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Iron-Sulfur (Fe-S) proteins are involved in many biological functions such as electron transport, photosynthesis, regulation of gene expression and enzymatic activities. Biosynthesis and transfer of Fe-S clusters depend on Fe-S clusters assembly processes such as ISC, SUF, NIF, and CIA systems. Unlike other eukaryotes which possess ISC and CIA systems, amitochondriate Entamoeba histolytica has retained NIF & CIA systems for Fe-S cluster assembly in the cytosol. In the present study, we have elucidated interaction between two proteins of E. histolytica CIA system, Cytosolic Fe-S cluster deficient 1 (Cfd1) protein and Nucleotide binding protein 35 (Nbp35). In-silico analysis showed that structural regions ranging from amino acid residues (P33-K35, G131-V135 and I147-E151) of Nbp35 and (G5-V6, M34-D39 and G46-A52) of Cfd1 are involved in the formation of protein-protein complex. Furthermore, Molecular dynamic (MD) simulations study suggested that hydrophobic forces surpass over hydrophilic forces between Nbp35 and Cfd1 and Van-der-Waal interaction plays crucial role in the formation of stable complex. Both proteins were separately cloned, expressed as recombinant fusion proteins in E. coli and purified to homogeneity by affinity column chromatography. Physical interaction between Nbp35 and Cfd1 proteins was confirmed in vitro by co-purification of recombinant Nbp35 with thrombin digested Cfd1 and in vivo by pull down assay and immunoprecipitation. The insilico, in vitro as well as in vivo results prove a stable interaction between these two proteins, supporting the possibility of its involvement in Fe-S cluster transfer to target apo-proteins through CIA machinery in E. histolytica. Our study indicates that initial synthesis of a Fe-S precursor in mitochondria is not necessary for the formation of Cfd1-Nbp35 complex. Thus, Cfd1 and Nbp35 with the help of cytosolic NifS and NifU proteins can participate in the maturation of non-mitosomal Fe-S proteins without any apparent assistance of mitosomes.

The critical importance of long-chain fatty acids in cellular homeostasis demands an efficient uptake system for these fatty acids and their metabolism in tissues. Increasing evidence suggests that the plasma-membrane-associated and cytoplasmic fatty-acid-binding proteins are involved in cellular fatty acid uptake, transport and metabolism in tissues. These binding proteins may also function in the fine tuning of cellular events by modulating the metabolism of long-chain fatty acids implicated in the regulation of cell growth and various cellular functions. Several membrane-associated fatty-acid-binding/transport proteins such as plasma membrane fatty-acid-binding protein (FABPpm, 43 kDa), fatty acid translocase (FAT, 88 kDa) and fatty acid transporter protein (FATP, 63 kDa) have been identified. In the feto-placental unit, preferential transport of maternal plasma arachidonic and docosahexaenoic acids across the placenta is of critical importance for fetal growth and development. Our studies have shown that arachidonic and docosahexaenoic acids are preferentially taken up by placental trophoblasts for fetal transport. The existence of a fatty-acid-transport system comprising multiple membrane-binding proteins (FAT, FATP and FABPpm) in human placenta may be essential to facilitate the preferential transport of maternal plasma fatty acids in order to meet the requirements of the growing fetus. The preferential uptake of arachidonic and docosahexaenoic acids by the human placenta has the net effect of shunting these maternal plasma fatty acids towards the fetus. The roles of plasma membrane-associated binding/transport proteins (FABPpm, FAT and FATP) in tissue-specific fatty acid uptake and metabolism are discussed.

To understand the placental role in the processes responsible for the preferential accumulation of maternal long-chain polyunsaturated fatty acids (LCPUFAs) in the fetus, we investigated fatty acid uptake and metabolism in the human placenta. A preference for LCPUFAs over nonessential fatty acids has been observed in isolated human placental membranes as well as in BeWo cells, a human placental choriocarcinoma cell line. A placental plasma membrane fatty acid binding protein (p-FABP(pm)) with a molecular mass of approximately 40 kDa was identified. The purified p-FABP(pm) preferentially bound with essential fatty acids (EFAs) and LCPUFAs over nonessential fatty acids. Oleic acid was taken up least and docosahexaenoic acid (DHA) most by BeWo cells, whereas no such discrimination was observed in HepG2 liver cells. Studies on the distribution of radiolabeled fatty acids in the cellular lipids of BeWo cells showed that DHA is incorporated mainly into the triacylglycerol fraction, followed by the phospholipid fraction; the reverse is true for arachidonic acid (AA). The greater cellular uptake of DHA and its preferential incorporation into the triacylglycerol fraction suggests that both uptake and transport modes of DHA by the placenta to the fetus are different from those of AA. p-FABP(pm) antiserum preferentially decreased the uptake of LCPUFAs and EFAs by BeWo cells compared with preimmune serum. Together, these results show the preferential uptake of LCPUFAs by the placenta that is most probably mediated via the p-FABP(pm).

Vitamin E (alpha-tocopherol) is a lipid-soluble antioxidant which is present in cellular membranes where it plays an important role in the suppression of free radical-induced lipid peroxidation. There are eight naturally occurring homologues of vitamin E which differ in their structure and in biological activity in vivo and in vitro. Various studies have suggested that the tocopherol distribution system favours the accumulation of alpha-tocopherol both in the plasma and different tissues. Mechanisms involved in the preferential accumulation of alpha-tocopherol are not yet well established; however, recent data indicate that both intracellular and membrane alpha-tocopherol-binding proteins may be involved in these processes. A 30 kDa alpha-tocopherol-binding protein (TBP) in the liver cytoplasm is now known to regulate plasma vitamin E concentrations by preferentially incorporating alpha-tocopherol into nascent very low density (VLDL) whereas the 15 kDa TBP may be responsible for intracellular distribution of alpha-tocopherol. The 30 kDa TBP is unique to the hepatocyte whereas the 15 kDa TBP is present in all major tissues. The 15 kDa TBP specifically binds alpha-tocopherol in preference to the delta- and gamma-tocopherol and may exclusively transport alpha-tocopherol to these intracellular sites. In addition, the presence of a membrane TBP (TBPpm) in tissues may regulate their alpha-tocopherol levels. Activity of erythrocyte TBPpm appears to be reduced in smokers, which may lead to reduced levels of alpha-tocopherol in these cells despite smokers have similar plasma levels of vitamin E as in non-smokers. The current status of the evidence for this directed flow of alpha-tocopherol through interactions with these proteins (TBP and TBPpm) is discussed.

The aim of this study was to investigate location and the types of membrane-associated and cytoplasmic fatty acid-binding proteins in human placental trophoblasts using monospecific polyclonal antibodies. Western blot analysis demonstrated the presence of multiple membrane and cytoplasmic fatty acid transport/binding proteins in human placenta. In addition to previously reported placental membrane fatty acid-binding (p-FABPpm, 40 kDa), fatty acid translocase (FAT, 88 kDa) and fatty acid transport protein (FATP, 62 kDa) were detected in both microvillous and basal membranes of the human placenta. Among the cytoplasmic proteins, heart (H) and liver (L) type FABP were detected in the cytosol of the human placental primary trophoblasts as well as in human placental choriocarcinoma (BeWo) cells. The immunoreactivity of epidermal type (E)-FABP was not detected in trophoblasts or BeWo cells despite its presence in human placental cytosol. Location of FAT and FATP on the both sides of the bipolar placental cells may favour transport of free fatty acids (FFA) pool in both directions i.e. from the mother to the fetus and vice versa. However, p-FABPpm, because of its exclusive location on the microvillous membranes, may favour the unidirectional flow of maternal plasma long-chain polyunsaturated fatty acids present in the FFA pool to the fetus, due to binding specificity for these fatty acids. Although the roles of these proteins in placental fatty acid uptake and metabolism are yet to be understood fully, their complex interaction may be involved in the uptake of maternal FFA by the placenta for delivery to the fetus.

To elucidate further the role of placental membrane fatty acid-binding protein (p-FABPpm) in preferential transfer of maternal plasma long chain polyunsaturated fatty acids (LCPUFA) across the human placenta, direct binding of the purified protein with various radiolabelled fatty acids (docosahexaenoic, arachidonic, linoleic and oleic acids) was investigated. Binding of these fatty acids to the protein revealed that p-FABPpm had higher affinities and binding capacities for arachidonic and docosahexaenoic acids compared with linoleic and oleic acids. The apparent binding capacities (Bmax) values for oleic, linoleic, arachidonic and docosahexaenoic acids were 2.0 +/- 0.14, 2.1 +/- 0.17, 3.5 +/- 0.11, 4.0 +/- 0.10 mol per mol of p-FABPpm whereas the apparent dissociation constant (Kd) values were 1.0 +/- .0.07, 0.73 +/- 0.04, 0.45 +/- 0.03 and 0.4 +/- 0.02 microM, respectively (n=3). In the case of human serum albumin, the Kd and Bmax values for all fatty acids were around 1 microM and 5 mol/mol of protein, respectively. These data provide direct evidence for the role of p-FABPpm in preferential sequestration of maternal arachidonic and docosahexaenoic acids by the placenta for transport to the fetus by virtue of its preferential binding of these fatty acids.

In order to understand the mechanisms by which fatty acids are taken up by the placenta, the uptake of oleic, linoleic, arachidonic, and docosahexaenoic acids by cultured human placental choriocarcinoma (BeWo) cells was examined. Fatty acid uptake by BeWo cells was temperature-dependent and exhibited saturable kinetics. Oleic acid was taken up least and docosahexaenoic acid most by these cells. Moreover, competitive studies of fatty acid uptake by BeWo cells also indicated preferential uptake compared with oleic acid in the order of docosahexaenoic acid, arachidonic acid, and linoleic acid. Western blot analysis demonstrated that BeWo cells express a protein immunoreactive with antibodies to the human placental plasma membrane fatty acid-binding protein (p-FABPpm). Furthermore, pre-treatment of BeWo cells with these antibodies inhibited most of the uptake of docosahexaenoic (64%) and arachidonic acids (68%) whereas oleic acid uptake was inhibited only 32% compared with the controls treated with preimmune serum. These results clearly demonstrate that the pFABPpm may be involved in the preferential uptake of essential fatty acids (EFA) and their long chain polyunsaturated fatty acids (LCPUFA) by these cells. Studies on the distribution of radiolabeled fatty acids in the cellular lipids of BeWo cells showed that docosahexaenoic acid was incorporated mainly in the triacylglycerol fraction, followed by the phospholipid fraction, whereas for arachidonic acid the reverse was true. The preferential incorporation of docosahexaenoic acid into triacylglycerol suggests that triacylglycerol may play an important role in the placental transport of docosahexaenoic acid to the fetal circulation. Together these results demonstrate the preferential uptake of EFA/LCPUFA by BeWo cells that is most probably mediated via the pFABPpm. We thus propose that the p-FABPpm may be involved in the sequestration of maternal plasma LCPUFA by the placenta.

We reported earlier the presence of a 40 kDa plasma membrane fatty acid-binding protein (FABPpm) in human placenta. This protein is thought to be involved in the sequestration of unesterified free fatty acids bound to albumin from the maternal plasma for delivery to the fetus. However, its location in human placental syncytiotrophoblasts is not known. These cells are bipolar; one side facing maternal circulation (microvillous membranes), and the other side facing fetal circulation (basal membranes). Therefore, it is important to resolve the location of this protein in trophoblast membranes in order to understand fatty acid transport and metabolism in human placenta. Isolated plasma membranes vesicles were prepared respectively from the maternal facing microvillous and fetal facing surface of the human full-term placental syncytiotrophoblast. Using these membrane preparations, fatty acid binding activity, the polyacrylamide gel electrophoresis radiobinding assay for FABPpm, and Western blot analysis of FABPpm were carried out to determine the location of this protein in these membranes. Based on the above studies we conclude that the FABPpm is located exclusively in the microvillous membranes. Since FABPpm may be responsible for FFA uptake, its location in the microvillous membranes favours the unidirectional flow of maternal FFA to the fetus.

A plasma membrane fatty-acid-binding protein (FABPpm) with a molecular mass of approximately 40 kDa has been identified in human placenta. Binding of both [14C] oleate and [14C] linoleate to human placental membranes was found to be time and temperature dependent. Sulphobromophthalein and alpha-tocopherol did not show competition with the [14C] fatty acid binding. These data suggest that the binding sites are specific for fatty acids. incubation of the membranes with trypsin reduced fatty acid binding activity, indicating that the binding sites were protein in nature. A FABPpm was then solubilized from placental membranes and purified to electrophoretic homogeneity. The fatty acid binding activity of the purified protein was confirmed by autoradioblotting. Polyclonal antiserum raised to FABPpm reduced fatty acid binding to placental membrane significantly compared with preimmune serum. The pI value and the amino acid composition of the protein suggest that the placental FABPpm is different from the previously identified hepatic FABPpm.

We previously reported the identification of a new alpha-tocopherol-binding protein (approximately 15 kDa) in the cytosol of rat liver and heart and in rabbit heart (A.K. Dutta-Roy et al., J. Nutr. Biochem. 5, 562-570, 1994). This protein specifically binds alpha-tocopherol and enhances its transfer between separate membranes. In the present paper we have purified and characterized the alpha-tocopherol-binding protein from bovine heart cytosol and compared its various structural and functional properties with the similar size (approximately 15 kDa) cytosolic fatty acid-binding protein of this tissue. alpha-Tocopherol-binding protein was purified to electrophoretic homogeneity from bovine heart cytosol by a procedure involving precipitation with 70% ammonium sulfate, followed sequentially by gel filtration chromatography and chromatofocusing. The purified protein migrated as a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis with an apparent molecular mass of 16 kDa. Isoelectric focusing of the purified protein showed that the pI value is around 4.5. The binding of alpha-tocopherol to the purified protein was rapid, reversible, and saturable. The alpha-tocopherol-binding protein did not bind oleate as assessed by direct radiolabeled fatty acid binding and fluorescence enhancement assay. Amino acid analysis showed the presence of a large number of Ala, Gly, Ser, Lys, and Pro residues and a lesser number of aromatic residues in this protein. Anti-bovine heart fatty acid-binding protein antibody did not recognize the alpha-tocopherol-binding protein in the Western blot. The Western blot, ligand affinity, molecular size, and amino acid analysis data suggest that the alpha-tocopherol-binding protein is different from the cytosolic fatty acid-binding protein and that it may be involved in intracellular transport and metabolism of alpha-tocopherol.

We have examined the expression of insulin-like growth factor 1 (IGF-1), IGF binding protein-1 (IGFBP-1), and IGF binding protein-3 (IGFBP-3) in the rat distal nephron during increased cell work load and hypertrophy, induced by the diuretic, furosemide. Furosemide was given for six days to increase distal sodium delivery and uptake. To mitigate salt loss, the animals drank 0.8% NaCl and 0.1% KCl. Control rats were infused with vehicle (0.9% saline) and drank tap water. Furosemide increased urinary volume (13-fold) and sodium excretion (eightfold), and decreased urine osmolarity (fourfold). By immunocytochemistry, staining for IGF-1 and IGFBP-1 was markedly increased in distal convoluted tubules and cortical collecting ducts; both segments also underwent hypertrophy. Increased staining for the peptides was evident early (1 hr, 18 hr) after furosemide, prior to hypertrophy of cells. Whereas transcripts of IGF-1 and IGFBP-3 mRNA showed little or no increase in extracts from furosemide-treated kidney cortices, IGFBP-1 mRNA was increased threefold 18 hours after furosemide. Alterations of IGF-1 and IGFBP-1 were independent of changes in plasma aldosterone, glucocorticoids or arginine vasopressin. That IGFBP-1 mRNA increased threefold without significant changes in IGF-1 mRNA suggests that hypertrophic stimuli might initially induce the synthesis of IGF binding protein followed by the trapping of extracellular IGF-1. The present study raises the possibility of IGF-1 and IGFBP-1 being involved in processes that lead to tubular hypertrophy. IGFBP-1 may regulate these effects by binding to and interaction with IGF-1.

Fatty acid-binding protein (FABPpm) has been identified and characterised from sheep placental membranes. Binding of [14C]oleate to placental membranes was found to be time- and temperature-dependent. Addition of a 20-fold excess unlabelled oleic, palmitic, or linoleic acid reduced the binding of [14C]oleate to the membranes to around 50% of total binding, whereas D-alpha-tocopherol at similar concentrations did not affect [14C]oleate binding. This indicates that the binding sites are specific to fatty acids. Specific binding of [14C]oleate was reduced by heat denaturation or trypsin digestion of the membranes, suggesting that the fatty acid-binding sites are protein in nature. FABPpm was then solubilised from sheep placental membranes, and subsequently purified to electrophoretic homogeneity using an oleate-agarose affinity column. The purified FABPpm had an apparent molecular mass of 40 kDa, as determined by SDS-PAGE and by gel permeation chromatography. The [14C]oleate-binding activity of the purified protein was also confirmed by PAGE followed by autoradioblotting. The specific binding for oleate was around 1.5 nmol per mg of membrane protein. Our data indicate the presence of FABPpm in sheep placental membranes.

An alpha-tocopherol-binding protein (TBP) with a molecular mass of 14.2 kDa has been identified from the cytosol of rat heart and liver and purified to electrophoretic homogeneity by precipitation with 70% ammonium sulphate, followed by gel filtration and ion-exchange chromatography. In addition to the 14.2 kDa TBP, liver also contains the previously described 30 kDa TBP. The concentrations of the 14.2 kDa TBP in heart and liver were 12.3 micrograms and 17.5 micrograms per g of tissue, respectively. The purified protein specifically binds d alpha-tocopherol in preference to the delta- and gamma-homologues but does not bind oleate. The TBP stimulated the transfer of d alpha-tocopherol from liposomes to mitochondria in vitro by 8-10 fold. These results suggest that low molecular mass TBPs may play a role in intracellular vitamin E transport.

Fatty acid-binding proteins (FABPs) have been identified and purified from interscapular brown adipose tissue of the rat. The proteins were characterized and their properties compared with the FABP present in white adipose tissue. FABP was purified to electrophoretic homogeneity from brown adipose tissue by a procedure involving precipitation with 70% ammonium sulphate, followed sequentially by ion-exchange chromatography and gel filtration chromatography. The purified fraction migrated as a single band on SDS-PAGE with an apparent molecular mass of 14,200. Scatchard analysis of [14C]oleate-binding to purified FABP gave a Kd value of approx. 0.80 +/- 0.02 microM and a maximal binding of 0.65 +/- 0.03 mol per mol of protein; these values were similar to that found with the FABP purified from white fat. The FABP concentration in brown adipose tissue was almost twice that of FABP in white adipose tissue. Fatty acid analysis of FABP from brown adipose tissue revealed that the intrinsic arachidonic acid content was proportionately higher than that present in FABP of white adipose tissue. Isoelectric focusing of delipidated FABP indicated that it existed with two charge isoforms (pI 6.85 and 7.35). The purified FABP additionally emerged in two peaks (FABP-I and FABP-II) from a reverse phase HPLC column. Amino acid analysis showed that Gly, Thr, and Ser residues in FABP-I were almost twice as high as in FABP-II. The N-terminals of both FABP-I and -II were not blocked. These components have been partially sequenced and showed a sequence homology only between 25-31 residues from the N-terminal. Further studies are required to elucidate the precise function of the two different isoforms of FABP in brown adipose tissue.

An alpha-tocopherol-binding protein has been isolated and purified from rabbit heart cytosol. The purified protein had an apparent molecular mass of 14,200, as derived from SDS-PAGE. The content of the protein in rabbit heart was around 11.8 micrograms per g of tissue. The binding of alpha-tocopherol to the purified protein was rapid, reversible, and saturable. Neither gamma nor delta tocopherol could displace the bound alpha-tocopherol from the protein, suggesting a high specificity for alpha-tocopherol. alpha-Tocopherol-binding protein did not bind oleate. Transfer of alpha-tocopherol from liposomes to mitochondria was stimulated 8-fold in the presence of the binding protein, suggesting that this protein may be involved in the intracellular transport of alpha-tocopherol in the heart.

Epidemiological studies suggest the existence of a strong relationship between the incidence of prostatic cancer and the intake of dietary lipids in humans. However, very little information is available on intracellular fatty acid metabolism in human prostatic tissue. The objective of this study was to identify and subsequently characterize a fatty acid binding protein of human prostatic tissue. A fatty acid binding protein (FABP) was purified and characterized from human prostatic tissue. The purified FABP had an apparent molecular mass of 15.0 +/- 1.0 kDa as averaged from three different methods, sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE), gel filtration and amino acid analysis. The pI value of the protein was determined to be 6.8. Scatchard analysis of fatty acid binding to the purified FABP from malignant prostatic tissue showed a Kd value of 0.53 +/- 0.02 microM for arachidonic acid (n = 5). The Kd values of FABP purified from benign prostatic tissue were 0.57 +/- 0.02 microM for oleic acid and 0.51 +/- 0.04 microM for arachidonic acid (n = 5). Fatty acid analysis revealed that the level of endogenously bound arachidonic acid was about 2.5-fold higher in FABP from malignant than from benign tissue. In addition, both malignant and benign tissues contained the same concentration of FABP. The concentrations of FABP in malignant and benign tissues were 19.2 +/- 1.8 and 21.4 +/- 2.1 micrograms per mg of total cytosolic protein, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

We have studied the effects of Efamol evening primrose oil (EPO) on fatty acid-binding proteins (L-FABP) of rat liver. EPO contains 72% cis-linoleic acid and 9% cis-gamma linolenic acid. EPO has been clinically used for treatment of a number of diseases in humans and animals. EPO is also known to lower cholesterol level in humans and animals. Feeding of an EPO supplemented diet to rats (n = 9) for 2 months decreases the oleate binding capacity of purified L-FABP of rat liver whereas the palmitate binding activity was increased by 38%. However, EPO feeding did not alter the L-FABP concentrations significantly as measured by using the fluorescence fatty acid probe, dansylamino undecanoic acid. Endogenous fatty acid analysis of L-FABPs revealed significant qualitative and quantitative changes in fatty acid pattern after EPO feeding. EPO feeding decreased the endogenous palmitate level by 53% and oleate level by 64% in L-FABPs and also EPO feeding decreased the total endogenous fatty acid content from 62 nanomole per mg of protein to 42 nanomole per mg of L-FABP (n = 3).

Feeding rats a diet deficient in choline results in fatty liver within 1 d. We studied the effect of short-term (1-3 d) choline deficiency on rat liver Z protein (fatty acid-binding protein). Groups of three females Sprague-Dawley rats were fed ad libitum a purified diet lacking choline and L-methionine or were supplemented with 0.2% choline chloride and 0.82% L-methionine. Animals were killed after 1, 2 or 3 d of consuming control or experimental diets and hepatic Z protein was prepared. Z protein in livers from experimental and control rats were estimated with the fluorescent probe dansylamino undecanoic acid. The corresponding fatty acid-binding activity was also determined. One day of choline-deficient diet increased Z protein concentration threefold, reaching a plateau on the second and third day. Fatty acid-binding activity of Z protein remained unchanged.

Z protein or fatty-acid-binding protein is abundant in the cytosol of many cell types including liver cells. It is considered to play an important role in intracellular transport and metabolism of long-chain fatty acids and other organic anions. We studied the role of Z protein in the metabolism of prostaglandin E1 (PGE1). Binding of tritiated prostaglandin E1 to this fatty-acid-binding protein (Z protein) purified from rat liver was determined. The binding of [3H]prostaglandin E1 to Z protein is rapid, saturable and reversible. Scatchard analysis of [3H]PGE1 binding to Z protein showed a single class of binding sites with a dissociation constant (Kd) of 37 nM. The binding capacity is 110 nmol/mg Z protein. Optimal [3H]PGE1 binding occurred at pH 7.4. The presence of 3 mM MgCl2 stimulated the prostaglandin E1 binding to Z protein. Competition experiments show that the binding of this autacoid to Z protein is highly specific. It could not be displaced by other prostaglandins (PGA1, PGA2, PGE2, PGB1, PGI2, PGD2, PGF2 alpha, and 6-keto PGF1 alpha). Z protein might be involved in the metabolism of prostaglandins in the cytosol.