Heart FABP Research Articles

Detection and cellular localization of plasma membrane-associated and cytoplasmic fatty acid-binding proteins in human placenta

The aim of this study was to investigate location and the types of membrane-associated and cytoplasmic fatty acid-binding proteins in human placental trophoblasts using monospecific polyclonal antibodies. Western blot analysis demonstrated the presence of multiple membrane and cytoplasmic fatty acid transport/ binding proteins in human placenta. In addition to previously reported placental membrane fatty acid-binding (p-FABPPm, 40 kDa), fatty acid translocase (FAT, 88 kDa) and fatty acid transport protein (FATP, 62 kDa) were detected in both microvillous and basal membranes of the human placenta. Among the cytoplasmic proteins, heart (H) and liver (L) type FABP were detected in the cytosol of the human placental primary trophoblasts as well as in human placental choriocarcinoma (BeWo) cells. The immunoreactivity of epidermal type (E)-FABP was not detected in trophoblasts or BeWo cells despite its presence in human placental cytosol. Location of FAT and FATP on the both sides of the bipolar placental cells may favour transport of free fatty acids (FFA) pool in both directions i.e. from the mother to the fetus and vice versa. However, p-FABPPm, because of its exclusive location on the microvillous membranes, may favour the unidirectional flow of maternal plasma long-chain polyunsaturated fatty acids present in the FFA pool to the fetus, due to binding specificity for these fatty acids. Although the roles of these proteins in placental fatty acid uptake and metabolism are yet to be understood fully, their complex interaction may be involved in the uptake of maternal FFA by the placenta for delivery to the fetus.

Fatty acid binding protein, a major protein in the flight muscle of migrating Western Sandpipers

Migratory flight in birds is fueled primarily by fatty acid oxidation imposing a requirement for high rates of fatty acid: (a) transport; (b) uptake; and (c) delivery to intracellular sites of β-oxidation. Muscle fatty acid binding protein (M-FABP) is a cytosolic protein involved in the intracellular transport of fatty acids. Its expression appears to be correlated with muscle fatty acid oxidation capacity. The M-FABP was isolated for the first time from a long distance migrant bird using: (i) size exclusion; (ii) anion exchange; and (iii) hydroxyapatite chromatography. M-FABP has a molecular weight of approximately 14 000 Da and an isoelectric point of pH 4.8. A partial amino acid sequence of the protein demonstrated homology to M-FABPs from other species (80% identical to human heart FABP). It was estimated that M-FABP comprises approximately 14 and 21% of total cytosolic protein of the pectoralis and heart, respectively; the highest values yet reported from any vertebrate muscle. The abundance of M-FABP in these tissues suggests that the protein may play a key role in fatty acid supply during endurance flight. Thus, it is proposed that a seasonal increase in M-FABP expression could be a component of physiological preparation for migration.

Bound water in apo and holo bovine heart fatty-acid-binding protein determined by heteronuclear NMR spectroscopy.

Two- and three-dimensional heteronuclear NMR experiments have been performed to identify internally bound water molecules in the solution structure of bovine heart fatty-acid-binding protein (heart FABP). NOE and rotating-frame NOE (ROE) cross peaks between protein protons and protons of bound water molecules were observed in two-dimensional H2O-ROE/NOE-1H,15N-heteronuclear single quantum coherence spectra recorded from a uniformly 13C/15N-enriched sample of bovine heart FABP. Contacts between water protons and 23 NH protons of the protein backbone were identified. The protein structure consists of 10 antiparallel beta-strands (betaA-betaJ), forming two nearly orthogonal beta-sheets, and a short helix-turn-helix motif connecting beta-strands A and B. The spatial folding resembles a beta-barrel. Most of the water molecules are localized in the gap between beta-strands D and E, and near the two alpha-helices. In the delipidated heart FABP additional contacts between water molecules and NH protons could be observed using a three-dimensional rotating frame Overhauser 1H,15N heteronuclear single quantum coherence experiment obtained with a 15N-labeled sample of apo-heart FABP.

Identification of a human heart FABP pseudogene located on chromosome 13

The fatty acid-binding proteins (FABPs) constitute a conserved group of cytosolic low molecular mass proteins, which consists of several types: liver, heart, myelin, epidermal, adipocyte, brain, intestinal and ileal type. The FABP gene structure is well conserved during evolution and exhibits a four-exon/three-intron structure. In the past, multiple hybridizing fragments were detected upon Southern blot analysis using heart FABP (H-FABP) cDNA as a probe. The origin of these fragments was not clear. We screened a human genomic library and isolated an intronless gene ( FABP3-ps) with 85% similarity to the human H-FABP cDNA and high similarity (76 and 79%) to the H-FABP cDNAs of mouse and bovine, respectively. By means of fluorescence in situ hybridization this processed pseudogene could be assigned chromosome 13q13-q14, whereas the gene for human H-FABP ( FABP3) resides on chromosome 1p32-p33. No expression of the processed pseudogene could be detected in skeletal muscle or fetal brain.

Human intestinal fatty acid binding protein: Report of an assay with studies in normal volunteers and intestinal ischemia

Background. Human intestinal fatty acid binding protein (hIFABP) is a cytoplasmic protein of mature small intestinal epithelium. Work with the rat demonstrated that serum levels of IFABP correlated with early phases of intestinal mucosal injury. The aim of this study was to develop an assay for hIFABP and assess its usefulness as a marker for intestinal mucosal injury in human beings. Methods. Recombinant hIFABP (r-hIFABP) was used to produce rabbit anti-hIFABP. Specificity and avidity of binding were tested with immunoprecipitation and Scatchard analysis. r-hIFABP was labeled with 125I, and a competitive assay was developed. Urine and serum from normal volunteers and from patients with necrotizing enterocolitis (NEC), acute thromboembolic related intestinal ischemia, and systemic inflammatory response syndrome were tested for hIFABP. Results. Molecular weight was 10 -12 kd, limit of detection was 1.87 ng/ml, and no cross-reactivity occurred when tested against rat IFABP or human heart FABP. Mean levels of hIFABP (ng/ml) were controls (serum less than 1.87, urine less than 1.87), NEC (serum 14.7 ng/ml), intestinal ischemia (serum 50 ng/ml, urine 52.3 ng/ml), systemic inflammatory response syndrome (serum 5.3 ng/ml, urine 13.2 ng/ml). Conclusions. This assay is quantitative for hIFABP in serum and urine. Results from both normal persons and those with various causes of intestinal ischemia parallel our previous findings in the rat. Preliminary findings suggest that hIFABP may serve as a diagnostic marker for early intestinal mucosal compromise and, in addition, that it should prove useful as a tool in developing rationale therapeutic regimens to treat these complex clinical problems.

Regulation of gene expression in adipose cells by polyunsaturated fatty acids.

In fat cells polyunsaturated fatty acids are both substrates for, and products of, triacylglycerol metabolism. Dietary fatty acids are efficiently incorporated into the triacylglycerol droplet under lipogenic conditions while rapidly mobilizing them during lipolytic stimulation. Hence, the flux and magnitude of the fatty acid pool in adipocytes is constantly changing in response to hormonal, metabolic and genetic determinants. Due to the rapidly changing flux of fatty acids, the majority of genes encoding enzymes and proteins of lipid metabolism are largely refractory to long-term regulatory control by fatty acids. Only at extremes of high or low lipid levels, or under pathophysiological conditions, do adipose genes respond by up- or down-regulating gene expression. Despite the lack of responsiveness to lipids in adipose tissue, a surprisingly large number of genes have been characterized recently as lipid responsive when assayed in heterologous systems. These observations suggest an endogenous negative element exists in the lipid signaling pathway in adipocytes. The major intracellular lipid binding protein in adipose cells is the adipocyte lipid binding protein (ALBP), the product of the aP2 gene. This protein is 15 kDa, abundant and found exclusively in the cytoplasm of adipocytes. The protein binds fatty acids and related lipids in a 1:1 stoichiometry within a large water filled interior cavity. The lipid binding protein forms high affinity associations with polyunsaturated fatty acids such as arachidonic acid (Kd approximately 250 nM) but not with prostaglandins of the E, D or J series (Kd > 4 microM). The upstream region of the aP2 gene contains a peroxisome-proliferator activated receptor response element which associates with PPARs to regulate its expression. A positive autoregulatory circuit exists to upregulate lipid binding protein expression when polyunsaturated fatty acid levels are increased. Analysis of adipose tissue from aP2 null animals generated by a targeted disruption revealed that the partial loss of ALBP expression in heterozygotes and complete lack of ALBP in the nulls was accompanied by a compensatory up-regulation of the keratinocyte lipid binding protein. However, the total amount of lipid binding protein in the nulls was less than 15% that in the wild type littermates. No evidence was found for upregulation of other lipid binding proteins such as the heart FABP or liver FABP. In aP2 nulls, the fatty acid composition was unaltered but the mass of fatty acid per gram tissue more than doubled relative to wild type. In heterozygotes, the level of fatty acid was intermediate to that of wild-type and nulls, consistent with an intermediate level of lipid binding protein. These results indicate that the fatty acid pool level in adipocytes is inversely correlated with the amount of lipid binding protein. Since prostaglandin biosynthesis is dependent upon polyunsaturated fatty acid substrates, the intracellular lipid binding proteins control accessibility of substrates of the prostanoid pathway. Intracellular lipid binding proteins therefore are negative elements in polyunsaturated fatty acid control of gene expression.

The NMR Solution Structure of Intestinal Fatty Acid-binding Protein Complexed with Palmitate: Application of a Novel Distance Geometry Algorithm

The three-dimensional solution structure of rat intestinal fatty acid-binding protein (I-FABP) complexed with palmitate has been determined using multidimensional triple-resonance NMR methods. The structure is based on 3889 conformational restraints derived mostly from 3-D 13C- and 15N-resolved nuclear Overhauser (NOESY) experiments. The 3-D NOESY data for this 15.4 kDa complex contained an average of nine possible interpretations per cross-peak. To circumvent this ambiguity, an eight-stage iterative procedure was employed to gradually interpret and introduce unambiguous distance restraints during subsequent rounds of structure calculations. The first stage of this procedure relied critically upon an initial structural model based on the consensus 1H/ 13C chemical shift-derived secondary structure and a set of symmetry-checked restraints derived from the 3-D 13C-resolved NOESY spectrum. The structures were calculated using DISTGEOM, a program that implements a novel distance geometry algorithm with pairwise Gaussian metrization. A central feature of this algorithm is the use of an iteratively optimized Gaussian distribution for the selection of trial distances, which overcomes the tendency of metrization to produce crushed structures. In addition, this algorithm randomly selects pairwise elements of the distance matrix, which results in an improved sampling of conformational space for a given computational effort. The final family of 20 distance geometry/simulated annealing structures exhibited an average pairwise C αroot-mean-square deviation of 0.98 Å, and their stereochemical quality, as assessed by PROCHECK, was comparable to that of 2.5 Å X-ray crystal structures. The NMR structure was compared with the X-ray crystal structure of the same ligand/protein complex and was found to be essentially identical within the precision of the results. The NMR structure was also compared with that of the palmitate complex with bovine heart FABP, which shares 30% sequence identity with rat I-FABP. The overall folds were the same, but differences were noted with respect to the presence or absence of apparent conformational heterogeneity and the location and conformation of the bound fatty acid.

Flexibility is a likely determinant of binding specificity in the case of ileal lipid binding protein

Background: The family of lipid binding proteins (LBPs) includes a large number of fatty acid binding proteins (FABPs) but only two proteins (ileal lipid binding protein, ILBP, and liver fatty acid binding protein) that can bind both fatty acids and bile acids. Bile acid transport is medically and pharmacologically important, but is poorly understood. To understand the binding properties of ILBP, we studied its solution structure with and without bound lipids and compared these with known structures of FABPs. Results The sequence-specific 1H resonance assignments for porcine ILBP have been determined by homonuclear two-dimensional (2D) NMR spectroscopy for the apo-protein as well as for ILBP complexes with fatty acid and bile acid ligands. From NOE spectra and hydrogen exchange data, similar secondary structure elements were identified for all three protein forms. ILBP is composed of ten antiparallel β strands arranged in two nearly orthogonal β sheets (a fold seen in other FABPs, and dubbed the ‘ β-clam shell’), covered on one side by two short, nearly parallel α helices. Binding of fatty acids or bile acids to ILBP alters mainly the side-chain proton resonances of amino acids within the protein cavity, indicating that both bile acids and fatty acids can bind in the interior of the protein between the two β sheets; binding of bile acids stabilizes the protein backbone by a small amount. Fast hydrogen exchange rates for the backbone amide protons of ILBP indicate that the hydrogen-bonding network of the β sheet in ILBP is weaker than the corresponding network in rat intestinal and bovine heart FABPs. Conclusion 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. It is likely that this flexibility is important in allowing bile acids, which are larger and more rigid than fatty acids, to enter the central cavity of the protein.

Development and characterization of antibodies to human heart fatty acid binding proteins

Development and characterization of antibodies to human heart fatty acid binding proteins

Association between breast cancer patient survival and levels of creatine kinase BB in tumor cytosols

Fatty acid-binding proteins (FABPs) are abundant low-molecular-weight cytosolic proteins in tissues involved in fatty acid (FA) metabolism. Because epidermis is also an active lipogenic tissue, we examined cytosols from murine and porcine epidermis and cultured human keratinocytes and fibroblasts for FABPs. High-affinity FA-binding activity was present in both epidermis and differentiated keratinocytes, whereas no high-affinity FA-binding activity was found in cultured human fibroblasts or undifferentiated keratinocytes. By column chromatography, a single binding peak was identified in the high (90–100 kDa)-molecular-weight range and no binding activity was evident in the low (14–15 kDa)-molecular-weight range, where conventional FABPs elute. Moreover, rabbit anti-rat heart FABP, anti-rat intestine FABP, and anti-rat liver FABP antisera did not identify proteins in the 14–15-kDa range in murine epidermal cytosol by Western immunoblots, whereas the anti-rat-heart antibody recognized a protein of approximately 32 kDa. Isoelectric focusing of differentiated keratinocyte cytosol demonstrated a single FA-binding peak having a pI of ≈ 4.0. Analysis of this binding peak by SDS-PAGE revealed peptides of approximately 66 and 38 kDa. These findings suggest the possibility that the FA-binding protein in keratinocyte cytosol normally exists as a heterodimer. Western immunoblots of both differentiated keratinocyte cytosol and keratinocyte-conditional media stained with a rabbit anti-human serum albumin antibody identified a protein of approximately 67 kDa, but the electrofocused fraction did not react with this antibody. Thus, epidermis and differentiated keratinocytes possess high-affinity cytosolic FA-binding activity that cannot be ascribed either to conventional low-molecular-weight FABPs or to albumin.

Prostate specific antigen gene expression in ovarian cancer post-liver transplantation

Fatty acid-binding proteins (FABPs) are abundant low-molecular-weight cytosolic proteins in tissues involved in fatty acid (FA) metabolism. Because epidermis is also an active lipogenic tissue, we examined cytosols from murine and porcine epidermis and cultured human keratinocytes and fibroblasts for FABPs. High-affinity FA-binding activity was present in both epidermis and differentiated keratinocytes, whereas no high-affinity FA-binding activity was found in cultured human fibroblasts or undifferentiated keratinocytes. By column chromatography, a single binding peak was identified in the high (90–100 kDa)-molecular-weight range and no binding activity was evident in the low (14–15 kDa)-molecular-weight range, where conventional FABPs elute. Moreover, rabbit anti-rat heart FABP, anti-rat intestine FABP, and anti-rat liver FABP antisera did not identify proteins in the 14–15-kDa range in murine epidermal cytosol by Western immunoblots, whereas the anti-rat-heart antibody recognized a protein of approximately 32 kDa. Isoelectric focusing of differentiated keratinocyte cytosol demonstrated a single FA-binding peak having a pI of ≈ 4.0. Analysis of this binding peak by SDS-PAGE revealed peptides of approximately 66 and 38 kDa. These findings suggest the possibility that the FA-binding protein in keratinocyte cytosol normally exists as a heterodimer. Western immunoblots of both differentiated keratinocyte cytosol and keratinocyte-conditional media stained with a rabbit anti-human serum albumin antibody identified a protein of approximately 67 kDa, but the electrofocused fraction did not react with this antibody. Thus, epidermis and differentiated keratinocytes possess high-affinity cytosolic FA-binding activity that cannot be ascribed either to conventional low-molecular-weight FABPs or to albumin.

Immunohistochemical localization of two types of fatty acid-binding proteins in rat ovaries during postnatal development and in immature rat ovaries treated with gonadotropins.

The ovary of adult rats expresses two types of cytoplasmic fatty acid binding proteins (FABP), i.e., heart FABP (H-FABP) and intestinal 15 kDa protein (I-15P). We studied immunohistochemically the cellular localizations of these FABPs in the ovaries of rats at various postnatal ages and in the ovaries of immature (3-week-old) rats treated with pregnant mare serum gonadotropin (PMSG) and human chorionic gonadotropin (HCG). The cryosections of ovaries were incubated with polyclonal antibodies against H-FABP and I-15P, and the immunoreactions were visualized at both light and electron microscopic levels. The immunoreactivity for H-FABP occurred temporarily in the follicular epithelial (granulosa) cells from 3 days to 2 weeks post partum, and then was localized exclusively to the theca/interstitial gland cells from 2 weeks to adulthood. In contrast, the immunoreactivity for I-15P appeared temporarily in a small subset of theca/interstitial gland cells from 2 to 3 weeks, disappeared at 4 weeks, and was localized exclusively to the corpus luteum cells after the onset of ovulation in the animal around 5 weeks. In the immature rat ovaries induced to ovulate by treatment with gonadotropins, I-15P-immunoreactive cells were first recognized in the luteinized granulosa layer of large preovulatory follicles, and increased in number progressively in the developing corpora lutea after the ovulation. Two types of FABPs are expressed in distinct steroid-producing cell types of rat ovary, and their expressions seem to be regulated in coincidence with the expressions of respective steroid hormones. These results suggest that FABPs play specific roles in the ovarian hormone synthesis.

Regulation of fluorescent fatty acid transfer from adipocyte and heart fatty acid binding proteins by acceptor membrane lipid composition and structure.

Adipocyte and heart fatty acid binding proteins (A-FABP and H-FABP) are closely related members of the FABP family. Unlike the more distantly related liver FABP, these FABP have been proposed to transfer free fatty acids to model membranes by a collisional mechanism (Wootan, M. G., Bernlohr, D. A., and Storch, J. (1993) Biochemistry 32, 8622-8627; Kim, H. K., and Storch, J. (1992) J. Biol. Chem. 267, 20051-20056). Collisional transfer requires that the acceptor membranes interact with FABP during the transfer process. We, therefore, examined whether the acceptor membrane structure and lipid composition regulate the rate of anthroyloxy-labeled palmitate (2AP) transfer from A- and H-FABP, using a fluorescence resonance energy transfer assay. The results showed that 2AP transfer from A- and H-FABP was more rapid to acceptor vesicles containing acidic phospholipids and was slower to positively charged membranes. In addition, the rate of 2AP transfer from A- and H-FABP was enhanced by unsaturation of the phosphatidylcholine acyl chains and was slowed by the presence of cholesterol or sphingomyelin in the acceptor membranes. These latter changes were small but of a similar magnitude and together suggest that fatty acid transfer from A- and H-FABP was slower to membranes of greater lipid order. Since transfer by an aqueous diffusion mechanism would be unaffected by acceptor membrane properties, these studies strengthen the hypothesis that free fatty acid transfer from A- and H-FABP to membranes occurs via a collisional mechanism.

Purification, characterization and partial sequencing of the heart fatty acid-binding protein from Bufo arenarum

A 15.7 kDa fatty acid-binding protein from toad heart was purified by gel-filtration chromatography on Sephadex G-75 followed by anion-exchange chromatography on a Mono-Q column. Purity was confirmed by gel electrophoresis and isoelectric focusing. Molecular mass, isoelectric point, amino acid composition and partial internal sequence showed that the protein is related to mammalian heart fatty acid-binding proteins.

Molecular Cloning of a cDNA Encoding a Novel Fatty Acid-Binding Protein from Rat Skin

A novel skin-type fatty acid-binding protein, termed cutaneous(C)-FABP, has been purified from rat skin and a cDNA clone for this protein has been identified. The purified protein had the ability to bind long chain fatty acids like other rat FABPs. The deduced amino acid sequence of the cDNA clone comprises residues yielding a molecular mass for the polypeptide of 15.1 kDa and exhibits around 50% identity to myelin P2 protein, adipocyte FABP and heart FABP. Our results propose that C-FABP is a new member of the FABP family.

Fatty acid-binding protein from rat heart is phosphorylated on Tyr19 in response to insulin stimulation

The cytosolic fatty acid-binding protein from rat heart (H-FABP, M(r) 15,000) as well as FABP from mouse adipocytes (A-FABP, 62% homologous with H-FABP) contain a recognition sequence for protein tyrosine kinases, Asn-Phe-Asp-Asp-Tyr19. A-FABP has been shown by others to be partly phosphorylated on Tyr19, thus encouraging experiments designed to search for phosphotyrosine in H-FABP. For this purpose isolated cardiac myocytes were incubated with [32P]orthophosphate and analyzed by two-dimensional gel electrophoresis. A 15 kDa phosphoprotein present in the cytosolic protein fraction was specifically precipitated by a polyclonal antibody against rat heart FABP. Characterization of the phosphorylated FABP was facilitated by the development of an immunoaffinity purification procedure capable of isolating more than 200 micrograms FABP from four rat hearts in one step. Phosphoamino acid analysis and radiosequencing of the major tryptic phosphopeptide from immunopurified FABP revealed Tyr19 as the phosphorylated amino acid. Stimulation of cardiac myocytes with insulin in the presence of tyrosine phosphatase inhibitors led to a several-fold increase in the amount of phosphorylated FABP compared with a nearly undetectable level found without insulin stimulation, indicating that FABP may be a substrate for the insulin receptor tyrosine kinase. Phosphorylated FABP constitutes only a minor fraction compared to the large pool of FABP in the cardiac myocyte, thus obscuring the significance of this modification. However, as the phosphorylated Tyr19 residue is positioned within a helix-turn-helix-related domain of FABP, this modification may modulate a hitherto unknown DNA binding activity of FABP. A hypothesis is discussed in which phosphorylated FABP serves as a signalling molecule in the insulin signal transduction cascade.

Expression of Fatty Acid-Binding Proteins in the Developing Mouse Mammary-Gland

Proteins of 14,000 daltons (SLP-14) were isolated and partially characterized from mouse mammary glands of different developmental stages. The purified proteins were partially sequenced at the amino acid level. The SLP-14 belong to the family of fatty acid-binding (FABP) proteins. The major SLP-l4 expressed was a reflection of the differentiation stage of the mammary gland. Within the regions sequenced for each protein, virgin mouse mammary gland (primarily adipocytes) expressed a protein 100% homologous to adipocyte lipid binding protein whereas pregnant and lactating mouse mammary glands expressed a protein highly homologous to mouse heart fatty acid binding protein. In the lactating gland, the isolated protein was 97% identical to the heart FABP over a 98 amino acid stretch. We could not detect the 14Kd protein identified as mammary gland growth inhibitor (MDGI) which is also a member of the FABP family and highly homologous to heart FABP. A rabbit antiserum to the rat mammary SLP-14 recognized the SLP-14 proteins in mouse and rat mammary gland, skeletal muscle and heart, whereas it failed to recognize the SLP-14 in liver, intestine and other organs. These data indicate that the SLP-14 detected in rodent mammary gland is of the fatty acid-binding protein family. On the basis of amino acid sequence, the major form in the differentiated mouse mammary gland is apparently FABP and not MDGI. MDGI may he a protein in tow abundance and/or localized to a specific group of mammary epithelial cells.

Diversity of fatty acid-binding protein structure and function: studies with fluorescent ligands.

The mammalian fatty acid-binding proteins (FABP) are localized in many distinct cell types. They bind long chain fatty acids in vitro, however, their functions and mechanisms of action in vivo remain unknown. The present studies have sought to understand the relationships among these proteins, and to address the possible role of FABP in cellular fatty acid traffic. A series of anthroyloxy-labeled fluorescent fatty acids have been used to examine the physicochemical properties of the fatty acid-binding sites of different members of the FABP family. The fatty acid probes have also been used to study the rate and mechanism of fatty acid transfer from different FABP types to phospholipid membranes. The results of these studies show a number of interesting and potentially important differences between FABP family members. An examination of adipocyte and heart FABP (A- and H-FABP) shows that their fatty acid-binding sites are less hydrophobic than the liver FABP (L-FABP) site, and that the bound ligand experiences less motional constraint within the A- and H-FABP binding sites than within the L-FABP binding site. In keeping with these differences in structural properties, it was found that anthroyloxy-fatty acid transfer from A- and H-FABP to membranes is markedly faster than from L-FABP. Moreover, the mechanism of fatty acid transfer was found to be similar for the highly homologous A- and H-FABP, whereby transfer to phospholipid membranes appears to occur via transient collisional interactions between the FABP and membranes.(ABSTRACT TRUNCATED AT 250 WORDS)

High-Affinity Fatty Acid-Binding Activity in Epidermis and Cultured Keratinocytes Is Attributable to High-Molecular-Weight and Not Low-Molecular-Weight Fatty Acid-Binding Proteins

Fatty acid-binding proteins (FABPs) are abundant low-molecular-weight cytosolic proteins in tissues involved in fatty acid (FA) metabolism. Because epidermis is also an active lipogenic tissue, we examined cytosols from murine and porcine epidermis and cultured human keratinocytes and fibroblasts for FABPs. High-affinity FA-binding activity was present in both epidermis and differentiated keratinocytes, whereas no high-affinity FA-binding activity was found in cultured human fibroblasts or undifferentiated keratinocytes. By column chromatography, a single binding peak was identified in the high (90-100 kDa)-molecular-weight range and no binding activity was evident in the low (14-15 kDa)-molecular-weight range, where conventional FABPs elute. Moreover, rabbit anti-rat heart FABP, anti-rat intestine FABP, and anti-rat liver FABP antisera did not identify proteins in the 14-15-kDa range in murine epidermal cytosol by Western immunoblots, whereas the anti-rat-heart antibody recognized a protein of approximately 32 kDa. Isoelectric focusing of differentiated keratinocyte cytosol demonstrated a single FA-binding peak having a pI of approximately 4.0. Analysis of this binding peak by SDS-PAGE revealed peptides of approximately 66 and 38 kDa. These findings suggest the possibility that the FA-binding protein in keratinocyte cytosol normally exists as a heterodimer. Western immunoblots of both differentiated keratinocyte cytosol and keratinocyte-conditional media stained with a rabbit anti-human serum albumin antibody identified a protein of approximately 67 kDa, but the electrofocused fraction did not react with this antibody. Thus, epidermis and differentiated keratinocytes possess high-affinity cytosolic FA-binding activity that cannot be ascribed either to conventional low-molecular-weight FABPs or to albumin.

Mechanism of free fatty acid transfer from rat heart fatty acid-binding protein to phospholipid membranes. Evidence for a collisional process.

Fatty acid binding proteins (FABP) are a family of low molecular weight proteins found in many tissues that actively utilize free fatty acids (ffa). FABP would be expected to have a particularly important role in the heart, where over 80% of energy requirements are derived from oxidation of long chain fatty acids. The precise physiological function of heart FABP (H-FABP) has not been definitively identified, although it is thought to play a role in intracellular ffa transport. To examine the possible role of H-FABP in cardiac myocyte transfer of ffa, we examined the transfer of fluorescent anthroyloxy ffa (AOffa) from H-FABP to model phospholipid membranes, using a resonance energy transfer assay. In contrast to previous observations of ffa transfer from liver FABP and from membranes, transfer from H-FABP to membranes appears to occur by a different mechanism. AO-palmitate (16:0) transfer was 1.5-fold slower than AO-stearate (18:0) transfer, and mono-unsaturation did not affect the transfer rate. The AOffa transfer rate from H-FABP increased with increasing ionic strength and decreased slightly between pH 7 and 9. These results suggest that the rate of ffa transfer from H-FABP to membranes is independent of the ffa aqueous solubility. Thermodynamic analysis showed that the free energy of activation for the ffa transfer process arises primarily from an enthalpic component, with only a small entropic contribution, again suggesting the lack of an aqueous phase route of ffa delivery. Finally, the ffa transfer rate was found to be directly dependent on the concentration of acceptor membranes. These data therefore suggest that transfer of AOffa from H-FABP to membranes may occur via collisional interactions between the protein and membranes.