodies at 37 degrees C or in soluble form at 22 degrees C. Both B-FABP renatured from inclusion bodies and the solubly expressed protein could be purified to homogeneity by anion exchange chromatography and gel filtration in a functional conformation as they bound oleic acid with high affinity. None of the five cysteines of B-FABP was involved in disulphide bond formation. Isoelectric focusing revealed heterogeneity of the renatured protein but not of the solubly expressed protein. By Western blotting using affinity purified rabbit antibodies raised against the recombinant B-FABP it was demonstrated that in adult mice, B-FABP is predominantly expressed in the olfactory bulb.
TL;DR: Results herein show the presence of another two FABP in which partial amino acid sequences reveal great similarity with the corresponding sequences of other already known FABPs belonging to the heart type.
Abstract: A basic fatty acid binding protein (FABP), closely related to that of chicken liver, was isolated and characterized from catfish (Rhamdia sapo) liver in a previous work. Results herein show the presence of another two FABPs in which partial amino acid sequences reveal great similarity with the corresponding sequences of other already known FABPs belonging to the heart type. The purification procedures for both proteins involve gel filtration, anion-exchange chromatography, and sodium dodecyl sulfate – polyacrylamide gel electrophoresis (as a last step). Because both FABP N-termini were blocked, they were submitted to in-gel tryptic digestion and the resulting peptides were separated by high performance liquid chromatography, and sequenced by Edman degradation. One of these proteins presented the highest identity percentage when compared with those of the human and bovine heart and bovine brain (81%), and the other when compared with those of chicken retina (75%) and mouse and bovine heart FABP (70%). The ...