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1.
Human serum albumin is the most abundant protein in the circulatory system, and one of its principal functions is to transport fatty acids. Binding of octanoate, decanoate, laurate and myristate was studied by a rate-of-dialysis technique. The primary association constants increased, but not linearly, with chain length. The number of high-affinity sites also increased with chain length; octanoate and decanoate bind to one such site, whereas laurate and myristate most probably bind to two sites. Albumin is composed of three homologous helical domains (I-III), which can be subdivided into two subdomains (A and B). For getting information about the positions of the high-affinity sites we produced 13 recombinant isoforms mutated in four different subdomains. Results obtained with these albumins are in accordance with the following model: octanoate and decanoate bind to a single site in subdomain IIIA, laurate binds to sites in subdomains IIIA and IIIB, whereas myristate binds in subdomains IB and IIIB. The results also showed that primary fatty acid binding is sensitive to amino acid substitutions in other parts of the protein. This is in contrast to the effect of amino acid substitutions of genetic albumin variants (alloalbumins). Usually these substitutions, which are situated at the surface of the protein, have no effect on fatty acid binding. Binding of fatty acid anions to different high-affinity sites and the sensitivity of these sites to amino acid substitutions elsewhere in the protein (and perhaps also to other types of modifications) are important factors that could effect simultaneous binding of other ligands, e.g. in patients treated with albumin-binding drugs.  相似文献   

2.
Insights into binding of fatty acids by fatty acid binding proteins   总被引:10,自引:0,他引:10  
Members of the phylogenetically related intracellular lipid binding protein (iLBP) are characterized by a highly conserved tertiary structure, but reveal distinct binding preferences with regard to ligand structure and conformation, when binding is assessed by the Lipidex method (removal of unbound ligand by hydrophobic polymer) or by isothermal titration calorimetry, a true equilibrium method. Subfamily proteins bind retinoids, subfamily II proteins bind bulky ligands, examples are intestinal bile acid binding protein (I-BABP) and liver fatty acid binding protein (L-FABP) which binds 2 ligand molecules, preferably monounsaturated and n-3 fatty acids. Subfamily III intestinal fatty acid binding protein (I-FABP) binds fatty acid in a bent conformation. The fatty acid bound by subfamily IV FABPs has a U-shaped conformation; here heart (H-) FABP preferably binds n-6, brain (B-) FABP n-3 fatty acids. The ADIFAB-method is a fluorescent test for fatty acid in equilibrium with iLBP and reveals some correlation of binding affinity to fatty acid solubility in the aqueous phase; these data are often at variance with those obtained by the other methods. Thus, in this review published binding data are critically discussed, taking into account on the one hand binding increments calculated for fatty acid double bonds on the basis of the solubility hypothesis, on the other hand the interpretation of calorimetric data on the basis of crystallographic and solution structures of iLBPs.  相似文献   

3.
The development of a single-step, separation-free method for measurement of low concentrations of fatty acid using a surface plasmon resonance-enhanced fluorescence competition assay with a surface-bound antibody is described. The assay behavior was unexpectedly complex. A nonlinear coverage-dependent self-quenching of emission from surface-bound fluorescent label was deduced from the response kinetics and attributed to a surface plasmon-mediated energy transfer between adsorbed fluorophores, modified by the effects of plasmon interference. Principles of assay design to avoid complications from such effects are discussed. An anti-fatty acid mouse monoclonal antibody reacting to the alkyl chain was prepared and supported on a gold chip at a spacing appropriate for surface-plasmon field-enhanced fluorescence spectroscopy (SPEFS), by applying successively a self-assembled biotinylated monolayer, then streptavidin, then biotinylated protein A, and then the antibody, which was crosslinked to the protein A. Synthesis of a fluorescently (Cy5) tagged C-11 fatty acid is reported. SPEFS was used to follow the kinetics of the binding of the labeled fatty acid to the antibody, and to implement a competition assay with free fatty acid (undecanoic acid), sensitive at the 1 μM scale, a sensitivity limit caused by the low affinity of antibodies for free fatty acids, rather than the SPEFS technique itself. Free fatty acid concentration in human serum is in the range 0.1-1 mM, suggesting that this measurement approach could be applied in a clinical diagnostic context. Finally, a predictive, theoretical model of fatty acid binding was developed that accounted for the observed “overshoot” kinetics.  相似文献   

4.
In this study, lysophosphatidylcholine (lysoPC) was shown to bind to a fatty acid binding protein isolated from rat liver. To demonstrate the binding, lysoPC was incorporated into multilamellar liposomes and incubated with protein. For comparison, binding of both lysoPC and fatty acid to liver fatty acid binding protein, albumin, and heart fatty acid binding protein were measured. At conditions where palmitic acid bound to liver fatty acid binding protein and albumin at ligand to protein molar ratios of 2:1 and 5:1, respectively, lysoPC binding occurred at molar ratios of 0.4:1 and 1:1. LysoPC did not bind to heart fatty acid binding protein under conditions where fatty acid bound at a molar ratio of 2:1. Competition experiments between lysoPC and fatty acid to liver fatty acid binding protein indicated separate binding sites for each ligand. An equilibrium dialysis cell was used to demonstrate that liver fatty acid binding protein was capable of transporting lysoPC from liposomes to rat liver microsomes, thereby facilitating its metabolism. These studies suggest that liver fatty acid binding protein may be involved in the intracellular metabolism of lysoPC as well as fatty acids, and that functional differences may exist between rat liver and heart fatty acid binding protein.  相似文献   

5.
Fatty acid binding protein was purified from skeletal muscle of the spadefoot toad (Scaphiopus couchii), an estivating species. While estivating, this animal relies on the fatty acid oxidation for energy. Hence we were interested in the behaviour of fatty acid binding protein under conditions of elevated urea (up to 200 mM) and potassium chloride such as exist during estivation. Also we examined whether there were interactions between glycolytic intermediates and the binding ability of the protein. The amount of bound fatty acid (a fluorescence assay using cis-parinarate) was not affected (P < 0.05) by glucose, fructose 6-phosphate or phosphoenolpyruvate at physiological concentrations. By contrast, glucose 6-phosphate increased the amount of bound cis-parinarate but the apparent dissociation constant was not different from the control. Fructose 1,6-bisphosphate but not fructose 2,6-phosphate decreased cis-parinarate binding by 40%, commensurate with doubling the apparent dissociation constant (1.15-2.62 microM). Urea, guanidinium and trimethylamine N-oxide at 200 mM increased cis-parinarate binding 60% over controls. Urea (1 M) and KCl (200 mM) did not affect cis-parinarate binding compared to controls. The interaction of this fatty acid transporter with fructose 1,6-bisphosphate is discussed in terms of reciprocal interaction with phosphofructokinase since fatty acid is also an inhibitor of phosphofructokinase.  相似文献   

6.
A protein fraction with fatty acid binding activity has been isolated from mammary tissue from lactating rats by a process involving DEAE-cellulose ion-exchange chromatography, heat treatment, CM-cellulose ion-exchange chromatography and finally ammonium sulphate precipitation. The purified fraction migrated as a single band on SDS/polyacrylamide-gel electrophoresis with an apparent molecular mass of 14400. However, when this protein fraction was electrophoresed under non-dissociating conditions, two species were observed in a 4:1 ratio. The two components were separated using h.p.l.c. Both bind fatty acids and appear to have similar amino acid compositions although exhibiting different pI values of 4.8 and 4.9. The mammary fatty acid binding proteins appear to be very similar to the fatty acid binding protein isolated from rat heart based on the electrophoretic mobilities and amino acid composition. The major mammary form (pI 4.9) has been partially sequenced and the amino acid sequences obtained can be aligned with 67 residues of the revised rat heart amino acid sequence [Heuckeroth, Birkenmeier, Levin & Gordon (1987) J. Biol. Chem. 262, 9709-9717]. Both mammary species also showed immunochemical identity to rat heart fatty acid binding protein when tested with an anti-serum raised against the heart protein. Anti-sera raised against the minor mammary form (pI 4.8) specifically precipitated this form under non-denaturing conditions but both forms after they had been denatured. Quantitative immunoassays using the anti-(heart fatty acid binding protein) serum showed that concentrations of the fatty acid binding proteins present in mammary cytosols increase during lactation and increase further after feeding a high-fat diet.  相似文献   

7.
Conventional ESR spectra of 16-doxyl-stearic acid bound to bovine and human serum albumin were recorded at different temperatures in order to investigate the status of spin-labeled fatty acid in the interior of the protein globule. A computer spectrum simulation of measured spectra, performed by non-linear least-squares fits, clearly showed two components corresponding to strongly and weakly immobilized fatty acid molecules. The two-component model was verified on spectra measured at different pH. Thermodynamic parameters of the spin probe exchange between two spin probe states were analyzed. It was concluded that at physiological conditions, fatty acid molecules permanently migrate in the globule interior between the specific binding sites and a space among albumin domains.  相似文献   

8.
N C Fournier  M Rahim 《Biochemistry》1985,24(9):2387-2396
The quantitative subcellular distribution of the fatty acid binding protein (FABP) in heart muscle is reported for the first time. A gradient-like distribution according to the following pattern was observed: 6.96 mg X mL-1 on the myofibrils, 2.77 mg X mL-1 in the spaces surrounding the mitochondria, and 2.21 mg X mL-1 in the mitochondria. This heterogeneous distribution suggests that the local in vivo concentration of FABP might fluctuate as a function of time. The consequences of these possible fluctuations, particularly in the mitochondrial vicinity, were analyzed in an in vitro system containing a fixed concentration of cardiac mitochondria and stearic acid but variable concentrations of FABP. Competition for the fatty acid was observed between the mitochondrial membranes and the binding sites on the protein. Maximal binding of fatty acid to FABP was detected in the range of FABP concentration between 1 and 3 mg X mL-1. Remarkably, in this concentration range, two emerging peaks of beta-oxidative activity were also detected. As a major conclusion, it appears that the fatty acid pool, bound to FABP, is the source of fatty acid providing the beta-oxidative system with substrate. The mechanism of fatty acid transfer from this pool toward the beta-oxidative system remains an open question. However, it is suggested that a gradient-like distribution of FABP in the mitochondrial vicinity leads to the coexistence of multispecies of the protein by self-aggregation. Only two of these species seem to be involved in this fatty acid transfer. As a consequence, a strong modulation of fatty acid beta-oxidation rate is observed in isolated mitochondria when the concentrations of these two species are allowed to fluctuate. In conclusion, this unique cardiac fatty acid carrier, via its self-aggregation capacity and its in vivo gradient-like distribution, may act as a powerful effector in the regulation of heart energy.  相似文献   

9.
The product of the fadL gene (FadL) of Escherichia coli is a multifunctional integral outer-membrane protein required for the specific binding and transport of exogenous long-chain fatty acids [C12-C18]. FadL also serves as a receptor for the bacteriophage T2. In order to define regions of functional importance within FadL, the fadL gene has been mutagenized by the insertion of single-stranded hexameric linkers into the unique SalI restriction site that lies towards the 3' end of the gene and into four HpaII restriction sites distributed throughout the coding region. The five insertion mutants were classified into three groups based on their specific growth rates (alpha) in minimal media containing the long-chain fatty acid oleate (C18:1) as a sole carbon and energy source: Oleslow, alpha = 0.035-0.045; Ole +/-, alpha = 0.020-0.035; and Ole-, alpha less than or equal to 0.005 (wild-type, alpha = 0.07-0.10). The hexameric insertion at the SalI site (fadL allele termed S1; insertion after amino acid 410) conferred an Oleslow phenotype and resulted in a reduction of long-chain fatty acid transport (36% the wild-type level). This insertion mutant, however, bound oleic acid at wild-type levels and was fully functional as a receptor for the bacteriophage T2. The modified FadL-S1 protein did not have the heat-modifiable property characteristic of wild-type FadL. Insertions in the four HpaII sites (fadL alleles termed H1, H2, H3, and H5; after amino acids 41, 81, 238, and 389, respectively) resulted in all three classes of mutants. The fadL insertion mutant H5 was defective for long-chain fatty acid transport but bound oleic acid at significant levels. Together with the S1 allele, these data suggest that the carboxyl terminus of FadL is crucial for long-chain fatty acid transport. The insertion mutants H1 and H2 were defective for both oleic acid binding and transport suggesting that the amino terminus of FadL is important for long-chain fatty acid binding and transport. The fadL linker mutant H3 was defective in oleic acid binding yet had significant levels of oleic acid transport. These studies delineated for the first time different regions of the fadL gene that encode domains of FadL implicated in the binding and transport of long-chain fatty acids.  相似文献   

10.
The structure and dynamics of the fatty acid binding cavity in I-FABP (rat intestinal fatty acid binding protein) were analyzed. In the crystal structure of apo I-FABP, the probe occupied cavity volume and surface are 539+/-8 A3 and 428 A2, respectively (1.4 A probe). A total of 31 residues contact the cavity with their side chains. The side-chain cavity surface is partitioned according to the residue type as follows: 36-39% hydrophobic, 21-25% hydrophilic, and 37-43% neutral or ambivalent. Thus, the cavity surface is neither like a typical protein interior core, nor is like a typical protein external surface. All hydrophilic residues that contact the cavity-with the exception of Asp74-are clustered on the one side of the cavity. The cavity appears to expand its hydrophobic surface upon fatty acid binding on the side opposite to this hydrophilic patch. In holo I-FABP the fatty acid chain interactions with the hydrophilic side chains are mediated by water molecules. Molecular dynamics (MD) simulation of fully solvated apo I-FABP showed global conformational changes of I-FABP, which resulted in a large, but seemingly transient, exposure of the cavity to the external solvent. The packing density of the side chains lining the cavity, studied by Voronoi volumes, showed the presence of two distinctive small hydrophobic cores. The MD simulation predicts significant structural perturbations of the cavity on the subnanosecond time scale, which are capable of facilitating exchange of I-FABP internal water.  相似文献   

11.
Rat liver fatty acid-binding protein (FABP) can function as a fatty acid donor protein for both peroxisomal and mitochondrial fatty acid oxidation, since 14C-labeled palmitic acid bound to FABP is oxidized by both organelles. FABP is, however, not detected in peroxisomes and mitochondria of rat liver by ELISA. Acyl-CoA oxidase activity of isolated peroxisomes was not changed by addition of FABP or flavaspidic acid, an inhibitor of fatty acid binding to FABP, nor by disruption of the peroxisomal membranes. These data indicate that FABP may transfer fatty acids to peroxisomes, but is not involved in the transport of acyl-CoA through the peroxisomal membrane.  相似文献   

12.
Herein we report the first disclosure of biphenyl azoles that are nanomolar binders of adipocyte fatty acid binding protein (aFABP or aP2) with up to thousand-fold selectivity against muscle fatty acid binding protein and epidermal fatty acid binding protein. In addition a new radio-ligand to determine binding against the three fatty acid binding proteins was also synthesized.  相似文献   

13.
Intestinal enterocytes contain two homologous fatty acid-binding proteins, intestinal fatty acid-binding protein (I-FABP)2 and liver fatty acid-binding protein (L-FABP). Since the functional basis for this multiplicity is not known, the fatty acid-binding specificity of recombinant forms of both rat I-FABP and rat L-FABP was examined. A systematic comparative analysis of the 18 carbon chain length fatty acid binding parameters, using both radiolabeled (stearic, oleic, and linoleic) and fluorescent (trans-parinaric and cis-parinaric) fatty acids, was undertaken. Results obtained with a classical Lipidex-1000 binding assay, which requires separation of bound from free fatty acid, were confirmed with a fluorescent fatty acid-binding assay not requiring separation of bound and unbound ligand. Depending on the nature of the fatty acid ligand, I-FABP bound fatty acid had dissociation constants between 0.2 and 3.1 microM and a consistent 1:1 molar ratio. The dissociation constants for L-FABP bound fatty acids ranged between 0.9 and 2.6 microM and the protein bound up to 2 mol fatty acid per mole of protein. Both fatty acid-binding proteins exhibited relatively higher affinity for unsaturated fatty acids as compared to saturated fatty acids of the same chain length. cis-Parinaric acid or trans-parinaric acid (each containing four double bonds) bound to L-FABP and I-FABP were displaced in a competitive manner by non-fluorescent fatty acid. Hill plots of the binding of cis- and trans- parinaric acid to L-FABP showed that the binding affinities of the two sites were very similar and did not exhibit cooperativity. The lack of fluorescence self-quenching upon binding 2 mol of either trans- or cis-parinaric acid/mol L-FABP is consistent with the presence of two binding sites with dissimilar orientation in the L-FABP. Thus, the difference in binding capacity between I-FABP and L-FABP predicts a structurally different binding site or sites.  相似文献   

14.
The activities of the enzymes glycerol-3-phosphate dehydrogenase and fatty acid synthase are inhibited by palmitoyl-coenzyme A and oleate. The two isoforms of fatty acid binding proteins (PI 6.9 and PI 5.4) enhance the activities of glycerol-3-phosphate dehydrogenase and fatty acid synthase in the absence of palmitoyl-coenzyme A or oleate and also protect them against palmitoyl-coenzyme A or oleate inhibition. Levels of fatty acid binding proteins, the activities of the enzymes fatty acid synthase and glycerol-3-phosphate dehydrogenase increase with gestation showing a peak at term. However, the activity of fatty acid synthase showed the same trend up to the 30th week of gestation and then declined slightly at term. With the advancement of pregnancy when more lipids are required for the developing placenta, fatty acid binding proteins supply more fatty acids and glycerol-3-phosphate for the synthesis of lipids. Thus a correlation exists between glycerol-3-phosphate dehydrogenase, fatty acid synthase and fatty acid binding proteins in developing human placenta.  相似文献   

15.
The relationship between fatty acid binding proteins, ATP citrate lyase activity and fatty acid synthesis in developing human placenta has been studied. Fatty acid binding proteins reverse the inhibitory efect of palmitoyl-CoA and oleate on ATP citrate lyase and fatty acid synthesis. In the absence of these inhibitors fatty acid binding proteins activate ATP citrate lyase and stimulate [ 1-14 C] acetate incorporation into placental fatty acids indicating binding of endogenous inhibitors by these proteins. Thus these proteins regulate the supply of acetyl-CoA as well as the synthesis of fatty acids from that substrates. As gestation proceeds and more lipids are required by the developing placenta fatty acid binding protein content, activity of ATP citrate lyase and rate of fatty acid synthesis increase indicating a cause and efect relationship between the demand of lipids and supply of precursor fatty acids during human placental development.  相似文献   

16.
17.
The ability of purified rat liver and heart fatty acid binding proteins to bind oleoyl-CoA and modulate acyl-CoA synthesis by microsomal membranes was investigated. Using binding assays employing either Lipidex 1000 or multilamellar liposomes to sequester unbound ligand, rat liver but not rat heart fatty acid binding protein was shown to bind radiolabeled acyl CoA. Binding studies suggest that liver fatty acid binding protein has a single binding site acyl-CoA which is separate from the two binding sites for fatty acids. Experiments were then performed to determine how binding may influence acyl-CoA metabolism by liver microsomes or heart sarcoplasmic reticulum. Using liposomes as fatty acid donors, liver fatty acid binding protein stimulated acyl-CoA production, whereas that from heart did not stimulate production over control values. 14C-labeled fatty acid-fatty acid binding protein complexes were prepared, incubated with membranes, and acyl-CoA synthetase activity was determined. Up to 70% of the fatty acid could be converted to acyl-CoA in the presence of liver fatty acid binding protein but in the presence of heart fatty acid binding protein, only 45% of the fatty acid was converted. Liver but not heart fatty acid binding protein bound the acyl-CoA formed and removed it from the membranes. The amount of product formed was not changed by additional membrane, enzyme cofactors, or incubation time. Additional liver fatty acid binding protein was the only factor found that stimulated product formation. Acyl-CoA hydrolase activity was also shown in the absence of ATP and CoA. These studies suggest that liver fatty acid binding protein can increase the amount of acyl-CoA by binding this ligand, thereby removing it from the membrane and possibly aiding transport within the cell.  相似文献   

18.
Gel filtration on Sephadex G-75 of crude rat liver supernatant preincubated with [1-14C]oleic acid yields three peaks of radioactivity which are attributed to the presence in these fractions of fatty acid binding proteins. We have confirmed these observations with binding assays by phase partition, polyacrylamide gel electrophoresis, and thin layer electrofocusing. Peak I (mol. wt. 60,000 pI 5.01 was shown to be albumin, which mainly arises from a contamination of the liver preparation by blood. Peak II (mol. wt. 10,000, pI 5.9) is a fatty acid binding protein. Finally peak III (mol. wt. 1500, pI 5.7) is a fatty acid binding component, the chemical nature of which was not elucidated. These fatty acid binding fractions have no effect on the reaction of acyl-CoA synthetase whereas the crude liver supernatant does stimulate the activation of fatty acid as shown earlier. In consequence, the physiological role of these fatty acid binding fractions is not yet elucidated.  相似文献   

19.
Significant advances have been made in the past few years in our understanding of the mechanism of synthesis of fatty acids, the structural organization of fatty acid synthetase complexes and the mechanism of regulation of activity of these enzyme systems. Numerous fatty acid synthetase complexes have been purified to homogeneity and the mechanism of synthesis of fatty acids by these enzyme systems has been ascertained from tracer, and recently, kinetic studies. The results obtained by these methods are in complete agreement. Furthermore, the kinetic results have indicated that fatty acid synthesis proceeds by a seven-site ping-pong mechanism. Several of the fatty acid synthetases have been dissociated completely to nonidentical half-molecular weight subunit species and these have been separated by affinity chromatography. From one of these subunits acyl carrier protein has been obtained. Whether the nonidentical subunits can be dissociated into individual proteins or whether these subunits are each comprised of one peptide is still a matter of controversy. However, it appears to us that each of the half-molecular weight subunits is indeed comprised of individual proteins. Studies on the regulation of activity of fatty acid synthetase complexes of avian and mammalian liver have resulted in the separation by affinity chromatography of three species (apo, holo-a and holo-b) of fatty acid synthetase. Since these species have radically different enzyme activities they may provide a mechanism of short-term regulation of fatty acid synthetase activity. Other studies have shown that the quantity of avian and mammalian liver fatty acid synthetases is controlled by a change in the rate of synthesis of this enzyme complex. This change in the rate of synthesis of enzyme complex is under the control of insulin and glucagon. The former hormone increases the rate of enzyme synthesis, whereas the latter decreases it. Further studies on fatty acid synthetase complexes will undoubtedly concentrate upon more refined aspects of the structural organization of these enzyme systems, including the sequencing of acyl carrier proteins, the effects of protein-protein interaction on the kinetics of the partial reactions of fatty acid synthesis catalyzed by separated enzymes of the complex, the mechanism of hormonal regulation of fatty acid synthetase activity and x-ray diffraction analysis of subunits and complex.  相似文献   

20.
The intestinal mucosa metabolizes fatty acids differently when presented to the lumenal or basolateral membrane. Expression of both liver and intestinal fatty acid binding proteins (L- and I-FABPs) uniquely in the enterocyte offers a possible explanation of this phenomenon. An organ explant system was used to analyze the relative binding of fatty acids to each protein. More fatty acid was bound to L-FABP than to I-FABPs (28% vs. 6% of cytosolic radioactivity), no matter on which side the fatty acid was added. However, a 2-3-fold increase in fatty acid binding to the intestinal paralog was noted after apical addition of palmitic or oleic acid in mucosa from chow fed rats. When oleic acid was added apically, a 1.4-fold increase in binding to I-FABP was observed in mucosa derived from chronically fat fed rats, consistent with the previously observed 50% increase in the content of that protein. Immunocytochemical localization of both FABPs in vivo demonstrated an apical cytoplasmic localization in the fasting state, and redistribution to the entire cytoplasm after fat feeding. These data are consistent with the hypothesis that I-FABP may contribute to the metabolic compartmentalization of apically presented fatty acids in the intestine.  相似文献   

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