RESUMO
Mammalian liver has only one fatty acid-binding protein (L-FABP) while the liver of non-mammalian vertebrates expresses a liver basic FABP (Lb-FABP) in addition to other members of the FABP family. We explore the possibility that L-FABP isoforms accomplish, in the liver of mammals, the metabolic functions corresponding to the different FABPs present in the liver of non-mammalian vertebrates. We have isolated isoforms I and II which have a different residue 105, Asn in the former and Asp in the latter. We made a conformational comparison of the apo-isoforms by intrinsic fluorescence emission and fourth-derivative spectroscopy, native-state proteolysis and unfolding curves. Ligand affinity was studied by measuring cis-parinaric acid displacement by different ligands. They have differences in their molecular conformation, including the environment of the binding site. Isoform II has probably a more open conformation than isoform I, thus allowing the binding of a greater variety of ligands. The affinity of isoform II for lysophospholipids, prostaglandins, retinoids, bilirubin and bile salts is greater than that of isoform I. These characteristics of rat L-FABP isoforms I and II suggest that they may accomplish different functions as happens with those of the different FABP types in non-mammalian species.
Assuntos
Proteínas de Transporte/metabolismo , Fígado/metabolismo , Proteína P2 de Mielina/metabolismo , Proteínas de Neoplasias , Proteínas do Tecido Nervoso , Animais , Western Blotting , Proteínas de Transporte/química , Proteínas de Transporte/isolamento & purificação , Fracionamento Celular , Cromatografia Líquida de Alta Pressão , Eletroforese em Gel de Poliacrilamida , Endopeptidases , Proteína 7 de Ligação a Ácidos Graxos , Proteínas de Ligação a Ácido Graxo , Ácidos Graxos Insaturados , Corantes Fluorescentes , Focalização Isoelétrica , Masculino , Proteína P2 de Mielina/química , Proteína P2 de Mielina/isolamento & purificação , Dobramento de Proteína , Isoformas de Proteínas/química , Isoformas de Proteínas/metabolismo , Ratos , Ratos Sprague-Dawley , Espectrometria de Fluorescência , Espectrofotometria Ultravioleta , TripsinaRESUMO
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.
Assuntos
Proteínas de Transporte/química , Fígado/metabolismo , Proteína P2 de Mielina/química , Proteínas de Neoplasias , Tubarões/metabolismo , Sequência de Aminoácidos , Animais , Ligação Competitiva , Proteínas de Transporte/isolamento & purificação , Proteínas de Ligação a Ácido Graxo , Ácidos Graxos/metabolismo , Ácidos Graxos Insaturados/metabolismo , Dados de Sequência Molecular , Proteína P2 de Mielina/isolamento & purificação , Miocárdio/metabolismo , Fragmentos de Peptídeos/química , Isoformas de Proteínas , Alinhamento de Sequência , Análise de Sequência , TripsinaRESUMO
Up until now, the primary structure of fatty-acid-binding proteins (FABPs) from the livers of four mammalian (rat, human, cow and pig) and three nonmammalian (chicken, catfish and iguana) species has been determined. Based on amino acid sequence comparisons, it has been suggested that mammalian and nonmammalian liver FABPs may be paralogous proteins that originated by gene duplication, rather than as a consequence of mutations of the same gene. In this paper we report the isolation and amino acid sequence determination of two FABPs from axolotl (Ambistoma mexicanum) liver. One of them is similar to mammalian liver FABPs (L-FABPs) and the other to chicken, catfish and iguana liver FABPs (Lb-FABPs). The finding of both L-FABP and Lb-FABP in a single species, as reported here, indicates that they are paralogous proteins. The time of divergence of these two liver FABP types is estimated to be of approximately 694 million years ago. The ligand-binding properties of axolotl liver FABPs were studied by means of parinaric-acid-binding and parinaric-acid-displacement assays. L-FABP binds two fatty acids per molecule but Lb-FABP displays a fatty-acid-conformation-dependent binding stoichiometry; L-FABP shows a higher affinity for fatty acids, especially oleic acid, while Lb-FABP has a higher affinity for other hydrophobic ligands, especially retinoic acid. In addition, the tissue-expression pattern is different, L-FABP is present in liver and intestinal mucosa while the expression of Lb-FABP is restricted to liver. Data indicate distinct functional properties of both liver FABP types.
Assuntos
Ambystoma/genética , Proteínas de Transporte/genética , Evolução Molecular , Ácidos Graxos/metabolismo , Fígado/química , Proteína P2 de Mielina/genética , Proteínas de Neoplasias , Proteínas do Tecido Nervoso , Sequência de Aminoácidos , Animais , Ligação Competitiva , Proteínas de Transporte/química , Proteínas de Transporte/metabolismo , Proteína 7 de Ligação a Ácidos Graxos , Proteínas de Ligação a Ácido Graxo , Ácidos Graxos Insaturados/metabolismo , Expressão Gênica , Ligantes , Dados de Sequência Molecular , Proteína P2 de Mielina/química , Proteína P2 de Mielina/metabolismo , Análise de Sequência , Homologia de Sequência de Aminoácidos , Especificidade da Espécie , Distribuição TecidualRESUMO
Intestinal fatty acid binding protein (IFABP) undergoes a reversible thermal transition between 35 and 50 degreesC, as revealed by circular dichroism spectroscopy in the near-UV region. For the apoprotein, the molar ellipticity measured at 254 nm (possibly implicating the environment around F17 and/or F55) decreases significantly in this temperature range, while in the holoprotein (bound to oleic acid), this phenomenon is not observed. Concomitantly, an increase in the activity of binding to [14C]oleic acid occurs. Nevertheless, other spectroscopic evidence indicates that the beta-barrel structure, the major motif of this protein, is highly stable up to 70 degreesC. No changes associated with conformation were detected for both structures by fourth-derivative analysis of the UV absorption spectra, circular dichroism in the far-UV region, and intrinsic fluorescence measurements. Further structural information arises from experiments in which binding to the anionic fluorescent probes 1-anilinonaphthalene-8-sulfonic acid (ANS) and its dimer bisANS was examined. The fluorescence intensity of bound ANS diminishes monotonically, whereas that of bisANS increases slightly in the temperature range of 35-50 degreesC. Given the different size of these probes, model building suggests that ANS would be able to sense regions located deeply inside the cavity, while bisANS could also reach the vicinity of the small helical domain of this protein. In light of these results, we believe that this subtle conformational transition of IFABP, which positively influences the binding activity, would involve fluctuations at the peripheral "entry portal" region for the ligand. This interpretation is compatible with the discrete disorder observed in this place in apo-IFABP, as evidenced by NMR spectroscopy [Hodsdon, M. E., and Cistola, D. P. (1997) Biochemistry 36, 1450-1460].
Assuntos
Proteínas de Transporte/química , Proteínas de Transporte/metabolismo , Modelos Moleculares , Proteína P2 de Mielina/química , Proteína P2 de Mielina/metabolismo , Proteínas de Neoplasias , Proteínas do Tecido Nervoso , Temperatura , Animais , Radioisótopos de Carbono , Dicroísmo Circular , Proteína 7 de Ligação a Ácidos Graxos , Proteínas de Ligação a Ácido Graxo , Intestinos , Ligantes , Ácido Oleico/metabolismo , Ligação Proteica , Conformação Proteica , Ratos , Espectrometria de Fluorescência , Espectrofotometria Ultravioleta , Relação Estrutura-AtividadeRESUMO
The complete amino acid sequence of a basic liver fatty acid-binding protein (L-FABP) from catfish (Rhamdia sapo) was determined. Alignment of sequences shows that it has more similarity to chicken basic L-FABP than to mammalian L-FABP. The phylogenetic analysis suggests that basic L-FABP from catfish, chicken and iguana diverged from the mammalian protein before the fish-tetrapod divergence, thus implying that the two types are encoded by different genes. Supporting this conclusion, a 14-kDa protein, structurally closely related to mammalian L-FABP, was isolated from catfish intestine, indicating the presence of the two genes in the same species. The catfish basic L-FABP binds only one fatty acid/molecule, while mammalian L-FABP bind two. The former has more affinity for trans-parinaric acid than for cis-parinaric acid, in constrast to the latter proteins.
Assuntos
Proteínas de Transporte/química , Peixes-Gato/genética , Evolução Molecular , Ácidos Graxos/metabolismo , Fígado/metabolismo , Proteína P2 de Mielina/química , Proteínas de Neoplasias , Proteínas do Tecido Nervoso , Filogenia , Proteínas Supressoras de Tumor , Sequência de Aminoácidos , Animais , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Galinhas , Proteína 7 de Ligação a Ácidos Graxos , Proteínas de Ligação a Ácido Graxo , Humanos , Iguanas , Mucosa Intestinal/metabolismo , Cinética , Camundongos , Dados de Sequência Molecular , Proteína P2 de Mielina/genética , Proteína P2 de Mielina/metabolismo , Fragmentos de Peptídeos/química , Ratos , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , TubarõesRESUMO
The fatty acid-binding protein (FABP) from armadillo liver was purified to homogeneity by a procedure involving gel filtration and two anion exchange chromatography steps. The purified protein proved to have a pI between 5.0 and 5.2 and migrated by sodium dodecyl sulfate-polyacrilamyde gel electrophoresis as a single entity of approximately 14 kDa. The armadillo FABP cross-reacted with antiserum against rat liver FABP but not against rat intestinal FABP. The same as other members of the family, it has a blocked N-terminus. Amino acid sequencing of peptides obtained by cyanogen bromide cleavage and in-gel tryptic digestion shows that the armadillo, despite being one of the less evolved mammals, has a liver FABP of the same type as that of highly evolved mammals.
Assuntos
Tatus/metabolismo , Proteínas de Transporte/isolamento & purificação , Ácidos Graxos/metabolismo , Fígado/metabolismo , Proteína P2 de Mielina/isolamento & purificação , Proteínas de Neoplasias , Proteínas do Tecido Nervoso , Proteínas Supressoras de Tumor , Sequência de Aminoácidos , Aminoácidos/análise , Animais , Tatus/genética , Proteínas de Transporte/química , Proteínas de Transporte/genética , Reações Cruzadas , Citosol/metabolismo , Evolução Molecular , Proteína 7 de Ligação a Ácidos Graxos , Proteínas de Ligação a Ácido Graxo , Humanos , Imunoquímica , Ponto Isoelétrico , Dados de Sequência Molecular , Peso Molecular , Proteína P2 de Mielina/química , Proteína P2 de Mielina/genética , Filogenia , Ratos , Homologia de Sequência de AminoácidosRESUMO
A low-molecular-mass fatty acid-binding protein was isolated from the cytosol of the yeast Yarrowia lipolytica. Purification was achieved by a two-step procedure involving size-exclusion and cation-exchange chromatography. The isolated protein exists as a monomer of 15 kDa, is basic and has a blocked N-terminus. Internal amino acid sequencing suggests that this protein may belong to a novel class of fatty acid-binding proteins.
Assuntos
Proteínas de Transporte/química , Proteínas Fúngicas/química , Proteína P2 de Mielina/química , Proteínas de Neoplasias , Saccharomycetales/química , Sequência de Aminoácidos , Proteínas de Transporte/isolamento & purificação , Cromatografia em Gel , Cromatografia por Troca Iônica , Brometo de Cianogênio/metabolismo , Citoplasma/química , Eletroforese em Gel de Poliacrilamida , Proteínas de Ligação a Ácido Graxo , Proteínas Fúngicas/isolamento & purificação , Dados de Sequência Molecular , Peso Molecular , Proteína P2 de Mielina/isolamento & purificação , Ácido Palmítico/metabolismo , Fragmentos de Peptídeos/química , Análise de Sequência , Tripsina/metabolismoRESUMO
We report here the isolation of a fatty acid-binding protein (FABP) from the liver of the catfish Rhamdia sapo. The purification procedure involves gel filtration, anion-exchange chromatography and reverse-phase high-performance liquid chromatography. The purified protein is basic (pI > 8.7) and migrates on sodium dodecyl sulfate-gel electrophoresis as a single entity of about 15 kDa. Its amino acid composition resembles those of FABPs isolated from other animals. Unlike mammalian liver FABPs, catfish liver FABP contains at least one tryptophan residue per molecule. No significant cross-reactivity was observed between the purified protein and polyclonal antibodies against either rat liver FABP or rat heart FABP. Amino acid sequencing of peptides obtained by digestion with Lys-C revealed that the catfish protein is structurally more similar to chicken liver FABP (69% identity in a 67-residue overlap) than to human liver FABPs (36%), nurse shark (Ginglymostoma cirratum) liver FABP (30%) and human heart FABP (31%). Taken together, these results suggest that catfish liver FABP is far more closely related to chicken liver FABP than to the FABPs isolated from the liver of mammals or elasmobranchs.