RESUMO
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
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
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
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.