Isolation, characterization and binding properties of two rat liver fatty acid-binding protein isoforms.

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.

Acyl-CoA-binding protein in the armadillo Harderian gland: its primary structure and possible role in lipid secretion.

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.

Binding properties of Echinococcus granulosus fatty acid binding protein.

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.

Complex assembly of calgranulins A and B, two S100-like calcium-binding proteins from pig granulocytes.

Calgranulin A (CAGA) and calgranulin B (CAGB) are two S100-like calcium-binding proteins that in human, bovine and mouse granulocytes are associated into a heterocomplex. We have previously identified in pig granulocytes the porcine homologue of CAGA and a novel S100-like protein which was named calgranulin C (CAGC). As pig CAGA is not associated with CAGC, we herein investigate its possible association with other proteins. CAGA was purified from pig granulocytes by gel filtration followed by Mono Q chromatography. The purified fractions were analysed by SDS-polyacrylamide gel electrophoresis, isoelectric focusing, mass spectrometry, chemical cross-linking and hydrophobic interaction chromatography. The CAGA-associated protein was further characterized by amino acid sequencing. Two CAGA-containing fractions were isolated. One of them was identified as a CAGA homodimer. The other fraction consists of a heterocomplex containing CAGA and a pI 7.0 calcium-binding protein; this protein has a molecular mass of 15,877.9 +/- 3.8 Da (mean +/- SD) whereas it migrates on 10 and 16% polyacrylamide gels as a 24- and 20-kDa protein, respectively. The pI 7.0 protein was identified by internal amino acid sequencing as the porcine homologue of CAGB. The stoichiometry of the heterocomplex was estimated to be 1:1. Both the CAGA homodimer and CAGA/CAGB were found to be non-covalently associated. Unlike the homodimer, CAGA/CAGB was bound to a Phenyl Superose column in a calcium-dependent manner. Our results suggest that pig granulocytes contain, in addition to CAGC, a CAGA homodimer and a CAGA/CAGB heterodimer. It is proposed that CAGB/CAGB and the CAGA homodimer may play different roles in vivo.

Presence of intestinal, liver and heart/adipocyte fatty-acid-binding protein types in the liver of a chimaera fish.

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.

Presence of a fatty acid-binding protein in the armadillo Harderian gland.

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.

Purification and partial structural and kinetic characterization of an aromatic L-alpha-hydroxy acid dehydrogenase from epimastigotes of Trypanosoma cruzi.

An aromatic L-alpha-hydroxyacid dehydrogenase (AHADH) was purified to homogeneity from epimastigotes of Trypanosoma cruzi by a method involving chromatography on DEAE-cellulose, hydrophobic interaction chromatography on Phenyl-Sepharose and affinity chromatography on Affi-Gel Blue. The purified enzyme showed a single band in SDS-PAGE, with an apparent molecular mass of 36 kDa. Since the apparent molecular mass of the native enzyme, determined by gel filtration, is about 80 kDa, the native enzyme is a dimer of similar subunits. The amino acid composition was determined, as well as the sequences of 4 internal peptides obtained by CNBr cleavage at Met residues, and one peptide obtained after tryptic digestion. Three of the peptides presented considerable sequence similarity with the corresponding sequences of several malate dehydrogenases. The optimal pH for the enzyme reaction with p-hydroxyphenyl pyruvate and NADH as substrates was 7.5; that for the reverse reaction was 9.5. The apparent Km values for phenylpyruvate and p-hydroxyphenyl-pyruvate were 48 and 117 microM, respectively; that for L-phenyllactate in the reverse reaction was 420 microM. The enzyme was much less active with alpha-isocaproic acid as substrate, and other acids, including pyruvic and oxaloacetic, were not substrates at all. L-phenyllactic acid, but not the D-isomer, acted as substrate. The enzyme can therefore be considered as a general aromatic L-alpha-hydroxyacid dehydrogenase. The low apparent Km value for NADH (25 microM in the presence of phenylpyruvate) makes AHADH a candidate for the reoxidation of cytosolic NADH in T. cruzi.

Rat brain fatty acid-binding protein during development.

Cytosolic fatty acid-binding proteins (FABPs) have been described in rat and bovine whole brain. In the present study we investigated the distribution of FABP among white matter and gray matter as well as its changes during development. Fatty acid binding activity was similar in white and gray matter up to 40 days of age. In white matter it showed an age dependent increase thereafter, while in gray matter it remained constant throughout. Gel filtration (Sephadex G-75) of white matter cytosol of adult female rats resolved the fatty acid-binding activity in two peaks: A (Vo) and B (12-14 KDa; FABP). The specific binding activity in the FABP fraction was 10.4 pmol/micrograms of protein. The activity in peak A showed an age-dependent increase which paralleled myelin deposition. In contrast, the activity in the FABP fraction (peak B) remained undetectable up to 40 days of age, increasing thereafter. The differential distribution of cellular brain proteins with the capacity to bind fatty acids in gray matter and white matter suggests that this activity could be related to glial cells or to cell related structures such as myelin.

Production of aromatic alpha-hydroxyacids by epimastigotes of Trypanosoma cruzi, and its possible role in NADH reoxidation.

Epimastigotes of Trypanosoma cruzi in culture produce and excrete into the medium small amounts of phenyllactic acid and p-hydroxyphenyllactic acids, presumably arising from the catabolism of the aromatic amino acids phenylalanine and tyrosine, respectively. This production might constitute a minor pathway for the reoxidation of cytosolic NADH, through the concerted action of tyrosine aminotransferase and aromatic alpha-hydroxyacid dehydrogenase.

Presence and subcellular localization of tyrosine aminotransferase and p-hydroxyphenyllactate dehydrogenase in epimastigotes of Trypanosoma cruzi.

Cell-free extracts of epimastigotes of Trypanosoma cruzi contain tyrosine aminotransferase (TAT) and p-hydroxyphenyllactate dehydrogenase (pHPLDH). The TAT activity could be separated from aspartate aminotransferase (ASAT) by polyacrylamide gel electrophoresis or DEAE-cellulose chromatography; the latter procedure also allowed complete separation of pHPLDH. The subcellular localization of both T. cruzi enzymes, as determined by digitonin extraction, subcellular fractionation by differential centrifugation, and isopycnic ultracentrifugation in sucrose gradients, was mainly cytosolic, with low mitochondrial activities.

A recombinant tyrosine aminotransferase from Trypanosoma cruzi has both tyrosine aminotransferase and alanine aminotransferase activities.

Tyrosine aminotransferase purified from epimastigotes of Trypanosoma cruzi displays an additional activity of alanine aminotransferase, absent in all other tyrosine aminotransferases characterized so far. Since the parasite's genome contains a high number of copies of the tyrosine aminotransferase gene, we could not rule out the possibility that two very similar proteins, with changed specificity due to a few amino acid substitutions, might be responsible for the two activities. We have now expressed in Escherichia coli a recombinant tyrosine aminotransferase as a fusion protein with glutathione S-transferase. The purified fusion protein, intact or after thrombin cleavage, displays tyrosine aminotransferase and alanine aminotransferase activities with apparent Km values similar to those for the natural enzyme, thus proving that they belong to the same protein.

Aggregation of human neutrophils by heparin.

Commercial heparin preparations induce the aggregation of human polymorphonuclear neutrophils (PMNs). The minimum aggregating concentration (MAC) of different heparin lots was measured under standardized conditions. It was found to vary between 0.3 and 15 units/ml. Gel filtration of heparin showed that the aggregating activity eluted in a sharp peak with a pattern different from heparin. When heparin was bound to antithrombin III-Sepharose, the aggregating activity eluted totally in the high-affinity fraction. When PMNs are partially aggregated with heparin, further aggregation by N-formyl-methionyl-leucyl-phenylalanine (FMLP) or phorbol myristate-acetate (PMA) is not affected. Pretreatment of PMNs with a low-aggregating heparin fraction, inhibits further aggregation by a high-aggregating heparin fraction. These results suggest that the PMNs have binding sites for heparin different from those for PMA and FMLP. Binding of heparin is not enough for inducing aggregation. Certain structural requirements of the heparin molecule are probably essential for aggregation.

Presence of a fatty acid-binding protein and lipid stores in flight muscles of Dipetalogaster maximus (Hemiptera: Reduviidae).

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.

'In gel' cleavage with cyanogen bromide for protein internal sequencing.

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.

Purification and structural characterization of a fatty acid-binding protein from the liver of the catfish Rhamdia sapo.

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.

Purification and partial structural characterization of a fatty acid-binding protein from the liver of the South American armadillo Chaetophractus villosus.

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.

Inhibitory action of palmitic acid on the growth of Saccharomyces cerevisiae.

High concentrations of long-chain fatty acids have been found to be harmful to mammalian cells and prokaryotic organisms. This effect was investigated in Saccharomyces cerevisiae. Addition of 3 mmol/L palmitate to a yeast extract-peptone medium caused a significant inhibition of cell growth during the first 2 d of incubation, followed by renewed growth and palmitate utilization. Inhibition was also observed with palmitate concentrations down to 0.1 mmol/L. As inferred from catalase activity determinations, this effect was found to correlate with the absence of peroxisome proliferation. Finally, no inhibition was observed in exponential-phase cultures or in the presence of 0.1 g/L glucose, this suggesting that the physiological state of the cell may determine whether its growth will be inhibited by fatty acids.