Intracellular Lipid Transport Research Articles

Microarray analysis of peroxisome proliferator-activated receptor-γ induced changes in gene expression in macrophages

We used a combination of expression microarray and Northern blot analyses to identify target genes for peroxisome proliferator-activated receptor (PPAR) γ in RAW264.7 macrophages. PPARγ natural ligand 15-deoxy-Δ 12,14 prostaglandin and synthetic ligands ciglitazone and rosiglitazone increased the expression of scavenger receptor CD36 and ATP-binding cassette transporter A1, as well as adipophilin (a lipid droplet coating protein involved in intracellular lipid storage and transport), calpain (a protease implicated in ABCA1 protein degradation), and ADAM8 (a disintegrin and metalloprotease protein involved in cell adhesion). These findings are relevant to understanding the effect of PPARγ activation on gene expression and cognate pathways in macrophages.

StAR-related Lipid Transfer (START) Proteins: Mediators of Intracellular Lipid Metabolism

StAR-related lipid transfer (START) domains are 210-amino acid lipid binding domains implicated in intracellular lipid transport, lipid metabolism, and cell signaling events. The prototype is the steroidogenic acute regulatory protein (StAR), which transfers cholesterol to mitochondria in steroid hormone-producing cells (1). START domains are found in an extensive protein family, including START domain only and multidomain proteins, but lipid ligands have only been identified in a few cases (2). The human and mouse genomes each have 15 genes encoding START domains (Table I), and phylogenetic analysis divides the family into six subfamilies (Fig. 1, A and B) (3). X-ray crystal structures have been solved for the MLN64 START domain (4), StarD4 (5), and phosphatidylcholine transfer protein (PCTP) (6). All three share the same helix-grip fold (7), with -helices at the N and C termini separated by nine -strands and two shorter -helices. The curved -sheet forms a deep pocket with the C-terminal -helix acting as a lid, resulting in an internal hydrophobic cavity (Fig. 2). The START structure differs from other hydrophobic cavity lipid-binding proteins, such as sterol carrier protein 2 (SCP-2) (8), phosphatidylinositol transfer protein (PITP) (9), and the fatty acid-binding proteins (FABPs) (10). The PCTP structure was reported with a phosphatidylcholine (PC) molecule in the cavity (6), whereas the MLN64 and StarD4 structures contain cavities large enough ( 850 A) to accommodate a cholesterol ligand ( 740 A) (5). In each structure, lipid entry or egress would require a major conformational change, most likely opening or unfolding of the C-terminal -helix lid. In fact, this helix of PCTP has been implicated in membrane binding and PC extraction (11). Furthermore, StAR can form partially unfolded states (12) and loses helical content upon binding a cholesterol analogue (13). Recent modeling of StAR using structure-based thermodynamics showed that an open lid conformational state can exist at equilibrium and that cholesterol binding and lid closure would significantly stabilize the complex (14).

Retarded intracellular lipid transport associated with reduced expression of Cdc42, a member of Rho-GTPases, in human aged skin fibroblasts: a possible function of Cdc42 in mediating intracellular lipid transport.

Many cell types in atherosclerotic lesions are thought to have various biological abnormalities, such as impaired lipid homeostasis and slow cell proliferation, which may be related to senescence at cellular and individual levels. One of the common characteristics of senescent cells in vitro is the alteration of actin cytoskeletons, which have been reported to be involved in the intracellular transport of lipids. Recently, we raised the hypothesis that Cdc42, which is a member of the Rho-GTPase family and is known to play an important role in actin dynamics, might be important in cellular lipid transport. In the present study, we found that the protein expression levels and GTP-binding activities of Cdc42 were decreased in aged human skin fibroblasts. Moreover, we found the intracellular kinetics of Golgi-associated lipids to be retarded in these cells, which was demonstrated by the fluorescence recovery after photobleaching (FRAP) technique and the use of N-[7-(4-nitrobenzo-2-oxa-1,3-diazole)]-6-aminohexanoyl-D-erythro-sphingosine as a tracer. To correlate the decreased expression of Cdc42 with the retarded FRAP, we complemented the amount of wild-type c-myc-tagged Cdc42Hs (myc-Cdc42Hs-WT) by adenovirus-mediated gene transfer. We further tested the effect of the dominant-active form (myc-Cdc42Hs-DA, V12Cdc42Hs) or dominant-negative form (myc-Cdc42Hs-DN, N17Cdc42Hs) of Cdc42Hs on FRAP. Introduction of myc-Cdc42Hs-WT or myc-Cdc42Hs-DA recovered the retarded FRAP in the aged fibroblasts. Conversely, control fibroblasts infected with myc-Cdc42Hs-DN exhibited significantly retarded FRAP. These data clearly indicate that the expression of Cdc42, a small G protein, is decreased in the aged cells in close association with the retarded intracellular lipid transport. The present study demonstrates a possible function of Cdc42 in the mediation of intracellular lipid transport.

Biochemistry and genetics of interorganelle aminoglycerophospholipid transport.

The organelle specific reactions that constitute the biosynthetic pathway for aminoglycerophospholipid synthesis provide an important means for examining the biochemistry and genetics of intracellular lipid transport. Biochemical studies with intact and permeabilized cells, and isolated organelles have defined some of the essential features of lipid transport between the endoplasmic reticulum and mitochondria and Golgi/vacuole. Genetic screens have now also identified mutations and genes that are involved in aminoglycerophospholipid traffic between different membranes in mammalian cells, yeast and bacteria. Increasingly, studies focused upon intermembrane lipid movement are revealing important new information about this essential aspect of membrane biogenesis.

The cholesterol-regulated StarD4 gene encodes a StAR-related lipid transfer protein with two closely related homologues, StarD5 and StarD6.

Using cDNA microarrays, we identified StarD4 as a gene whose expression decreased more than 2-fold in the livers of mice fed a high-cholesterol diet. StarD4 expression in cultured 3T3 cells was also sterol-regulated, and known sterol regulatory element binding protein (SREBP)-target genes showed coordinate regulation. The closest homologues to StarD4 were two other StAR-related lipid transfer (START) proteins named StarD5 and StarD6. StarD4, StarD5, and StarD6 are 205- to 233-aa proteins consisting almost entirely of START domains. These three constitute a subfamily among START proteins, sharing approximately 30% amino acid identity with one another, approximately 20% identity with the cholesterol-binding START domains of StAR and MLN64, and less than 15% identity with phosphatidylcholine transfer protein (PCTP) and other START domains. StarD4 and StarD5 were expressed in most tissues, with highest levels in liver and kidney, whereas StarD6 was expressed exclusively in the testis. In contrast to StarD4, expression of StarD5 and MLN64 was not sterol-regulated. StarD4, StarD5, and StarD6 may be involved in the intracellular transport of sterols or other lipids.

Bile salt-stimulated carboxyl ester lipase influences lipoprotein assembly and secretion in intestine: a process mediated via ceramide hydrolysis.

Bile salt-stimulated carboxyl ester lipase (CEL), also called cholesterol esterase, is one of the major proteins secreted by the pancreas. The physiological role of CEL was originally thought to be its mediation of dietary cholesterol absorption. However, recent studies showed no difference between wild type and CEL knockout mice in the total amount of cholesterol absorbed in a single meal. The current study tests the hypothesis that CEL in the intestinal lumen may influence the type of lipoproteins produced. A lipid emulsion containing 4 mm phospholipid, 13.33 mm [(3)H]triolein, and 2.6 mm [(14)C]cholesterol in 19 mm taurocholate was infused into the duodenum of lymph fistula CEL(+/+) and CEL(-/-) mice at a rate of 0.3 ml/h. Results showed no difference between CEL(+/+) and CEL(-/-) mice in the rate of cholesterol and triglyceride transport from the intestinal lumen to the lymph. However, CEL(-/-) mice produced predominantly smaller lipoproteins, whereas the CEL(+/+) mice produced primarily large chylomicrons and very low density lipoprotein. The proximal intestine of CEL(-/-) mice was also found to possess significantly less ceramide hydrolytic activity than that present in CEL(+/+) mice. By using Caco2 cells grown on Transwell membranes as a model, sphingomyelinase treatment inhibited the secretion of larger chylomicron-like lipoproteins without affecting total cholesterol secretion. In contrast, the addition of CEL to the apical medium increased the amount of large lipoproteins produced and alleviated the inhibition induced by sphingomyelinase. Taken together, this study identified a novel and physiologically significant role for CEL, namely the promotion of large chylomicron production in the intestine. The mechanism appears to be mediated through CEL hydrolysis of ceramide generated during the lipid absorption process.

A caveolin dominant negative mutant associates with lipid bodies and induces intracellular cholesterol imbalance.

Recent studies have indicated a role for caveolin in regulating cholesterol-dependent signaling events. In the present study we have analyzed the role of caveolins in intracellular cholesterol cycling using a dominant negative caveolin mutant. The mutant caveolin protein, cav-3(DGV), specifically associates with the membrane surrounding large lipid droplets. These structures contain neutral lipids, and are accessed by caveolin 1-3 upon overexpression. Fluorescence, electron, and video microscopy observations are consistent with formation of the membrane-enclosed lipid rich structures by maturation of subdomains of the ER. The caveolin mutant causes the intracellular accumulation of free cholesterol (FC) in late endosomes, a decrease in surface cholesterol and a decrease in cholesterol efflux and synthesis. The amphiphile U18666A acts synergistically with cav(DGV) to increase intracellular accumulation of FC. Incubation of cells with oleic acid induces a significant accumulation of full-length caveolins in the enlarged lipid droplets. We conclude that caveolin can associate with the membrane surrounding lipid droplets and is a key component involved in intracellular cholesterol balance and lipid transport in fibroblasts.

Structure of Sterol Carrier Protein 2 at 1.8 Å Resolution Reveals a Hydrophobic Tunnel Suitable for Lipid Binding,

Sterol carrier protein 2, also known as nonspecific lipid transfer protein is a ubiquitous, small, basic protein of 13 kDa found in animals. Its primary structure is highly conserved between different species, and it has been implicated in the intracellular transport of lipids and in a wide range of other in vitro functions related to sterol and fatty acid metabolism. Sterol carrier protein 2 deficiency in mice leads to elevated concentrations of phytanic acid in the serum and causes hepatocarcinogenesis. However, its actual physiological role is still unknown. Although sterol carrier protein 2 has been studied extensively in the past 20 years, very little is known concerning its three-dimensional structure. The crystal structure of rabbit sterol carrier protein 2, determined at 1.8 A resolution with the MIRAS method, shows a unique alpha/beta-fold. The core of this protein forms a five-stranded antiparallel beta-sheet flanked by five helices. A C-terminal segment (residues 114-123), together with part of the beta-sheet and four alpha-helices, form a hydrophobic tunnel providing the environment for apolar ligands such as fatty acids and fatty acyl-coenzyme As. Structurally well-characterized nonspecific lipid transfer proteins from plants have hydrophobic tunnel-like cavities, which were identified as the binding site for fatty acids and related apolar ligands. Despite the fact that plant nonspecific lipid transfer proteins are smaller proteins than sterol carrier protein 2, show no sequence homology to sterol carrier protein 2, and are structurally unrelated, the cavities of these two classes of proteins are very similar with respect to size, shape, and hydrophobicity, suggesting a common functional role.

Anderson's disease: exclusion of apolipoprotein and intracellular lipid transport genes.

Anderson's disease is a rare, hereditary hypocholesterolemic syndrome characterized by chronic diarrhea, steatorrhea, and failure to thrive associated with the absence of apo B48-containing lipoproteins. To further define the molecular basis of the disease, we studied 8 affected subjects in 7 unrelated families of North African origin after treatment with a low-fat diet. Lipid loading of intestinal biopsies persisted, but the pattern and extent of loading was variable among the patients. Electron microscopy showed lipoprotein-like particles in membrane-bound compartments, the densities (0.65 to 7.5 particles/mu(2)) and the mean diameters (169 to 580 nm) of which were, in general, significantly larger than in a normal fed subject (0.66 particles/mu(2), 209 nm mean diameter). There were also large lipid particles having diameters up to 7043 nm (average diameters from 368 to 2127 nm) that were not surrounded by a membrane. Rarely, lipoprotein-like particles 50 to 150 nm in diameter were observed in the intercellular spaces. Intestinal organ culture showed that apo B and apo AIV were synthesized with apparently normal molecular weights and that small amounts were secreted in lipid-bound forms (density <1.006 g/mL). Normal microsomal triglyceride transfer protein (MTP) and activity were also detected in intestinal biopsies. Segregation analyses of 4 families excluded, as a cause of the disease, significant regions of the genome surrounding the genes for apo AI, AIV, B, CI, CII, CIII, and E, as were the genes encoding 3 proteins involved in intracellular lipid transport, MTP, and fatty acid binding proteins 1 and 2. The results suggest that a factor other than apoproteins and MTP are important for human intestinal chylomicron assembly and secretion.

Sex and drugs and HCV?

<h3>ABSTRACT</h3> Lipid metabolism is spatiotemporally regulated within cells, yet intervention into lipid functions at subcellular resolution remains difficult. Here we report a method that enables site-specific release of sphingolipids and...

Requirement for the heart-type fatty acid binding protein in cardiac fatty acid utilization.

Nonenzymatic cytosolic fatty acid binding proteins (FABPs) are abundantly expressed in many animal tissues with high rates of fatty acid metabolism. No physiological role has been demonstrated for any FABP, although these proteins have been implicated in transport of free long-chain fatty acids (LCFAs) and protection against LCFA toxicity. We report here that mice lacking heart-type FABP (H-FABP) exhibit a severe defect of peripheral (nonhepatic, non-fat) LCFA utilization. In these mice, the heart is unable to efficiently take up plasma LCFAs, which are normally its main fuel, and switches to glucose usage. Altered plasma levels of LCFAs, glucose, lactate and beta-hydroxybutyrate are consistent with depressed peripheral LCFA utilization, intensified carbohydrate usage, and increased hepatic LCFA oxidation; these changes are most pronounced under conditions favoring LCFA oxidation. H-FABP deficiency is only incompletely compensated, however, causing acute exercise intolerance and, at old age, a localized cardiac hypertrophy. These data establish a requirement for H-FABP in cardiac intracellular lipid transport and fuel selection and a major role in metabolic homeostasis. This new animal model should be particularly useful for investigating the significance of peripheral LCFA utilization for heart function, insulin sensitivity, and blood pressure.

Regulated turnover of a cell surface-associated pool of newly synthesized apolipoprotein E in HepG2 cells

Newly synthesized apolipoprotein E (apoE) is incompletely secreted and partially degraded by hepatocytes. To evaluate current models concerning apoE's role in remnant lipoprotein clearance by the liver, we performed a detailed and quantitative pulse-chase analysis of apoE secretion, subcellular distribution, and proteolytic degradation by the human hepatoma cell line HepG2. Only about 35% of newly synthesized apoE were found to be secreted to the culture medium. As determined by a protease-protection assay, a substantial amount of newly synthesized apoE was transported to the cell surface, constituting more than half of the cellular apoE under steady-state conditions. A subpopulation representing almost 40% of newly secreted apoE was rapidly rebound to the cell surface, indicating a dynamic equilibrium between cell surface and secreted apoE. These pools of newly synthesized apoE were subject to proteolytic turnover that occurred in lysosomes, presumably after re-endocytosis. We found that the proteolytic turnover of cell surface and secreted apoE was regulated by the availability of apoE ligands, being almost completely suppressed by the presence of lipoprotein-containing human serum or isolated lipoproteins, namely LDL and HDL. The characteristics of regulated degradation of cell surface apoE shed new light on potential physiological functions of this pool of apoE. Our results provide evidence that hepatocytes are capable of forming a large pool of cell surface-associated apoE, thereby supporting the previously proposed secretion-recapture model for apoE.

透過性ＣＨＯ細胞を用いたホスファチジルセリンの細胞内輸送の研究 蛍光標識脂質による顕微鏡観察

The mechanisms of intracellular transport of lipids and asymmetric distribution of membrane lipids were still unknown. The intracellular transport of fluorescent phosphatidylserine analogues, 1-palmitoyl-2- (N- [12 [(7-nitrobenz-2-oxa-1, 3-diazole-4-yl) amino] dodecanoyl]) -phosphatidylserine (C12-NBD-PS) and 1-palmitoyl-2- (N- [6 [(7-nitrobenz-2-oxa-1, 3-diazole-4-yl) amino] caproyl]) -phosphatidylserine (C6-NBD-PS) was investigated in CHO cells. When CHO cells were incubated with liposomes containing C12-NBD-PS for 30 min at 37°C, fluorescent PS appeared in the Golgi apparatus. On the other hand, C6-NBD-PS was observed in the intracellular membrane systems but not in the Golgi apparatus under the same experimental conditions.To investigate transport of C12-NBD-PS to the Golgi apparatus further, the plasma membranes of CHO cells were selectively perforated with θ-toxin from Clostridium perfringens and the cytosol was replaced by rat brain cytosol, ATP and Ca2+. In order to establish the optimal condition of cell permeabilization, release of lactate dehydrogenase (LD) activity from the CHO cells was determined as function of temperature, time of incubation and θ-toxin concentration. Then C12-NBD-PS transport to the Golgi apparatus was examined under various conditions.Incubation of the perforated cells with rat brain cytosol, ATP and Ca2+ results in reconstitution of transport of C12-NBD-PS to the Golgi apparatus. However in the absence of rat brain cytosol or ATP, this transport was not recovered. The results suggest that PS specific transport protein is involved in the transport of phosphatidylserine to the Golgi apparatus in CHO cells.

Mechanisms and functional features of polarized membrane traffic in epithelial and hepatic cells.

Epithelial cells express plasma-membrane polarity in order to meet functional requirements that are imposed by their interaction with different extracellular environments. Thus apical and basolateral membrane domains are distinguished that are separated by tight junctions in order to maintain the specific lipid and protein composition of each domain. In hepatic cells, the plasma membrane is also polarized, containing a sinusoidal (basolateral) and a bile canalicular (apical)-membrane domain. Relevant to the biogenesis of these domains are issues concerning sorting, (co-)transport and regulation of transport of domain-specific membrane components. In epithelial cells, specific proteins and lipids, destined for the apical membrane, are sorted in the trans-Golgi network (TGN), which involves their sequestration into cholesterol/sphingolipid 'rafts', followed by 'direct' transport to the apical membrane. In hepatic cells, a direct apical transport pathway also exists, as revealed by transport of sphingolipids from TGN to the apical membrane. This is remarkable, since in these cells numerous apical membrane proteins are 'indirectly' sorted, i.e. they are first transferred to the basolateral membrane prior to their subsequent transcytosis to the apical membrane. This raises intriguing questions as to the existence of specific lipid rafts in hepatocytes. As demonstrated in studies with HepG2 cells, it has become evident that, in hepatic cells, apical transport pathways can be regulated by protein kinase activity, which in turn modulates cell polarity. Finally, an important physiological function of hepatic cells is their involvement in intracellular transport and secretion of bile-specific lipids. Mechanisms of these transport processes, including the role of multidrug-resistant proteins in lipid translocation, will be discussed in the context of intracellular vesicular transport. Taken together, hepatic cell systems provide an important asset to studies aimed at elucidating mechanisms of sorting and trafficking of lipids (and proteins) in polarized cells in general.

Cytokine regulation of low-density lipoprotein receptor gene transcription in human mesangial cells.

The intracellular transport of lipids through regulation of the LDL receptor (LDLr) may be important in the progression of renal dysfunction. The present study was undertaken to investigate whether cytokines have any major effects on LDLr regulation and lipid-mediated glomerular injury in human mesangial cells (HMC). We explored the effects of 50 ng/ml of tumour necrosis factor alpha (TNF alpha), 5 ng/ml of transforming growth factor beta (TGF beta), platelet-derived growth factor (PDGF), and interleukin-1beta (IL-1beta) on the regulation of LDLr gene transcription in a human mesangial cell line (HMCL) using cell proliferation, LDL binding, northern blot and LDLr promoter activity assays. TNF alpha, TGF beta, PDGF or IL-1beta did not significantly stimulate HMCL proliferation at the concentrations given above, but maximally stimulated LDLr mRNA expression and increased LDLr promoter activity by 167.48+/-23.56%, 150.47+/-24.41%, 127.71+/-24.65% and 163.01+/-31.91% respectively, at 24 h. An increased LDL binding was observed in parallel with increased LDLr mRNA. The tyrosine kinase transduction pathway was involved in LDLr upregulation induced by all four cytokines. Additionally, TGF beta involved serine/threonine kinase and G-protein pathways, and IL-1beta involved calmodulin, serine/threonine kinase and PKC pathways in upregulating LDLr. A high concentration of LDL (250 microg/ml) inhibited promoter activity, but TNF alpha, TGF beta, PDGF and IL-1beta co-incubated with LDL could override transcriptional inhibition by LDL. TNF alpha, TGF beta, PDGF and IL-1beta increased LDLr gene expression by increasing sterol-independent and mitogenesis-independent gene transcription. This process may contribute to lipid deposition and foam cell formation in HMC.

A Genetic Screen for Aminophospholipid Transport Mutants Identifies the Phosphatidylinositol 4-Kinase, Stt4p, as an Essential Component in Phosphatidylserine Metabolism

In an effort to understand molecular mechanisms of intracellular lipid transport, we have focused upon specific events required for de novo aminophospholipid synthesis in the yeast Saccharomyces cerevisiae. A genetic system for examining the steps between phosphatidylserine (PtdSer) synthesis in the endoplasmic reticulum and its transport to and decarboxylation by PtdSer decarboxylase 2 in the Golgi/vacuole has been developed. We have isolated a mutant, denoted pstB1, that accumulates PtdSer and has diminished phosphatidylethanolamine formation despite normal PtdSer decarboxylase 2 activity. The lesion in PtdSer metabolism is consistent with a defect in interorganelle lipid transport. A genomic DNA clone that complements the mutation was isolated, and sequencing revealed that the clone contains the STT4 gene, encoding a phosphatidylinositol 4-kinase. The pstB1 mutant exhibits a defect in Stt4p-type phosphatidylinositol 4-kinase activity, and direct gene replacement indicates that STT4 is the defective gene in the mutant. Creation of an STT4 null allele (stt4Delta::HIS3) demonstrates the gene is essential. These results provide evidence that implicates phosphoinositides in the regulation of intracellular aminophospholipid transport.

Lipid-transfer proteins: a puzzling family of plant proteins

Lipid-transfer proteins are small, basic proteins, and have been purified from various plant sources. They are able to transfer lipids between membranes <i>in vitro</i> and, on the basis of this, were initially thought to participate in the intracellular flux of lipids during membrane synthesis. However, the finding that these proteins are located in the cell wall and can be secreted has led to the suggestion that they are not required for intracellular lipid transport. Instead, they may be involved in cutin biosynthesis, surface wax formation, pathogen-defence reactions, or the adaptation of plants to environmental changes.

細胞内脂質輸送総論

細胞内脂質輸送総論

Mammalian Cdc42 Is a Brefeldin A-sensitive Component of the Golgi Apparatus

In this study, we have used immunocytochemical and fractionation approaches to provide a description of the localization of the mammalian Cdc42 protein (designated Cdc42Hs) in vivo. A specific anti-peptide antibody was generated against the C-terminal region of Cdc42Hs. Using affinity-purified preparations of this antibody in indirect immunofluorescence experiments, Cdc42Hs was found to be localized to the Golgi apparatus. Similar to the well-characterized non-clathrin coat proteins ADP-ribosylation factor (ARF) and beta-COP, the perinuclear clustering of Cdc42Hs is rapidly dispersed upon exposure of the cells to the drug brefeldin A, suggesting that it too may play a role in the processes of intracellular lipid and protein transport. Employing cell lines possessing inducible forms of ARF, we demonstrate here a tight coupling of the nucleotide-bound state of ARF and the subcellular localization of Cdc42Hs. Specifically, the expression of wild-type ARF had no effect on the brefeldin A sensitivity of Cdc42Hs while, as is the case for ARF and beta-COP, expression of a GTPase-deficient form of ARF (ARF(Q71L)) renders these Golgi-localized proteins resistant to brefeldin A treatment (; ). Moreover, the induced expression of a mutant form of ARF with a low affinity for nucleotide resulted in constitutive redistribution of Cdc42Hs in the absence of brefeldin A treatment. These results suggest that Cdc42Hs may play a role in cell morphogenesis by acting on targets in the Golgi that direct polarized growth at the plasma membrane.

One-step purification of rat heart-type fatty acid-binding protein expressed in Escherichia coli

Heart-type fatty acid-binding protein (H-FABP) is a member of a family of 14–15 kDa lipid binding proteins which are believed to enhance intracellular transport of lipids by facilitating their cytoplasmic diffusion. To obtain sufficient amounts of protein for in vitro studies, we expressed rat H-FABP in Escherichia coli and compared its biochemical properties with the protein isolated from rat heart. An effective method was developed to purify recombinant rat H-FABP from cell lysates in a single step using anion-exchange chromatography. This method also proved to be applicable for purifying heterologously expressed human H-FABP. Recombinant rat H-FABP, which made up approximately 25% of the soluble proteins in E. coli, was obtained in a yield of 30–40 mg/l culture. Characterization showed that recombinant rat H-FABP was indistinguishable from the protein isolated from rat heart regarding molecular mass and oleic acid binding. Some heterogeneity upon isoelectric focusing was observed, presumably due to differences in N-terminal processing of the proteins. In conclusion, a method is presented for efficient high-yield production of recombinant rat H-FABP.