RESUMO
EgFABP1 is a developmentally regulated intracellular fatty acid binding protein characterized in the larval stage of parasitic platyhelminth Echinococcus granulosus. It is structurally related to the heart group of fatty acid binding proteins (H-FABPs). Binding properties and ligand affinity of recombinant EgFABP1 were determined by fluorescence spectroscopy using cis- and trans-parinaric acid. Two binding sites for cis- and trans-parinaric acid were found (K(d(1)) 24+/-4 nM, K(d(2)) 510+/-60 nM for cis-parinaric acid and K(d(1)) 32+/-4 nM, K(d(2)) 364+/-75 nM for trans-parinaric). A putative third site for both fatty acids is discussed. Binding preferences were determined using displacement assays. Arachidonic and oleic acids presented the highest displacement percentages for EgFABP1. The Echinococcus FABP is the unique member of the H-FABP group able to bind two long chain fatty acid molecules with high affinity. Structure-function relationships and putative roles for EgFABP1 in E. granulosus metabolism are discussed.
Assuntos
Proteínas de Transporte/metabolismo , Echinococcus/metabolismo , Ácidos Graxos/metabolismo , Proteínas de Peixes , Sequência de Aminoácidos , Animais , Sítios de Ligação , Ligação Competitiva , Proteínas de Transporte/biossíntese , Proteínas de Transporte/química , Echinococcus/química , Echinococcus/embriologia , Escherichia coli/metabolismo , Proteínas de Ligação a Ácido Graxo , Ácidos Graxos/química , Ácidos Graxos Insaturados/química , Larva/metabolismo , Dados de Sequência Molecular , Plasmídeos , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Espectrometria de Fluorescência , Relação Estrutura-AtividadeRESUMO
The fatty acid-binding proteins (FABPs) are a very well known protein family which includes the liver basic FABPs (Lb-FABPs), a subgroup so far characterized in several vertebrates but not in mammals. The most important difference recognized between the proteins in this subgroup and the better known mammalian liver FABPs (L-FABPs) is the stoichiometry of ligand binding: two fatty acid molecules in L-FABPs compared with one in Lb-FABPs. The only Lb-FABP with a known three-dimensional structure is that of chicken Lb-FABP, but the details of ligand binding are still unresolved as the crystals of the protein are grown at an acidic pH and the protein has been shown to lose its ligand under these conditions. The two proteins whose crystallizations are reported here are the second and third members of this subfamily to be crystallized. The crystals of axolotl Lb-FABP belong to either space group P4(1)2(1)2 or P4(3)2(1)2, with unit-cell parameters a = b = 65.38, c = 60.90 A, and diffract to a resolution of 2.0 A on a conventional source at room temperature. The crystals of toad Lb-FABP belong to either space group P4(1)22 or P4(3)22, with unit-cell parameters a = b = 48.14, c = 135.23 A, and diffract to 2.5 A resolution under the same conditions. It is expected that the solution of these two structures will help to clarify the structural differences between Lb-FABPs and L-FABPs and will possibly explain the different binding stoichiometries observed in these otherwise so similar protein subfamilies.
Assuntos
Proteínas de Transporte/química , Fígado/química , Proteínas de Neoplasias , Animais , Cristalização , Cristalografia por Raios X , Proteínas de Ligação a Ácido Graxo , Conformação Proteica , VertebradosRESUMO
Similar to those of other species, the Harderian glands of armadillo produce an abundant lipid secretion, most of which is composed of 1-alkyl-2,3-diacylglycerol. Biosynthesis of this component is apparently performed with the participation of one cytosolic pool of acyl-CoA and another of free fatty acids. The acyl-CoA-binding protein (ACBP) is present at a concentration at least 7-fold that of the heart-type fatty acid-binding protein (H-FABP), though lower than that in other armadillo organs such as liver and brain. The ACBP complete amino acid sequence was determined by Edman degradation of peptides generated by cleavage of the protein with cyanogen bromide, endopeptidase Glu-C, and trypsin. ACBP consists of 86 residues and has a calculated molecular mass of 9783 Da, taking into account that an acetyl group is blocking the N-terminus. Identity percentages between armadillo Harderian gland ACBP and other known ACBPs show that the protein belongs to the liver-specific ACBP isoform (L-ACBP). The fact that the ACBP concentration is higher than that of FABP suggests that the Harderian gland is able to store acyl-CoA amounts in ACBP larger than those of fatty acids in H-FABP for 1-alkyl-2,3-diacylglycerol synthesis.
Assuntos
Tatus/metabolismo , Proteínas de Transporte/química , Glândula de Harder/química , Metabolismo dos Lipídeos , Proteínas de Neoplasias , Proteínas Supressoras de Tumor , Sequência de Aminoácidos , Animais , Proteínas de Transporte/isolamento & purificação , Proteínas de Transporte/fisiologia , Cromatografia em Gel , Cromatografia em Camada Fina , Citosol/química , Inibidor da Ligação a Diazepam , Diglicerídeos/biossíntese , Proteína 7 de Ligação a Ácidos Graxos , Proteínas de Ligação a Ácido Graxo , Feminino , Glândula de Harder/metabolismo , Humanos , Mamíferos/metabolismo , Dados de Sequência Molecular , Especificidade de Órgãos , Alinhamento de Sequência , Especificidade da EspécieRESUMO
Only one fatty acid-binding protein (FABP) from the liver of the lungfish (Lepidosiren paradoxa) was isolated and characterized. The sequence comparison of lungfish FABP with that of the known members of the liver FABP (L-FABP) and liver basic FABP (Lb-FABP) subfamilies indicates that it is more closely related to chicken, iguana, frog, axolotl, catfish, and shark Lb-FABPs than to mammalian and axolotl L-FABPs. Lungfish liver expression of this single Lb-FABP contrasts with the other fish studied so far which coexpress an Lb-FABP with heart-adipocyte and/or intestinal FABP types. The lungfish liver FABP expression pattern resembles that of tetrapods, which only expresses liver type FABPs. Lungfish Lb-FABP is one of the two FABPs reported to have a disulfide bridge. The molecular modeling of lungfish Lb-FABP predicts that nine of the conserved residues of Lb-FABPs are oriented toward the binding cavity, thus suggesting they are related to the protein binding characteristics.
Assuntos
Proteínas de Transporte/química , Fígado/metabolismo , Proteínas de Neoplasias , Adipócitos/metabolismo , Sequência de Aminoácidos , Animais , Cromatografia , Citosol/metabolismo , Relação Dose-Resposta a Droga , Eletroforese em Gel de Poliacrilamida , Evolução Molecular , Proteínas de Ligação a Ácido Graxo , Peixes , Guanidina/farmacologia , Immunoblotting , Ligantes , Fígado/química , Espectrometria de Massas , Modelos Moleculares , Dados de Sequência Molecular , Ligação Proteica , Dobramento de Proteína , Estrutura Terciária de Proteína , Homologia de Sequência de Aminoácidos , Fatores de TempoRESUMO
A fatty acid-binding protein (FABP) from the cytosolic fraction of the triatomine Dipetalogaster maximus (Uhler) flight muscles was purified by a procedure based on gel filtration, reverse-phase high performance liquid chromatography, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The protein has an apparent molecular mass of 14 kDa, and its N-terminus is unblocked. Its N-terminal sequence was obtained by submitting an SDS-PAGE band blotted onto a polyvinylidene difluoride membrane to Edman degradation. The sequence obtained indicates that this FABP belongs to the heart type. This is the first time that a fatty acid-binding protein has been reported for a triatomine. The presence of said FABP, abundant mitochondria, and lipid stores in the flight muscles of D. maximus suggests that beta oxidation of fatty acids is used by the triatomine thoracic muscle as an energy source, and could be related to its dispersal capacity.
Assuntos
Proteínas de Transporte/análise , Lipídeos/análise , Músculo Esquelético/química , Proteínas de Neoplasias , Triatominae/química , Animais , Proteínas de Ligação a Ácido Graxo , Voo Animal , Músculo Esquelético/anatomia & histologia , Análise de Sequência de Proteína , Triatominae/anatomia & histologia , Asas de AnimaisRESUMO
Despite the evolutionary-tree data suggesting that gene duplication leading to the divergence of the three branches which heart, liver and intestinal fatty acid-binding proteins belong to must have occurred before the vertebrate/invertebrate split, only the heart fatty acid-binding protein has been reported for invertebrates. In an attempt to shed light on this apparent inconsistency the presence of the other two branch members was investigated in the Urochordata Molgula pedunculata, an ascidian species close to vertebrates. The mantle-, gonad- and digestive tube-cytosolic fractions, obtained by centrifugation at 106,000 g, were incubated separately with [1-(14)C]palmitic acid and then fractionated on a Sephadex G-75 column. In the case of gonads and digestive tube, radioactive peaks corresponding to a molecular mass of 14-16 kDa, characteristic of fatty acid-binding proteins, were detected. When the experiment was performed on the mantle, this peak showing fatty acid binding capacity was absent. Western Blot of the radioactive 14-16 kDa Sephadex fraction from the urochordate gonad cross-reacted with rat liver fatty acid-binding protein anti-serum but did not do so with anti-rat intestinal, adipocyte or heart fatty acid-binding protein antisera. The material from the digestive tube was not recognized by any of the antisera. The most abundant protein in said 14-16 kDa fraction was a protein disulphide isomerase-related protein. Its partial amino acid sequence was determined.
Assuntos
Citosol/metabolismo , Proteínas de Ligação a Ácido Graxo/metabolismo , Gônadas/metabolismo , Isomerases de Dissulfetos de Proteínas/metabolismo , Urocordados/metabolismo , Sequência de Aminoácidos , Animais , Proteínas de Ligação a Ácido Graxo/isolamento & purificação , Trato Gastrointestinal/metabolismo , Humanos , Dados de Sequência Molecular , Isomerases de Dissulfetos de Proteínas/isolamento & purificação , Alinhamento de SequênciaRESUMO
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
A fatty acid-binding protein from the cytosolic fraction of the armadillo Chaetophractus villosus Harderian gland was purified to homogeneity by a procedure based on gel filtration and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The protein has an apparent molecular mass of 14 kDa. N-terminal sequence analysis showed that the protein has a blocked N-terminus. For internal amino acid sequencing, the protein was digested in-gel and the resulting peptides were fractionated by reverse-phase high performance liquid chromatography and subjected to automated Edman degradation. Partial amino acid sequencing suggests that it belongs to the heart type. Moreover, it cross-reacted with anti-serum to rat heart fatty acid-binding protein but not with rat intestinal and liver anti-sera. A very slow cross-reaction was also found with anti-serum to rat ALBP. This is the first time that a fatty acid-binding protein has been reported in a Harderian gland.
Assuntos
Proteínas de Transporte/isolamento & purificação , Proteínas de Transporte/metabolismo , Glândula de Harder/metabolismo , Proteína P2 de Mielina/isolamento & purificação , Proteína P2 de Mielina/metabolismo , Proteínas de Neoplasias , Proteínas do Tecido Nervoso , Sequência de Aminoácidos , Animais , Tatus , Proteínas de Transporte/genética , Cromatografia Líquida de Alta Pressão , Proteína 7 de Ligação a Ácidos Graxos , Proteínas de Ligação a Ácido Graxo , Dados de Sequência Molecular , Proteína P2 de Mielina/genética , Ratos , Alinhamento de SequênciaRESUMO
NADP-linked glutamate dehydrogenase (NADP+-GluDH, EC 1.4.1.4) has been purified to homogeneity from epimastigotes of Trypanosoma cruzi by an improved procedure, and the amino acid sequences of 11 internal peptides obtained by digestion with trypsin, endopeptidase Lys-C, endopeptidase Arg-C or CNBr have been obtained. Using oligonucleotide primers synthesized according to the amino acid sequence of the N-terminus of the mature enzyme and to the nucleotide sequence of a clone corresponding to the C-terminus, obtained by immunological screening of an expression library, two complete open reading frames (TcGluDH1 and TcGluDH2) were isolated and sequenced. The sequences obtained are most similar to that of the NADP+-GluDH of Escherichia coli (70-72% identity), and less similar (50-56%) to those of lower eukaryotes. Using TcGluDH1 as a probe, evidence for the presence of several genes and developmental regulation of the expression of NADP+-GluDH in different parasite stages was obtained. TcGluDH1 encodes an enzymically active protein, since its expression in E. coli resulted in the production of a GluDH activity with kinetic parameters similar to those of the natural enzyme.
Assuntos
DNA de Protozoário/química , Desidrogenase de Glutamato (NADP+)/genética , Desidrogenase de Glutamato (NADP+)/isolamento & purificação , Trypanosoma cruzi/enzimologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Citosol/enzimologia , Escherichia coli/enzimologia , Evolução Molecular , Desidrogenase de Glutamato (NADP+)/metabolismo , Dados de Sequência Molecular , Proteínas Recombinantes/metabolismo , Trypanosoma cruzi/crescimento & desenvolvimentoRESUMO
Five fatty-acid-binding proteins from the liver of the elephant fish (Callorhynchus callorhynchus), a chimaera fish that belongs--together with the elasmobranchs--to the ancient chondrichthyes class were isolated and characterized. The purification procedures for these proteins involved gel filtration, anion-exchange chromatography, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis as a last step. They were submitted to "in gel" tryptic or cyanogen bromide digestion and the resulting peptides were separated by high performance liquid chromatography and then sequenced by Edman degradation. According to their partial amino acid sequences, one of them presents the highest identity with fatty-acid-binding proteins from human and catfish liver, another three with those from mammalian heart or adipose tissue and the fifth with the mammalian intestinal fatty-acid-binding protein. The presence of various members of this protein family, as now found in elephant fish and previously in catfish (Rhamdia sapo) liver, does not occur in mammalian liver which express only one a characteristic fatty-acid-binding protein.
Assuntos
Adipócitos/química , Proteínas de Transporte/análise , Peixes/metabolismo , Intestinos/química , Fígado/química , Proteína P2 de Mielina/análise , Miocárdio/química , Proteínas de Neoplasias , Proteínas do Tecido Nervoso , Proteínas Supressoras de Tumor , Sequência de Aminoácidos , Animais , Western Blotting , Peixes-Gato/metabolismo , Cromatografia por Troca Iônica , Proteína 7 de Ligação a Ácidos Graxos , Proteínas de Ligação a Ácido Graxo , Humanos , Soros Imunes , Dados de Sequência Molecular , Peso Molecular , RatosRESUMO
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
Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) is a powerful purification technique in protein chemistry research. This procedure is frequently used as a last step in protein purification for sequencing. For proteins which are N-terminal blocked, 'in gel' digestion offers a useful approach for the generation of internal sequence data from proteins purified by SDS-PAGE. In this study, we propose a procedure where proteins purified by this method are chemically cleaved 'in gel' by using CNBr and the resulting peptides are isolated in a second SDS-PAGE. After that, electroblotting is performed onto PVDF membranes and the electroblotted and Coomassie-stained peptide, from each band is then sequenced by Edman degradation. Proteins often have a small number of methionines whose cleavage allows the obtention of long peptides suitable to sequence a good deal of residues. Three standard proteins of different molecular mass have been assayed by this procedure and the 'in situ' cleavage profile compared with direct chemical digestion in a protein solution.
Assuntos
Eletroforese em Gel de Poliacrilamida/métodos , Proteínas/química , Análise de Sequência/métodos , Sequência de Aminoácidos , Western Blotting , Brometo de Cianogênio , Dodecilsulfato de SódioRESUMO
Calgranulin C (CAGC) from pig granulocytes has been crystallized and X-ray diffraction data have been collected to 2.6 A resolution. The crystals belong to the trigonal system, space group P3(1)21 or P3(2)21, cell parameters a = b = 54.35 (2), c = 141.32 (5) A and probably contain two molecules in the asymmetric unit. CAGC is amongst the first reported typical S100-1ike calcium-binding protein to be crystallized and studied by X-ray crystallography.
RESUMO
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.
Assuntos
Proteínas de Transporte/química , Proteínas de Transporte/isolamento & purificação , Peixes-Gato/metabolismo , Fígado/metabolismo , Proteína P2 de Mielina/química , Proteína P2 de Mielina/isolamento & purificação , Proteínas de Neoplasias , Proteínas do Tecido Nervoso , Ácidos Palmíticos/metabolismo , Proteínas Supressoras de Tumor , Sequência de Aminoácidos , Animais , Proteínas de Transporte/metabolismo , Galinhas , Citosol/metabolismo , Proteína 7 de Ligação a Ácidos Graxos , Proteínas de Ligação a Ácido Graxo , Humanos , Dados de Sequência Molecular , Proteína P2 de Mielina/metabolismo , Ácido Palmítico , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/isolamento & purificação , Ratos , Homologia de Sequência de Aminoácidos , Especificidade da EspécieRESUMO
A basic fatty acid binding protein (FABP), closely related to that of chicken liver, was isolated and characterized from catfish (Rhamdia sapo) liver in a previous work. Results herein show the presence of another two FABPs in which partial amino acid sequences reveal great similarity with the corresponding sequences of other already known FABPs belonging to the heart type. The purification procedures for both proteins involve gel filtration, anion-exchange chromatography, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (as a last step). Because both FABP N-termini were blocked, they were submitted to in-gel tryptic digestion and the resulting peptides were separated by high performance liquid chromatography, and sequenced by Edman degradation. One of these proteins presented the highest identity percentage when compared with those of the human and bovine heart and bovine brain (81%), and the other when compared with those of chicken retina (75%) and mouse and bovine heart FABP (70%). The presence of several FABPs plus the fact that they belong to different types, as found in the Rhamdia sapo liver, is unusual in mammals, which express a characteristic liver-type member of this protein family.
Assuntos
Proteínas de Transporte/isolamento & purificação , Fígado/química , Proteína P2 de Mielina/isolamento & purificação , Proteínas de Neoplasias , Proteínas do Tecido Nervoso , Proteínas Supressoras de Tumor , Sequência de Aminoácidos , Animais , Proteínas de Transporte/classificação , Peixes-Gato , Bovinos , Galinhas , Proteína 7 de Ligação a Ácidos Graxos , Proteínas de Ligação a Ácido Graxo , Humanos , Camundongos , Dados de Sequência Molecular , Peso Molecular , Proteína P2 de Mielina/classificação , Homologia de Sequência de AminoácidosRESUMO
The fatty acid binding protein (FABP) from toad liver cytosol was purified to homogeneity by a procedure involving gel filtration and anion exchange chromatography. The protein presented a molecular mass of 13 987 +/- 2 daltons determined by electrospray mass spectrometry. Native isoelectric focusing of the purified liver FABP revealed a single pI 6.8 band. On the other hand, the toad heart FABP showed a different mobility than that of toad liver FABP by both sodium dodecyl sulfate-polyacrylamide gel electrophoresis and isoelectric focusing. Moreover, toad liver FABP cross-reacted with antisera to mammalian liver FABP but not with antisera to heart FABP. The difference between toad liver and heart FABPs was further confirmed by partial amino acid sequencing. As the N-terminus of toad liver FABP was blocked, the protein was chemically and enzymatically cleaved and the resulting peptides were subjected to automated Edman degradation. Partial amino acid sequencing showed that the toad liver FABP is related to that of mammalian liver and is clearly different from the amphibian heart FABP as well as from the amphibian intestine FABP.