Nonenzymatic proteins mediating intracellular lipid transport and metabolism. Current status and emerging trends.
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Animals↗
Biomedical subjects
F Spener
Publications and source records attributed to F Spener.
Subcellular distribution of cardiac fatty acid-binding protein in bovine heart muscle and quantitation with an enzyme-linked immunosorbent assay.
Several types of the 14-15 kDa fatty acid-binding proteins (FABPs) are known to occur in the cytosol of mammalian cells. With antibodies raised against the cardiac-type protein from bovine heart, immunoblots indicated a more widespread distribution of the cardiac FABP in subcellular fractions, such as mitochondria and nuclei. A detailed view was obtained when the post-embedding protein A-gold labeling method was applied to cross-sections of heart cells and isolated subcellular fractions. Cardiac FABP in myocytes was associated with myofibrils and localized within mitochondria and nuclei. After subfractionation of mitochondria, the binding protein was recovered with matrix proteins only. A non-competitive enzyme-linked immunosorbent assay (ELISA) of the direct type was developed specifically for bovine cardiac FABP. This assay was sensitive in the range of 0.05 to 1 ng, and concentrations of cardiac FABP per mg protein were found for cytosol, matrix and nuclei to be around 3.18, 0.18 and 0.03 micrograms, respectively. The newly found compartmentation of cardiac FABP in the heart cell must be considered when the true functions of the protein, yet to be defined, are studied.
Animals↗
On the role of fatty acid binding proteins in fatty acid transport and metabolism.
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Animals↗
Compartmentation of hepatic fatty-acid-binding protein in liver cells and its effect on microsomal phosphatidic acid biosynthesis.
Fatty-acid-binding proteins are known to occur in the cytosol of mammalian cells and to bind fatty acids and their CoA-esters. Application of the postembedding protein A-gold labeling method with antibody against the hepatic type fatty-acid-binding protein (hFABP) to cross-sections of liver cells and a newly developed gel-chromatographic immunofluorescence assay established qualitatively (1) that hFABP in mitochondria was confined to outer mitochondrial membranes, (2) the presence of this protein in microsomes and (3) that nuclei were also filled with hFABP. Quantitative data elaborated with a non-competitive ELISA confirmed these results. A significant difference to the distribution of cardiac FABP in heart muscle cells, where this type of protein was found in cytosol, matrix and nuclei, was observed (Börchers et al. (1989) Biochim. Biophys. Acta, in the press). hFABP-containing rat liver microsomes were incubated with long-chain acyl-CoAs in the presence of hFABP (isolated from rat liver cytosol) in a study on the acylation of sn-glycerol-3-phosphate and lysophosphatidic acid. Both acyltransferases were stimulated by addition of hFABP to the incubation medium. The morphological, immunochemical as well as kinetic data infer a direct interaction of hFABP with microsomal membranes in liver cells.
Acyltransferases↗
Cloning of a full-length complementary DNA for fatty-acid-binding protein from bovine heart.
A full-length cDNA for bovine heart fatty-acid-binding protein (H-FABP) was cloned from a lambda gt11 cDNA library established from bovine heart muscle. The cDNA sequence shows an open reading frame coding for a protein with 133 amino acids. Colinearity with the amino acid sequences of four tryptic peptides was asserted. H-FABP isolated from bovine heart begins with an N-acetylated valine residue, however, as derived from analysis of the tryptic, amino-terminal-blocked peptide and the molecular mass of the peptide obtained via secondary-ion mass spectrometry. The molecular mass of the total protein is 14673 Da. Bovine H-FABP is 89% homologous to rat H-FABP and 97% homologous to the bovine mammary-derived growth-inhibition factor described recently by Böhmer et al. [J. Biol. Chem. 262, 15137-15143 (1987)]. Significant homologies were also found with bovine myelin protein P2 and murine adipocyte protein p422. Secondary-structure predictions were proposed for these proteins, based on computer analysis, which reveal striking similarities.
Acetylation↗
Interactions of fatty acids with neutral fatty-acid-binding protein from bovine liver.
Hepatic-type fatty-acid-binding protein (hFABP) from the cytosol of bovine liver is a 14.4-kDa neutral protein with a blocked N-terminus and a disulfide system located on the surface of the protein. It binds two molecules of fatty acid in one binding site, apparent dissociation constants of the oleic acid/hFABP complex are 0.24 microM and 2.15 microM. Computer analysis of circular dichroic spectra predicts that hFABP contains about 12% alpha-helix, 45% beta-structure, 15% beta-turn and 27% unordered structure. Ellipticities indicative of secondary structure are not affected by fatty acid binding. Cationic amino acid residues of hFABP (1 His, 15 Lys, 2 Arg) were screened for ionic fatty acid/protein interactions. His was excluded, as 1H-NMR analysis of His-C2 and His-C4 protons indicated that binding of oleic acid shifts the pK of His from 6.9 to 7.1 only in hFABP with the disulfide system in the oxidized state; acylation of His with diethylpyrocarbonate does not affect the binding of the fatty acid. Acetylation of Lys reduces binding marginally, whereas modification of Arg with phenylglyoxal lowers the binding activity by 65%. From 1H-NMR investigations, conformational changes within the protein, due to a sort of disaggregation of hFABP upon fatty acid binding, were derived. Most of the proton resonances sharpen up with ligand binding, and some of the methyl resonances shift positions, possibly because they are directly involved in the fatty acid/protein interaction.
Animals↗
A novel acyl-CoA-binding protein from bovine liver. Effect on fatty acid synthesis.
Bovine liver was shown to contain a hitherto undescribed medium-chain acyl-CoA-binding protein. The protein co-purifies with fatty-acid-binding proteins, but was, unlike these proteins, unable to bind fatty acids. The protein induced synthesis of medium-chain acyl-CoA esters on incubation with goat mammary-gland fatty acid synthetase. The possible function of the protein is discussed.
Animals↗
Production and testing of a proposed reference preparation of the purified main allergen from castor beans.
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Allergens↗
Isolation and characterization of the fatty acid binding protein from human heart.
We have isolated in pure form a fatty acid binding protein (FABP) from human cardiac muscle. After preparation of a 100,000 g supernatant fraction, the procedure required only one gel chromatographic (Sephacryl S 200) and two cation exchange (CM-Sephadex C 50) steps. The recovery of FABP was 55%. Pure FABP (12.5 mg) was obtained from a 1-g of dry powder equivalent of the high-speed supernatant. The protein had an Mr of 15,500 +/- 1,000 Da and an isoelectric point of 5.3. The properties of human cardiac FABP, i.e., molecular mass, isoelectric point, amino acid composition, ultraviolet spectrum, and affinities for hydrophobic ligands, were close to those found for FABPs from bovine heart (Jagschies et al. 1985. Eur. J. Biochem. 152: 537-545). In addition, immunological cross-reactivities showed a relationship between FABPs from several mammalian heart tissues. The data elaborated by us and others support the existence of a cardiac-type FABP that is distinct from the well-defined hepatic-type and gut-type FABPs.
Amino Acids↗
Bovine fatty acid binding proteins. Isolation and characterisation of two cardiac fatty acid binding proteins that are distinct from corresponding hepatic proteins.
When a 100,000 X g supernatant from bovine heart was incubated with [1-14C]oleic acid and subjected to isoelectric focusing, two fatty acid binding proteins (FABPs) with isoelectric points at 4.9 and 5.1 were detected. The proteins were purified on a large scale first by heat and acid precipitation of a postmitochondrial supernatant, as well as fractionation with ammonium sulfate, then by alternate application of ion-exchange and gel chromatography. The procedure afforded around 60 mg pure proteins from 1.5 kg fresh heart muscle. Relative molecular masses of 15 300 +/- 1600 for both proteins were derived from sodium dodecyl sulfate/polyacrylamide gel electrophoresis, gel chromatography, sedimentation velocity as well as from amino acid analysis. Up to 50% of the proteins' secondary structures consisted of beta-sheet. N-termini of the peptide chains were blocked; the amino acid compositions of the two proteins were similar, but differed considerably from those of the two FABPs isolated from bovine liver [Haunerland et al. (1984) Hoppe Seyler's Z. Physiol. Chem. 365, 365-376]. Whereas hepatic FABPs changed their pI upon binding fatty acids, cardiac FABPs did not. Cardiac FABPs were immunologically identical, but did not cross-react with hepatic proteins. A reversible, concentration-dependent self-association reported for FABP from pig heart [Fournier et al. (1983) Biochemistry 22, 1863-1872] was not observed for FABP from bovine heart. Changes of concentration did not alter secondary structure, intrinsic fluorescence or the sedimentation coefficient of the protein.
Amino Acids↗
Spectroscopic investigations on the binding site of bovine hepatic fatty acid binding protein. Evidence for the existence of a single binding site for two fatty acid molecules.
The hydrophobic region of the binding site of a bovine fatty acid binding protein (pI 7.0-FABP) has been characterized using fluorescence and circular dichroism (CD) spectroscopy. Blue-shifts of fluorescence emission maxima and increased lifetimes of naphthylamine dyes, anthroyloxy-fatty acids, pyrene nonanoic acid and trans-parinaric acid indicated a hydrophobic interaction with FABP. The fluorescence quenching of various anthroyloxy-fatty acids by iodide and acrylamide showed lower accessibility to the fluorophore linked to the carbon adjacent to the carbonyl group and towards the methyl end of the fatty acid. Binding stoichiometries were different for fatty acids and their bulky fluorescent analogues. trans-Parinaric acid when bound to FABP showed a complex induced CD-spectrum, which is explained by a close proximity of two ligands in the same binding site. Fluorescent derivatives of phosphatidylcholine with trans-parinaric acid and cholesteryl trans-parinarate did not bind to FABP. Thus, the binding site appears to be constructed for high affinity binding of long chain fatty acids.
Animals↗
X-ray studies on triclinic crystals of fatty acid binding protein. Examples of an extremely X-ray-resistant protein.
Fatty acid binding protein (pI 7.0) from bovine liver cytosol was crystallized using polyethylene glycol 4000 and 6000 as precipitating agents. The crystals are triclinic, space group P1. One molecule of 14 kDa occupies the unit cell with constants a = 33.5 A, b = 39.4 A, c = 30.6 A, alpha = 113.6 degrees, beta = 113.8 degrees, gamma = 88.8 degrees. Crystal diffraction extends to at least 2.25 A resolution and the crystals are stable in the X-ray beam for more than 450 h. One native data set to 2.5 A resolution has been collected.
Animals↗
Fatty-acid-binding proteins. Occurrence of two fatty-acid-binding proteins in bovine liver cytosol and their binding of fatty acids, cholesterol, and other lipophilic ligands.
Fatty-acid-binding proteins (FABPs) are known as cytosolic binding sites for fatty acids and their CoA esters. Radioactively labeled and fluorescent fatty acids were used to locate and identify these proteins in bovine liver cytosol. The occurrence of two species of FABPs was demonstrated and these were designated pI6.0-FABP and pI7.0-FABP according to their isoelectric points in the delipidated state. Oleic acid/FABP binding ratios were 1 with pI6.0-FABP and 2 with pI7.0-FABP. Upon binding of oleic acid the isoelectric points of liganded FABPs shifted to pH 5.0-5.1 in each case. Both proteins were purified by removing nonbinding proteins by acid and heat denaturation and subsequent gel filtration. By making use of the pI shifts observed upon lipidation and delipidation of the binding proteins with ligand fatty acids, final purification was achieved in two fractionations by isoelectric focusing. The binding proteins (Mr 11 800 +/- 1 000) had similar amino-acid compositions (no Trp) and were not covalently modified by carbohydrate and fatty acid. Fatty acids and their CoA esters were complexed by either FABP, cholesterol only by pI-7.0-FABP, though non-stoichiometrically. 16-(9-Anthroyloxy)palmitic acid was bound by pI-7.0-FABP in a 1:1 ratio and precluded the additional binding of a straight-chain fatty acid. Electrophoretic titration curves indicated dissociation of the oleic acid/pI7.0-FABP complex below pH 5.0. It appears that fatty acids and their CoA esters are the foremost binding partners of FABPs in vivo. The results are discussed in terms of a single binding site for fatty acids per molecule FABP.
Amino Acids↗
The analysis of fatty acid binding to protein using a modified equilibrium dialysis method: detailed analysis of chromophore-fatty acid-protein interactions.
In this paper we extend our previous analysis of fatty acid-chromophore-protein interactions using a modified equilibrium dialysis method described previously. A more rigorous mathematical treatment is combined with a micro-dialysis method using a maximum volume of dialyzate of between 250 microliters and 400 microliters to examine the suitability of different chromophores (mepacrine, quinine, chloroquine, chlorpromazine, methylene blue, rhodamine 6G, 6-carboxyfluorescein) for studying the binding of fatty acid to protein. The macro- and micro-methods of dialysis are compared, and the binding of fatty acid to bovine serum albumin and beta-lactoglobulin discussed as examples of the method. Problems associated with propagated errors in the measurements and obtaining the number of binding sites and the binding constants from curve-fitting are also considered.
Binding Sites↗
Dynamics of Galactolipids and Plastids in Nonphotosynthetic Cells of Glycine max Suspension Cultures : A Morphological and Biochemical Study.
The age-dependent interrelationship of galactolipids and plastids in heterotrophic cell suspension cultures of Glycine max (soybean) was studied with regard to aging of nonphotosynthetic cells. Cells were propagated in the dark and under illumination with white light, and were harvested at days 7 (end of logarithmic phase), 14, and 21 (extended stationary phase). Electron microscopy revealed in dark-grown cells a proliferating decay of the amyloplast-type plastids, which could be correlated to a decrease of galactolipids. This trend was dramatically reversed in irradiated cultures, where the plastids of day 21 cells appeared rejuvenated. A concomitant increase of galactolipid content in the cells was observed, yet chlorophyll synthesis and photosynthetic activity were not induced. The dynamics of galactolipid contents did not correlate with total lipid contents in dark-grown as well as in irradiated cultures. [(3)H]Galactose served as a radioactive probe for the subcellular localization of galactolipids by electron microscopic autoradiography. Apart from plastids, galactolipids may also be constituents of the plasma membrane. The results render the heterotrophic cell suspension culture a suitable model to study the impact of senescence on plastids of nonphotosynthetic cells.
Journal Article↗
Isolation and biosynthesis of cyclopentenylglycine, a novel nonproteinogenic amino acid in Flacourtiaceae.
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Carbon Radioisotopes↗
Localisation and characterization of the fatty acid synthesizing system in cells of Glycine max (soubean) suspension cultures.
In course of a study of fatty acid synthetase in higher plants, non-green cell suspension cultures of Glycine max (soybean) served as model tissues. For the first time, a fatty acid synthesizing system was characterized in cell cultures of higher plants and was found to be solely located in proplastids of the cells. Optimum activity of the fatty acid synthesizing system in proplastids was observed between pH 8.0 and 8.2; with [1-14C]acetate as substrate, cofactors required were CoA, ATP, Mn2+, Mg2+, HCO3-, NADH and NADPH. The system was more sensitive towards NADH than NADHP. [1-14C]Acetate,[2-14C]-malonate and [3-14C]pyruvate served as precursors for fatty acids, indicating the presence of pyruvate dehydrogenase activity in proplastids. In disrupted proplastids, [2-14C]malonylCoA was a better precursor than [1-14C]acetylCoA. After incubation of proplastids with [2-14C]malonate, a small shift, from palmitic acid to higher homologs, of label incorporated was observed, as compared to incorporation of label from [1-14C]acetate and [3-14C]pyruvate. Under the conditions of the experiment, only small amounts of polyunsaturated fatty acids, the main fatty acid components of this organelle, were synthesized. In respect to fatty acid synthesis, the non-green cell suspension culture resembles photosynthetic leaf tissue.
Cells, Cultured↗
Biosynthesis of cyclopentenyl fatty acids. Cyclopentenylglycine, a non-proteinogenic amino acid as precursor of cyclic fatty acids in Flacourtiaceae.
In seeds of Hydnocarpus anthelminthica of Flacourtiaceae, cyclopentenylglycine and cyclopentenyl fatty acids are found naturally. The non-proteinogenic amino acid may serve as precursor of cyclopentenyl fatty acids via aleprolic acid, the starter molecule for these long-chain compounds. After administration of cyclopentenyl[2-14C]glycine to maturing seeds of H. anthelminthica, labelled cyclopentenyl fatty acids were synthesized. Comparative activities were observed, when [1-14C]aleprolic acid was supplied to the seeds. Incorporation studies with [1-14C]acetate revealed that the chain-lengthening systems for straight-chain and cyclic fatty acids were still functioning in mature seeds. Endosperm and embryo of H. Anthelminthica seeds synthesized cyclopentenyl fatty acids from cyclopentenyl[2-14C]glycine, [1-14C]aleprolic acid and [1-14C]acetate. In embryonic tissue, a dilution experiment proved the following path for cyclopentenyl fatty acid biosynthesis: cyclopentenylglycine leads to aleprolic acid leads to cyclopentenyl fatty acids. The conversion of cyclopentenylglycine to aleprolic acid may occur via transamination and oxidative decarboxylation; activated aleprolic acid is then lengthened by C2-units to cyclopentenyl fatty acids.
Acetates↗