Primary structure and cellular distribution of two fatty acid-binding proteins in adult rat kidneys

Fatty acid-binding proteins (FABPs) were purified from the kidneys of female and male rats and characterized by primary structure and histological distribution in the kidney. Two FABPs (14 and 15.5 kDa) were found in male rat kidney cytosol whereas only 14-kDa FABP could be recognized in female rat kidneys throughout the purification steps. The amino acid sequence of the 14-kDa FABP was identical to that of rat heart FABP deduced from the cDNA sequence (Heuckeroth, R. O., Birkenmeier, E. H., Levin, M. S., and Gordon, J. I. (1987) J. Biol. Chem. 262, 9709-9717). Structural analysis of the male-specific 15.5-kDa FABP identified this second FABP as a proteolytically modified form of alpha 2u-globulin, an 18.7-kDa major urinary protein of adult male rats (Unterman, R. D., Lynch, K. R., Nakhasi, H. L., dolan, K. P., Hamilton, J. W., Cohn, D. V., and Feigelson, P. (1981) Proc. Natl. Acad. Sci. U.S.A. 78, 3478-3482) which shares a common ancestry with a number of hydrophobic ligand-binding proteins such as serum retinol-binding proteins. Immunohistochemical investigation disclosed that heart-type FABP (14-kDa FABP) is localized in the cytoplasm of the epithelia of the distal tubules in both male and female rat kidneys whereas 15.5-kDa FABP immunostaining was observed predominantly in the endosomes or lysosomes of proximal tubules in male rat kidneys. These results suggest strongly the functional divergence of two FABPs in the rat kidney.     

Characterization and binding properties of fatty acid-binding proteins from human, pig, and rat heart

Fatty acid-binding proteins (FABPs) were isolated from the cytosols of hearts of man, pig, and rat by gel filtration and anion-exchange chromatography. The heart FABPs had a Mr of about 15,000 (pig, rat) and 15,500 (man); pI values were 5.2, 4.9, and 5.0 for human, pig, and rat heart, respectively. In contrast to liver FABPs, tryptophan was present in the heart FABPs. Binding characteristics for long-chain fatty acids determined with the radiochemical Lipidex assay were comparable for all three proteins. Heart FABPs also bind palmitoyl-CoA and -carnitine with an affinity comparable to that for palmitic acid. Other ligands investigated, heme, bilirubin, cholesterol, retinoids, and prostaglandins, could not compete with oleic acid for binding by human heart FABP. Binding parameters of FABP for oleic acid from multilamellar liposomes were comparable to those from the Lipidex binding assay. Immunological interspecies cross-reactivity with antisera against the heart FABPs was much higher between man and pig than between rat and man or pig. None of the antisera reacted with liver FABPs. The IgG fraction of anti-human heart FABP serum inhibited fatty acid binding to human heart FABP.     

Characterization and binding properties of human fetal lung fatty acid-binding proteins

When delipidated Mr>10,000 cut-off human fetal lung cytosol was separated on gel filtration and ion-exchange chromatography on Auto-FPLC system, two fatty acid-binding proteins (FABPs) of pI 6.9 and pI 5.4 were purified to homogeneity. On Western blotting analysis with the anti-human fetal lung pI 6.9 FABP, these two proteins showed immunochemical cross reactivity with each other and with purified hepatic FABPs but not with cardiac or gut FABP. These two FABPs have identical molecular mass of 15.2 kDa, which is slightly higher than that of the hepatic proteins (14.2 kDa). Carbohydrate covalently linked to FABPs, that may substantially add to the molecular mass, was not detected in the purified protein preparations. Amino acid analysis revealed that both the proteins have same amino acid composition each containing one Trp residue that is lacking in hepatic FABP. Different isoforms of lung FABP exhibited different binding ability for their natural ligands. These proteins bind palmitoyl CoA with higher affinity than oleic acid. pI 6.9 FABP can more rapidly and efficiently transfer fatty acid than can pI 5.4 FABP from unilammelar liposomes. Thus these FABPs may play a critical role in fatty acid transport during human fetal lung development.Abbreviations AO anthroyloxy - 12-AS 12-(9-anthroyloxy)stearic acid - FABP fatty acid-binding protein - NBD-PE [N-(4-nitrobenzo-2-oxa-1,3-diazole)phosphatidylethanolamine - Pal-CoA palmitoyl coenzyme A - PITC phenylisothiocyanate - PBS phosphate-buffered saline - PtdCho phosphatidylcholine - SUV small unilamellar vesicle - Tris tris(hydroxymethyl) amino methane     

The binding affinity of fatty acid-binding proteins from human, pig and rat liver for different fluorescent fatty acids and other ligands

1. Two forms of fatty acid-binding proteins (FABPs) were isolated from human, pig and rat liver cytosols by gelfiltration and anion-exchange chromatography. 2. Both forms did not show physicochemical or chemical differences. They had an Mr of about 14.5 kDa for all species. pI Values were 5.8 for both forms of human and pig liver FABP and 6.4 for both forms of rat liver FABP. In contrast to heart FABPs no tryptophan was present in liver FABPs. 3. Liver FABPs show a much higher enhancement of fluorescence at binding of 11-dansylaminoundecanoic acid, 16-anthroyloxy-palmitic acid and 1-pyrene-dodecanoic acid than heart FABPs and additionally a blue shift in excitation and emission wavelengths with the first fatty acid. 4. The bulky side-chain did not affect fatty acid binding since binding constants of liver FABPs were comparable for these fluorescent fatty acids and oleic acid (0.3-0.7 microM). 5. A 1:1 binding stoichiometry was obtained for oleic acid binding with heart and liver FABPs. 6. Liver FABPs have a high binding affinity for C16-C22 saturated and unsaturated fatty acids, palmitoyl-CoA, bromo-substituted fatty acids, POCA, tetradecylglycidic acid and flavaspidic acid. 7. Fatty acid binding could be reduced to less than 50% by arginine modification with 2,3-butadione or by enzymatic degradation of FABPs with trypsin or pronase.     

Bovine fatty acid binding proteins. Isolation and characterisation of two cardiac fatty acid binding proteins that are distinct from corresponding hepatic proteins

When a 100,000 X g supernatant from bovine heart was incubated with [1-14C]oleic acid and subjected to isoelectric focusing, two fatty acid binding proteins (FABPs) with isoelectric points at 4.9 and 5.1 were detected. The proteins were purified on a large scale first by heat and acid precipitation of a postmitochondrial supernatant, as well as fractionation with ammonium sulfate, then by alternate application of ion-exchange and gel chromatography. The procedure afforded around 60 mg pure proteins from 1.5 kg fresh heart muscle. Relative molecular masses of 15 300 +/- 1600 for both proteins were derived from sodium dodecyl sulfate/polyacrylamide gel electrophoresis, gel chromatography, sedimentation velocity as well as from amino acid analysis. Up to 50% of the proteins' secondary structures consisted of beta-sheet. N-termini of the peptide chains were blocked; the amino acid compositions of the two proteins were similar, but differed considerably from those of the two FABPs isolated from bovine liver [Haunerland et al. (1984) Hoppe Seyler's Z. Physiol. Chem. 365, 365-376]. Whereas hepatic FABPs changed their pI upon binding fatty acids, cardiac FABPs did not. Cardiac FABPs were immunologically identical, but did not cross-react with hepatic proteins. A reversible, concentration-dependent self-association reported for FABP from pig heart [Fournier et al. (1983) Biochemistry 22, 1863-1872] was not observed for FABP from bovine heart. Changes of concentration did not alter secondary structure, intrinsic fluorescence or the sedimentation coefficient of the protein.     

Amino acid sequence and some ligand binding properties of fatty acid-binding protein from bovine brain

Summary A fatty acid-binding protein (FABP) from the cytosol of bovine brain was purified by Sephadex G-75 filtration and electrofocusing. The purified protein migrated as a single protein band in 15% polyacrylamide gel electrophoresis with an apparent molecular mass of 14.7 kDa. To ascertain that the purified protein was a FABP, it was submitted to fatty acid-binding tests. Oleic and palmitic acids bound to brain FABP but this was not the case for palmitoyl CoA. By Scatchard analysis the ligand binding values were: Kd = 0.28 µM, Bmax (mol/mol) = 0.6 for oleic acid and Kd = 0.8 µM, Bmax (mol/mol) = 2.1 for palmitic acid. The complete amino acid sequence of the brain FABP was determined and a microheterogeneity was observed. Sequence comparison with other FABPs of known sequence and the observed microheterogeneity demonstrated the presence in brain of several homologous FABPs closely related to heart FABP.This paper corresponds to a communication at the first international workshop on fatty acid binding proteins (Maastricht, the Netherlands, September 4–5, 1989).     

Fatty acid binding proteins from different tissues show distinct patterns of fatty acid interactions

Fatty acid binding proteins (FABP) form a family of proteins displaying tissue-specific expression. These proteins are involved in fatty acid (FA) transport and metabolism by mechanisms that also appear to be tissue-specific. Cellular retinoid binding proteins are related proteins with unknown roles in FA transport and metabolism. To better understand the origin of these tissue-specific differences we report new measurements, using the acrylodated intestinal fatty acid binding protein (ADIFAB) method, of the binding of fatty acids (FA) to human fatty acid binding proteins (FABP) from brain, heart, intestine, liver, and myelin. We also measured binding of FA to a retinoic acid (CRABP-I) and a retinol (CRBP-II) binding protein and we have extended to 19 different FA our characterization of the FA-ADIFAB and FA-rat intestinal FABP interactions. These studies extend our previous analyses of human FABP from adipocyte and rat FABPs from heart, intestine, and liver. Binding affinities varied according to the order brain approximately myelin approximately heart > liver > intestine > CRABP > CRBP. In contrast to previous studies, no protein revealed a high degree of selectivity for particular FA. The results indicate that FA solubility (hydrophobicity) plays a major role in governing binding affinities; affinities tend to increase with increasing hydrophobicity (decreasing solubility) of the FA. However, our results also reveal that, with the exception of the intestinal protein, FABPs exhibit an additional attractive interaction for unsaturated FA that partially compensates for their trend toward lower affinities due to their higher aqueous solubilities. Thermodynamic potentials were determined for oleate and arachidonate binding to a subset of the FABP and retinoid binding proteins. FA binding to all FABPs was enthalpically driven. The DeltaH degrees values for paralogous FABPs, proteins from the same species but different tissues, reveal an exceptionally wide range of values, from -22 kcal/mol (myelin) to -7 kcal/mol (adipocyte). For orthologous FABPs from the same tissue but different species, DeltaH degrees values were similar. In contrast to the enthalpic dominance of FA binding to FABP, binding of FA to CRABP-I was entropically driven. This is consistent with the notion that FA specificity for FABP is determined by the enthalpy of binding. Proteins from different tissues also revealed considerable heterogeneity in heat capacity changes upon FA binding, DeltaC(p) values ranged between 0 and -1.3 kcal mol(-1) K(-1). The results demonstrate that thermodynamic parameters are quite different for paralogous but are quite similar for orthologous FABP, suggesting tissue-specific differences in FABP function that may be conserved across species.     

Analyzing the structures, functions and evolution of two abundant gastrointestinal fatty acid binding proteins with recombinant DNA and computational techniques

The structures of intestinal and liver fatty acid binding proteins (FABPs) have been determined from an analysis of the nucleotide sequences of cloned cDNAs. The primary translation product of intestinal FABP mRNA contains 132 residues (Mr = 15 124). Liver FABP mRNA encodes a 127 amino acid polypeptide (Mr = 14 273). In vitro co-translational cleavage and translocation assays showed that neither sequence has a cleavable signal peptide or signal peptide equivalent - suggesting that the FABPs do not enter the secretory apparatus but rather are targeted to the cytoplasm. A variety of computational techniques were used to compare the two FABP sequences. The results indicate that liver and intestinal FABP are paralogous homologues. A superfamily of proteins was defined which includes the FABPs, the cellular retinol and retinoic acid binding proteins, the P2 protein of peripheral nerve myelin, and a polypeptide known as 422 whose synthesis is induced during differentiation of 3T3-L1 cells to adipocytes. No sequence homologies were noted between any of these small molecular weight cytosolic proteins and nonspecific lipid transfer protein (sterol carrier protein 2), phosphatidylcholine transfer protein, serum albumin or apolipoprotein AI. The FABPs may have structural features responsible for lipid-protein interactions that are not present in these non-homologous sequences. The distribution of intestinal and liver FABP mRNAs in adult rat tissues and the changes in FABP gene expression which occur during gastrointestinal development support the notion that these proteins are involved in fatty acid uptake, transport and/or compartmentalization. However, differences in tissue distribution and periods of non-coordinate expression during gastrointestinal ontogeny suggest that the two FABPs have distinct functions. The relationship between intestinal and liver FABPs and similar sized cytosolic FABPs isolated from brain, skeletal and cardiac muscle remains unclear. Recombinant DNA techniques combined with comparative sequence analyses offer a useful approach for defining unique as well as general structure-function relationships in this group of fatty acid binding proteins.     

The fatty acid-binding protein from human skeletal muscle

Fatty acid-binding protein (FABP) was isolated from human skeletal muscle by gel filtration and anion- and cation-exchange chromatography. The isolation procedure, however, with rat and pig skeletal muscle gave mostly inactive preparations. Rat muscle FABP preparations contained parvalbumin as a contaminant. FABP from human muscle had a Mr of about 15 kDa, a pI value of 5.2, and a Kd value with oleic acid of 0.50 microM. Skeletal muscle and heart FABPs and their antisera showed a strong cross-reactivity on Western blots and in enzyme-linked immunosorbent assays (ELISA). No cross-reactivity was observed with liver FABP and its antiserum. On the basis of amino acid composition, electrophoretic behavior, fatty acid binding, and immunochemical properties, human skeletal muscle FABP must be similar or closely related to human heart FABP. The FABP content determined by ELISA was comparable in various human muscles and cultured muscle cells, but lower than that in rat muscles.     

Fatty acid binding protein 4 in human skeletal muscle

The mechanisms that regulate intramyocellular triglycerol (IMTG) storage and mobilization are largely unknown. However, during the last decades several intracellular fatty acid binding proteins (FABPs) have been identified. FABP3 is the dominating FABP in skeletal muscle. Expression of additional FABPs is suggested from findings in FABP3-null mutated mice. In the present study, our aims were to investigate if FABP4 is expressed within skeletal muscle fibers and if FABP3 and FABP4 are more abundant in skeletal muscle fibers in endurance-trained than in control subjects. We show that FABP4 protein is expressed within the skeletal muscle fibers and that FABP4 mRNA and protein are more abundant in the endurance trained subjects. Still, FABP4 is markedly less expressed than FABP3, which is the generally accepted dominating FABP in skeletal muscle tissue.     

Tissue specific expression and developmental regulation of two genes coding for rat fatty acid binding proteins

We have examined the tissue distribution and developmental regulation of two low molecular weight cytosolic fatty acid binding proteins. Based on their initial site of isolation, they have been referred to as liver and intestinal fatty acid binding proteins (FABP). Cloned cDNAs were used to probe blots of RNAs extracted from a wide variety of adult rat tissues as well as small intestine and liver RNA obtained from fetal, suckling, and weaning animals. The highest concentrations of "liver" FABP mRNA were found in small intestine and liver. "Intestinal" FABP mRNA is most abundant in small bowel RNA while only trace amounts were encountered in liver. Both mRNAs were detectable in stomach, colon, pancreas, spleen, lung, heart, testes, adrenal, and brain RNA at 1-8% the concentrations observed in small intestine. Accumulation of both mRNAs in the small intestinal epithelium increases during development. The mRNAs are first detectable between the 19th and 21st day of gestation. They undergo a coordinated 3-4-fold increase in concentration within the first 24 h after birth. Thereafter, gut levels of intestinal FABP mRNA remain constant during the suckling period while liver FABP mRNA increases an additional 2-fold. Liver FABP mRNA levels are also induced in hepatocytes during the first postnatal day but subsequently do not change during the suckling and weaning phase, despite marked alterations in hepatic fatty acid metabolism. These observations support the concept that the major role of these proteins is to facilitate the entry of lipids into cells and/or their subsequent intracellular transport and compartmentalization. The data also raise questions about the identity of extragastrointestinal FABPs.     

Isolation and characterization of the fatty acid binding protein from human heart

We have isolated in pure form a fatty acid binding protein (FABP) from human cardiac muscle. After preparation of a 100,000 g supernatant fraction, the procedure required only one gel chromatographic (Sephacryl S 200) and two cation exchange (CM-Sephadex C 50) steps. The recovery of FABP was 55%. Pure FABP (12.5 mg) was obtained from a 1-g of dry powder equivalent of the high-speed supernatant. The protein had an Mr of 15,500 +/- 1,000 Da and an isoelectric point of 5.3. The properties of human cardiac FABP, i.e., molecular mass, isoelectric point, amino acid composition, ultraviolet spectrum, and affinities for hydrophobic ligands, were close to those found for FABPs from bovine heart (Jagschies et al. 1985. Eur. J. Biochem. 152: 537-545). In addition, immunological cross-reactivities showed a relationship between FABPs from several mammalian heart tissues. The data elaborated by us and others support the existence of a cardiac-type FABP that is distinct from the well-defined hepatic-type and gut-type FABPs.     

Does fatty acid-binding protein play a role in fatty acid transport?

Summary The possible property of fatty acid-binding proteins (FABPs) to transport fatty acid was investigated in various model systems with FABP preparations from liver and heart. An effect of FABP, however, was not detectable with a combination of oleic acid-loaded mitochondria and vesicles or liposomes due to the rapid spontaneous transfer. Therefore, the mitochondria were separated from the vesicles in an equilibrium dialysis cell. The spontaneous fatty acid transfer was much lower and addition of FABP resulted in an increase of fatty acid transport. Oleic acid was withdrawn from different types of monolayers by FABP with rates up to 10%/min. When two separate monolayers were used, FABP increased fatty acid transfer between these monolayers and an equilibrium was reached.Abbreviations FABP(s) fatty acid-binding protein(s) - PC phosphatidylcholine - PS phosphatidylserine - PE phosphatidylethanolamine     

Role of fatty acid-binding protein in lipid metabolism of insect flight muscle

Since insect flight muscles are among the most active muscles in nature, their extremely high rates of fuel supply and oxidation pose interesting physiological problems. Long-distance flights of species like locusts and hawkmoths are fueled through fatty acid oxidation. The lipid substrate is transported as diacylglycerol in the blood, employing a unique and efficient lipoprotein shuttle system. Following diacylglycerol hydrolysis by a flight muscle lipoprotein lipase, the liberated fatty acids are ultimately oxidized in the mitochondria. Locust flight muscle cytoplasm contains an abundant fatty acid-binding protein (FABP). The flight muscle FABP ofLocusta migratoria is a 15 kDa protein with an isoelectric point of 5.8, binding fatty acids in a 1:1 molar stoichiometric ratio. Binding affinity of the FABP for longchain fatty acids (apparent dissociation constant K_d=5.21±0.16 M) is however markedly lower than that of mammalian FABPs. The NH₂-terminal amino acid sequence shares structural homologies with two insect FABPs recently purified from hawkmoth midgut, as well as with mammalian FABPs. In contrast to all other isolated FABPs, the NH₂ terminus of locust flight muscle FABP appeared not to be acetylated. During development of the insect, a marked increase in fatty acid binding capacity of flight muscle homogenate was measured, along with similar increases in both fatty acid oxidation capacity and citrate synthase activity. Although considerable circumstantial evidence would support a function of locust flight muscle FABP in intracellular uptake and transport of fatty acids, the finding of another extremely well-flying migratory insect, the hawkmothAcherontia atropos, which employs the same lipoprotein shuttle system, however contains relatively very low amounts of FABP in its flight muscles, renders the proposed function of FABP in insect flight muscles questionable.     

Primary structure of locust flight muscle fatty acid binding protein.

The amino acid sequence of the fatty acid binding protein (FABP) from flight muscle of the locust, Schistocerca gregaria, has been determined. The sequence of the N-terminal 39 amino acid residues, determined by automated Edman degradation, was used to prepare a degenerate oligonucleotide that corresponded to amino acid residues 16-23. cDNA coding for FABP was constructed from flight muscle mRNA and amplified by the polymerase chain reaction using the degenerate oligonucleotide and an oligo dT-NotI primer adapter as primers. The amplification product was cloned and sequenced. Additionally, a cDNA library of flight muscle mRNA was prepared and screened with a 414-bp probe prepared from the clone. The primary structure of locust FABP was compared with the proteins in the Swiss protein databank and found to have significant homology with mammalian FABPs over the entire 133-residue sequence. The best match was versus human heart FABP (41% identity), attesting to the highly conserved nature of this protein. The results suggest that locust muscle FABP is a member of the lipid binding protein superfamily and may provide valuable insight into the evolution of this abundant protein class.     

Function and regulation of hepatic and intestinal fatty acid binding proteins

Two structurally different fatty acid binding proteins (FABP) have been isolated from rat liver and small intestinal epithelium. hFABP is a 14 184 Da protein found in abundance in both liver and small intestine, whereas gFABP (15 063 Da) is abundantly present only in small intestine. This review discusses studies which have provided insight into the physiological functions of these proteins. These include analyses of endogenous and exogenous ligand binding to FABP in vitro; examination of the modulating effect of FABP preparations on enzyme activities in vitro; exploration of relationships between alterations in cytosolic FABP content in response to hormonal, pharmacological, and dietary manipulations and changes in the rates of cellular fatty acid uptake and utilization; and studies of hFABP turnover and the mechanisms of FABP regulation. These experiments provide compelling evidence for a broad role of the FABPs in the transport, utilization and cellular economy of free fatty acids in the liver and small intestine, and also in protecting several aspects of cellular function against the modulatory effects of fatty acids, fatty acyl-CoA esters, and other ligands. Studies of FABP regulation also suggest a role in long-term rather than short-term modulation of hepatic fatty acid metabolism and indicate that hFABP and gFABP may perform different functions in the small intestine.     

Rapid methods for the characterization of unilamellar phospholipid vesicles. Application to studies on fatty acid donor and acceptor properties of membranes and fatty acid binding proteins

Vesicles having diameters from 20 to 200 nm were prepared from egg-yolk phosphatidylcholine (PC) and were separated as well as analyzed by methods that can be carried out with standard laboratory equipment. Gel-chromatography on Sephacryl S 1000 was adapted for expeditious size analysis of vesicles as well as for isolation of vesicle populations having a narrow range of diameters. The internal volume of vesicles was derived from enzymic tests for PC and for glucose encapsulated. Size analysis and enzymic determinations provided a convenient check for the lamellarity of membranes produced.Fatty acids and fatty acid binding proteins (FABPs) must interact in vivo in the presence of cellular membranes. As a model, interactions between unilamellar vesicles, anthroyloxypalmitic acid (A16:0) and FABPs were studied with the aid of gel-chromatographic methods elaborated and of fluorescence spectroscopy. FABP from bovine heart donated A16:0 to membranes, whereas FABP from bovine liver removed this fatty acid from vesicle membranes. The results revealed characteristic differences between cardiac and hepatic FABPs with regard to binding a fatty acid.     

The main fatty acid-binding protein in the liver of the shark (Halaetunus bivius) belongs to the liver basic type. Isolation, amino acid sequence determination and characterization.

Three fatty acid-binding proteins (FABPs) from the liver of the shark Halaetunus bivius were isolated and characterized: one of them belongs to the liver-type FABP family and the other two to the heart-type FABP family. The complete primary structure of the first FABP, and partial primary structures of the two others, were determined. The liver-type FABP constitutes 69% of the total FABPs, and its amino acid sequence presents the highest identity with chicken, catfish, iguana and elephant fish liver basic FABPs. The L-FABP protein has low affinity for palmitic and oleic acids and high affinity for linoleic and arachidonic acids and other hydrophobic ligands, all of them important for the metabolic functions of the liver. In contrast, both heart-type FABPs have the highest affinity for palmitic acid, the principal fatty acid mobilized from fat deposits for beta-oxidation.     

Historic overview of studies on fatty acid-binding proteins

Summary Fatty acid-binding proteins (FABPs) were first identified in the cytosol of rat intestinal mucosa during studies on the regulation of intestinal fatty acid uptake. The subsequent finding of FABP activity in the cytosol of many other tissues initially was believed to reflect a single protein. However, the FABPs are now recognized as products of an ancient gene family comprised of at least 9 structurally related, soluble intracellular members, a number of which exhibit high-affinity binding of long-chain fatty acids. Despite recent insights into regulation and tissue-specific expression suggesting FABPs to subserve diverse roles, their precise biological functions remain to be elucidated.