Identification of high affinity membrane-bound fatty acid-binding proteins using a photoreactive fatty acid

A photoaffinity labeling method was developed to identify and characterize high affinity fatty acid-binding proteins in membranes. The specific labeling of these sites requires the use of low concentrations (nanomolar) of the photoreactive fatty acid 11-m-diazirinophenoxy-[11-³H]undecanoate. It was delivered as a bovine serum albumin (BSA) complex which serves as a reservoir for fatty acid and thus allows precise control of unbound fatty acid concentrations. ThefadL protein ofE. coli, which is required for fatty acid permeation of its outer membrane, was labeled by the photoreactive fatty acid neither specifically nor saturably when the probe was added in the absence of BSA; however when a nanomolar concentration of the uncomplexed probe was maintained in the presence of BSA, the labeling of thefadL protein was highly specific and saturable. This photoaffinity labeling method was also used to characterize a 22 kDa, high affinity fatty acid-binding protein which we have recently identified in the plasma membrane of 3T3-L1 adipocytes. This protein bound the probe with a K_d of 216 nM. The approach described is easily capable of identifying membrane-bound fatty acid-binding proteins and can distinguish between those of high and low affinities for fatty acids. It represents a general method for the identification and characterization of fatty acid-binding proteins.Abbreviations BSA Bovine Serum Albumin - DAP m-Diazirinophenoxy - SDS-PAGE Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis     

Epidermal growth factor receptors in fetal and maternal rabbit lung

The pattern of morphologic and functional development of lung during intrauterine period is influenced by several endogenous compounds. Recently Epidermal Growth Factor (EGF), when administered in vivo, has been shown to accelerate pulmonary maturation in fetal rabbit and sheep. We sought evidence for EGF receptor occurrence in fetal and maternal rabbit lung plasma membranes. The percent specific binding (mean ± S.E.M.) (125-I) EGF to LPM in the mother (n=5) and the fetus at term (n=7) was 1.08 ± 0.08 and 2.25 ± 0.12 per 175 μg of LPM protein respectively. The number of receptor sites per mg of LPM protein in the mother were significantly less than that in the fetus (44 ± 11 and 250 ± 24 × 10^?10, p < 0.001) with no apparent differences in Kd (2.10 ± 0.39 and 2.47 ± 0.24 × 10⁹). Presence of high affinity receptors for EGF in fetal and maternal lung plasma membranes suggests a direct role of EGF in fetal lung maturation.     

Fatty acyl-CoA binding activity of the nuclear thyroid hormone receptor

Long-chain fatty acids and their acyl-CoA esters are potent inhibitors of nuclear thyroid hormone (T₃) receptor in vitro. In the present study, we obtained evidence for acyl-CoA binding activity in the nuclear extract from rat liver. The activity sedimented at a position (3.5 S) identical with that of the T₃ receptor, and the two activities sedimented together. Similarly, they coeluted on DEAE-Sephadex. After partial purification of the receptor, it was again inhibited strongly by acyl-CoAs. Heat stability and a partial trypsin digestion of the receptor both suggested that the action site of oleoyl-CoA overlapped the T₃-binding domain of the receptor. In addition, thyroid hormone receptor β1, synthesized in vitro, bound oleoyl-CoA specifically and its T₃-binding activity was inhibited. The dissociation constant for oleoyl-CoA binding to the partially purified receptor was 1.2 × 10^?7 M. This value as well as its molecular size distinguished the nuclear binding sites from the cytoplasmic fatty acid/acyl-CoA binding proteins. Oleoyl-CoA had no effect on the glucocorticoid receptor, another member of the nuclear hormone-receptor superfamily. From these results, we propose that thyroid hormone receptor is a specific acyl-CoA binding protein of the cell nucleus.     

Oxygen metabolism of mammalian spermatozoa. Generation of hydrogen peroxide by rabbit epididymal spermatozoa

Rabbit spermatozoa from the cauda epididymis produced 0.7–0.8nmol of H₂O₂/min per 10⁸ cells at cell concentrations below 10⁷ cells/ml with linear dependence on cell concentration. Above 2 × 10⁷ cells/ml, the rate again became linear with cell concentration but decreased to 0.1–0.2nmol/min per 10⁸ cells. Spermatozoa treated with amphotericin B, which makes the plasma membrane highly permeable to low-molecular-weight compounds, showed a similar dependence of H₂O₂ production rate on cell concentration; below 10⁷ cells/ml the rate was 0.3–0.4nmol/min per 10⁸ cells; above 2 × 10⁷ cells/ml, the rate was 0.1–0.2nmol/min per 10⁸ cells. Hypo-osmotically treated rabbit epididymal spermatozoa, a preparation useful for studying mitochondrial function in sperm [Keyhani & Storey (1973) Biochim. Biophys. Acta 305, 557–565] produced 0.1–0.2nmol/min per 10⁸ cells in the absence of added substrates. The dependence of rate on cell concentration was linear from 10⁷ to 2.2 × 10⁸ cells/ml. This endogenous rate was unaffected by rotenone, but stimulated 4-fold by antimycin A. Addition of the mitochondrial substrates lactate plus malate increased the rate of H₂O₂ production to 0.3nmol/min per 10⁸ cells. The decreased rate of H₂O₂ production observed with intact sperm at high cell concentrations is attributed to reaction of H₂O₂ with the cells, possibly with the plasma membrane, which is lost after hypo-osmotic treatment. Rabbit spermatozoa have glutathione peroxidase and glutathione reductase activities, but these seem to play little role in removal of H₂O₂ generated. The rate at low cell concentration is taken to be the unperturbed rate. The sources of H₂O₂ production in rabbit spermatozoa have been tentatively resolved into a low-molecular-weight component, lost after amphotericin treatment, a mitochondrial component and a rotenone-insensitive component that has not been identified.     

Calcium binding to placental plasma membranes as measured by rate of diffusion in a flow dialysis system

The Ca²⁺-binding properties of placental plasma membranes were studied using a flow dialysis system.Ca²⁺-binding was not detectable at pH 4.0, but increased at higher pH values to a maximum binding at pH 11.0.Two types of Ca²⁺-binding sites were identified: high-affinity sites with dissociation constant K_s = 3.1 · 10^{−5 M} and a capacity of 26 nmoles per mg protein; low-affinity sites with K_s = 1.1 · 10^{−3 M} and a capacity of 266 nmoles per mg protein.The affinities of Mg²⁺ and Sr²⁺ for the high-affinity sites were 10-fold lower than that of Ca²⁺, and for the low-affinity sites were 4- and 8-fold lower, respectively.The placental plasma membranes contain sites for Ca²⁺ with capacity, specificity and affinity within the range reported for other membranes involved in an active transport of Ca²⁺ (mitochondria, sarcoplasmic reticulum, cardiac microsomes). The presence of high-affinity Ca²⁺ sites as well as Ca²⁺-ATPase implicates these membranes in Ca²⁺ transport from the maternal to the fetal circulation.     

N-1-napthylphthalamic-acid-binding activity of a plasma membrane-rich fraction from maize coleoptiles

Summary Plasma membrane-rich fractions were prepared from maize coleoptiles by low-shear homogenization and differential and sucrose-gradient centrifugation. Plasma membrane fragments were identified using a specific cytochemical stain based on phosphotungstic acid prepared in chromic acid. In a comparison of 10 different cell fractions of varying plasma membrane content, the N-1-napthylphthalamic-acid (NPA)-binding activity of the fractions was directly proportional to the content of plasma membrane. The NPA binding appears to be strong K _M between 10^-8 and 10^-7 M) but non-covalent. NPA is known to inhibit auxin transport efficiently and quickly. Thus, the results are consistent with the localization of auxin transport sites at the plasma membrane of plant cells.Purdue University Agricultural Experiment Station Journal Paper No. 4355. This work was supported in part by a grant from the National Science Foundation GB-23183.Supported by National Science Foundation Postdoctoral Fellowship.     

cAMP Mediators of Pulsatile Insulin Secretion from Glucose-stimulated Single β-Cells

Pulsatile insulin release from glucose-stimulated β-cells is driven by oscillations of the Ca²⁺ and cAMP concentrations in the subplasma membrane space ([Ca²⁺]_pm and [cAMP]_pm). To clarify mechanisms by which cAMP regulates insulin secretion, we performed parallel evanescent wave fluorescence imaging of [cAMP]_pm, [Ca²⁺]_pm, and phosphatidylinositol 3,4,5-trisphosphate (PIP₃) in the plasma membrane. This lipid is formed by autocrine insulin receptor activation and was used to monitor insulin release kinetics from single MIN6 β-cells. Elevation of the glucose concentration from 3 to 11 mm induced, after a 2.7-min delay, coordinated oscillations of [Ca²⁺]_pm, [cAMP]_pm, and PIP₃. Inhibitors of protein kinase A (PKA) markedly diminished the PIP₃ response when applied before glucose stimulation, but did not affect already manifested PIP₃ oscillations. The reduced PIP₃ response could be attributed to accelerated depolarization causing early rise of [Ca²⁺]_pm that preceded the elevation of [cAMP]_pm. However, the amplitude of the PIP₃ response after PKA inhibition was restored by a specific agonist to the cAMP-dependent guanine nucleotide exchange factor Epac. Suppression of cAMP formation with adenylyl cyclase inhibitors reduced already established PIP₃ oscillations in glucose-stimulated cells, and this effect was almost completely counteracted by the Epac agonist. In cells treated with small interfering RNA targeting Epac2, the amplitudes of the glucose-induced PIP₃ oscillations were reduced, and the Epac agonist was without effect. The data indicate that temporal coordination of the triggering [Ca²⁺]_pm and amplifying [cAMP]_pm signals is important for glucose-induced pulsatile insulin release. Although both PKA and Epac2 partake in initiating insulin secretion, the cAMP dependence of established pulsatility is mediated by Epac2.     

Fatty acid transport and activation and the expression patterns of genes involved in fatty acid trafficking

These studies defined the expression patterns of genes involved in fatty acid transport, activation and trafficking using quantitative PCR (qPCR) and established the kinetic constants of fatty acid transport in an effort to define whether vectorial acylation represents a common mechanism in different cell types (3T3-L1 fibroblasts and adipocytes, Caco-2 and HepG2 cells and three endothelial cell lines (b-END3, HAEC, and HMEC)). As expected, fatty acid transport protein (FATP)1 and long-chain acyl CoA synthetase (Acsl)1 were the predominant isoforms expressed in adipocytes consistent with their roles in the transport and activation of exogenous fatty acids destined for storage in the form of triglycerides. In cells involved in fatty acid processing including Caco-2 (intestinal-like) and HepG2 (liver-like), FATP2 was the predominant isoform. The patterns of Acsl expression were distinct between these two cell types with Acsl3 and Acsl5 being predominant in Caco-2 cells and Acsl4 in HepG2 cells. In the endothelial lines, FATP1 and FATP4 were the most highly expressed isoforms; the expression patterns for the different Acsl isoforms were highly variable between the different endothelial cell lines. The transport of the fluorescent long-chain fatty acid C₁-BODIPY-C₁₂ in 3T3-L1 fibroblasts and 3T3-L1 adipocytes followed typical Michaelis–Menten kinetics; the apparent efficiency (k_cat/K_T) of this process increases over 2-fold (2.1 × 10⁶–4.5 × 10⁶ s⁻¹ M⁻¹) upon adipocyte differentiation. The V_max values for fatty acid transport in Caco-2 and HepG2 cells were essentially the same, yet the efficiency was 55% higher in Caco-2 cells (2.3 × 10⁶ s⁻¹ M⁻¹ versus 1.5 × 10⁶ s⁻¹ M⁻¹). The kinetic parameters for fatty acid transport in three endothelial cell types demonstrated they were the least efficient cell types for this process giving V_max values that were nearly 4-fold lower than those defined form 3T3-L1 adipocytes, Caco-2 cells and HepG2 cells. The same cells had reduced efficiency for fatty acid transport (ranging from 0.82 × 10⁶ s⁻¹ M⁻¹ to 1.35 × 10⁶ s⁻¹ M⁻¹).     

Effect of essential fatty acid deficiency on activity of liver plasma membrane enzymes in the rat

Liver plasma membranes (LPM) were isolated from rats fed an essential fatty acid-supplemented diet (+EFA) or from rats fed an essential fatty acid-deficient diet (-EFA). The proportions of linoleate and arachidonate in membrane total fatty acids in the ?EFA preparations were one-half or less than the values for the +EFA preparations. Basal, F^?, or glucagon-stimulated adenylate cyclase activities were significantly lower in EFA-deficient livers than in nondeficient ones. Addition of GTP significantly enhanced glucagon-stimulated adenylate cyclase in both groups, but extent of stimulation above basal was greater in EFA-deficient livers. Portal vein injection of glucagon in vivo resulted in significantly higher cAMP formation in +EFA livers than in ?EFA livers. When glucagon was used in vitro at 1–1,000 nM, stimulation of adenylate cyclase remained lower in EFA-deficient membranes, but extent of stimulation above basal activity was larger in ?EFA membranes than in +EFA. Total Na⁺, K⁺ (Mg²⁺)-ATPase from EFA-depleted LPM exhibited significantly higher values of apparent K_m and V_max. 5′-Nucleotidase activity, in contrast, was considerably decreased in EFA-deficient rats. These findings show that, in animals, changes in unsaturated fatty acid composition can affect the properties of membrane-bound enzymes. These alterations could be due to changes in membrane physical properties and/or prostaglandin formation.     

Kinetic analysis of the binding of immunoglobulins IgG1 and IgG2 to bovine mammary cells

Previous reports were confirmed that specific binding sites exist on bovine mammary cells near parturition presumably involved in the transfer of immunoglobulins IgG1 and IgG2 across the mammary gland at the time of colostrum formation. Determination of the kinetic parameters of these binding sites using ¹²⁵I-labeled IgG1 and IgG2 immunoglobulins indicated the presence of sites with association constants (K_a) of about 5 · 10⁸?10 · 10⁸ M^?1 for both subclasses during normal lactation with about 9000 and 3000 sites per cell for each, respectively. The number of IgG1 sites tended to increase as the time of parturition approached. In addition, a new group of sites numbering about 5000 per cell with very strong binding of IgG1 (K_a about 45 · 10⁸ M^?1) appeared on the cells about a week before parturition. The numbers and affinity of the IgG1 and IgG2 binding sites bear a relationship to the approximate 7:1 ratio of these immunoglobulin subclasses found in colostrum and normal milk and to the time of maximum colostrum formation. The results support the premise that a highly selective transport mechanism exists in the bovine mammary epithelial cell for the transfer of IgG1 and IgG2 immunoglobulins from blood to the lacteal secretions.     

Fatty acid transport across the cell membrane: Regulation by fatty acid transporters

Transport of long-chain fatty acids across the cell membrane has long been thought to occur by passive diffusion. However, in recent years there has been a fundamental shift in understanding, and it is now generally recognized that fatty acids cross the cell membrane via a protein-mediated mechanism. Membrane-associated fatty acid-binding proteins (‘fatty acid transporters’) not only facilitate but also regulate cellular fatty acid uptake, for instance through their inducible rapid (and reversible) translocation from intracellular storage pools to the cell membrane. A number of fatty acid transporters have been identified, including CD36, plasma membrane-associated fatty acid-binding protein (FABP_pm), and a family of fatty acid transport proteins (FATP1–6). Fatty acid transporters are also implicated in metabolic disease, such as insulin resistance and type-2 diabetes. In this report we briefly review current understanding of the mechanism of transmembrane fatty acid transport, and the function of fatty acid transporters in healthy cardiac and skeletal muscle, and in insulin resistance/type-2 diabetes. Fatty acid transporters hold promise as a future target to rectify lipid fluxes in the body and regain metabolic homeostasis.     

Direct interaction,instrumental for signaling processes,between LacCer and Lyn in the lipid rafts of neutrophil-like cells

Lactosylceramide [LacCer; β-Gal-(1-4)-β-Glc-(1-1)-Cer] has been shown to contain very long fatty acids that specifically modulate neutrophil properties. The interactions between LacCer and proteins and their role in cell signaling processes were assessed by synthesizing two molecular species of azide-photoactivable tritium-labeled LacCer having acyl chains of different lengths. The lengths of the two acyl chains corresponded to those of a short/medium and very long fatty acid, comparable to the lengths of stearic and lignoceric acids, respectively. These derivatives, designated C18-[³H]LacCer-(N₃) and C24-[³H]LacCer-(N₃), were incorporated into the lipid rafts of plasma membranes of neutrophilic differentiated HL-60 (D-HL-60) cells. C24-[³H]LacCer-(N₃), but not C18-[³H]LacCer-(N₃), induced the phosphorylation of Lyn and promoted phagocytosis. Incorporation of C24-[³H]LacCer-(N₃) into plasma membranes, followed by illumination, resulted in the formation of several tritium-labeled LacCer-protein complexes, including the LacCer-Lyn complex, into plasma membrane lipid rafts. Administration of C18-[³H]LacCer-(N₃) to cells, however, did not result in the formation of the LacCer-Lyn complex. These results suggest that LacCer derivatives mimic the biological properties of natural LacCer species and can be utilized as tools to study LacCer-protein interactions, and confirm a specific direct interaction between LacCer species containing very long fatty acids, and Lyn protein, associated with the cytoplasmic layer via myristic/palmitic chains.     

Fusicoccin Binding to Its Plasma Membrane Receptor and the Activation of the Plasma Membrane H-ATPase: I. Characteristics and Intracellular Localization of the Fusicoccin Receptor in Microsomes from Radish Seedlings

The characteristics of fusicoccin binding were investigated in microsomes from 24-h-old radish (Raphanus sativus L.) seedlings. The time course of fusicoccin binding depended on fusicoccin concentration: equilibrium was reached much faster at 10 nanomolar fusicoccin than at 0.3 nanomolar fusicoccin. Scatchard analysis of equilibrium binding as a function of fusicoccin concentration indicated a single class of receptor sites with a K_d of 1.8 nanomolar and a site density of 6.3 picomoles per milligram protein. Similar values (K_d 1.7 nanomolar and site density 7 picomoles per milligram protein) were obtained from the analysis of the dependence of equilibrium binding on membrane concentration at fixed fusicoccin concentrations. Fusicoccin binding comigrated with the plasma membrane H⁺-ATPase in an equilibrium sucrose density gradient: both activities formed a sharp peak (1.18 grams per milliliter) clearly distinct from that of markers of other membranes which all peaked at lower densities. The saturation profiles of fusicoccin binding and of fusicoccin-induced activation of the plasma membrane H⁺-ATPase, measured under identical conditions, were similar, supporting the view that fusicoccin-induced activation of the plasma membrane H⁺-ATPase is mediated by fusicoccin binding to its plasma membrane receptor.     

Modulation of fatty acid-binding protein content of adult rat heart in response to chronic changes in plasma lipid levels

The aim of this work was to study in the adult rat heart the effect of modifications of fatty acid (FA) supply on the content of cytoplasmic fatty acid-binding protein (H-FABP_c). To modify the amount of circulating lipids, three different treatments were chosen: (i) an hypolipidemic treatment with Clofibrate, administered daily through a gastric tube at a dose of 250 mg/kg per day for one week, (ii) a continuous intravenous infusion of 20% Intralipid, a fat emulsion, for one week at a dose of 96 ml/kg per day, and (iii) a normobaric hypoxia exposure (pO₂=10%) for three weeks. At the end of each treatment plasma lipids, myocardial H-FABP_c content and the activities of three key enzymes (citrate synthase, CS, fructrose-6-phosphate kinase, FPK and hydroxy-acyl CoA-dehydrogenase, HAD) were assessed. With each of the three treatments a decrease of plasma cholesterol and phospholipid levels was observed. Plasma FA concentration increased with Intralipid infusion and decreased with chronic hypoxia. The heart H-FABP_c content was increased by 20% with Clofibrate, decreased by 20% with chronic hypoxia and remained unaltered upon Intralipid treatment. The induced changes in H-FABP_c content were not related directly to changes in plasma lipid levels. CS activity was slightly decreased in the hypoxia group, FPK activity decreased in the Clofibrate group, and HAD activity decreased in the Intralipid group. Among the various groups heart H-FABP_c content was related to HAD activity. In conclusion, the H-FABP_c content of adult rat heart appears responsive to changes in plasma lipid levels.     

Influences of cell size and aging on l-[H]norepinephrine binding sites in rat epididymal fat cells

[³H]norepinephrine binding to isolated rat fat cells was studied as a function of adipose cell age and size. Rats aged from 4 to 78 weeks were used.Scatchard analysis of norepinephrine binding revealed in old fat cells like in young ones the existence of two orders of binding sites with respectively high and low affinity for norepinephrine. The apparent association constants Ka₁ and Ka₂ associated with these binding sites did not differ consistently in the different groups of fat cells studied (Ka₁ = 1.7 to 2.2 × 10⁶ × M⁻¹; Ka₂ = 1.9 to 2.5 × 10⁴ × M⁻¹), suggesting that age and cell size do not modify the apparent affinity of norepinephrine-binding sites in rat fat cells.On the contrary, the total amount of norepinephrine bound to each of these sites was dependent upon cell age and size. In fact, maximum binding of norepinephrine to the high affinity sites was 0.9 and 9 pmol/10⁵ cells in small (diameter: 35 μ) and large (diameter: 105 μ) adipocytes, respectively, the values found for the low affinity sites being 13 and 135 pmol/10⁵ cells. When expressed per unit of fat cell area, however, the total binding capacity for these sites appeared practically constant (2.4 — 2.8 pmol × 10⁻³/mm² and 34.2 — 38.2 pmol × 10⁻³/mm² for the high and low affinity sites respectively). These data suggest that the total norepinephrine binding capacity of the fat cell is directly proportional to its surface.     

13C NMR studies of fatty acid-protein interactions: comparison of homologous fatty acid-binding proteins produced in the intestinal epithelium

Summary A high-resolution, solution-state NMR method for characterizing and comparing the interactions between carboxyl ¹³C-enriched fatty acids (FA) and individual binding sites on proteins has been developed. The utility of this method results from the high degree of resolution of carboxyl from other carbon resonances and the high sensitivity of FA carboxyl chemical shifts to intermolecular environmental factors such as degree of hydrogen-bonding or hydration, degree of ionization (pH), and proximity to positively-charged or aromatic side-chain moieties in proteins. Information can be obtained regarding binding heterogeneity (structural as well as thermodynamic), binding stoichiometries, relative binding affinities, the ionization behavior of bound FA and protein side-chain moieties, the physical and ionization states of unbound FA, and the exchange rates of FA between protein binding sites and between protein and non-protein acceptors of FA, such as model membranes.Cytosolic fatty acid binding proteins represent an excellent model system for studying and comparing fatty acid-protein interactions. Prokaryotic expression vectors have been used to direct efficient synthesis of several mammalian intestinal FABPs in E. coli. This has enabled us to isolate gram-quantities of purified FABPs, to introduce NMR-observable isotopes, and to generate FABP mutants.The intestine is the only tissue known to contain abundant quantities of more than one FABP homologue in a single cell type. It is likely that these homologous FABPs serve distinct functional roles in intestinal lipid transport. This paper presents comparative ¹³C NMR results for FA interactions with FABP homologues from intestine, and the functional implications of these analyses are discussed.Abbreviations FA Fatty Acid(s) - FABP Fatty Acid Binding Protein(s) - I-FABP_c Cytosolic rat intestinal Fatty Acid Binding Protein - L-FABP_c Cytosolic rat liver Fatty Acid Binding Protein - CD Circular Dichroic spectroscopy Established Investigator of the American Heart Association     

Fatty acid metabolism in human breast cancer cells (MCF7) transfected with heart-type fatty acid binding protein

The human breast cancer cell line MCF7 does not express heart-type fatty acid binding protein (H-FABP), a marker protein for differentiated mammary gland. MCF7 cells transfected with the bovine H-FABP cDNA expressed the corresponding protein and were characterized by growth inhibition and lower tumorgenicity in nude mice [22]. By enzyme linked immunoassay we now determined the amount of bovine H-FABP in these cells as 638 ± 80 ng/mg protein and used the transfected cells to study the role of H-FABP in fatty acid metabolism. Compared to control cells the uptake of radioactively labelled palmitic acid and oleic acid into MCF7 cells after 30 or 60 min was increased by 67% in H-FABP expressing transfectants, demonstrating a stimulatory role for this FABP-type in fatty acid metabolism. However, preferential targeting of [¹⁴C]oleic acid into neutral or phospholipid classes was not observed by the criterion of high performance thin layer chromatography followed by autoradiography. A reason for the modest increase of fatty acid uptake in H-FABP transfected MCF7 cells may be the basal expression of epidermal-type FABP, which was detected for the first time in these cells. It appears that the small amount of E-FABP expressed in MCF7 cells fulfils the need of the cells for a cytosolic fatty acid carrier under culture conditions and that even high concentrations of another FABP do only slightly increase the uptake due to limitations of fatty acid transport through the plasma membrane or of metabolism.     

Binding of sulfosuccinimidyl fatty acids to adipocyte membrane proteins: Isolation and ammo-terminal sequence of an 88-kD protein implicated in transport of long-chain fatty acids

Summary We recently reported (Harmon et al., J. Membrane Biol. 124:261–268, 1991) that sulfo-N-succinimidyl derivatives of long-chain fatty acids (SS-FA) specifically inhibited transport of oleate by rat adipocytes. These compounds bound to an 85–90 kD membrane protein which was also labeled by another inhibitor of FA transport [³H]DIDS (4,4-diisothiocyanostilbene-2-2-sulfonate). These results indicated that the protein was a strong candidate as the transporter for long-chain fatty acids. In this report we determined that the apparent size of the protein is 88 kD and its isoelectric point is 6.9. We used [³H]SS-oleate (SSO), which specifically labels the 88-kD protein, to isolate it from rat adipocyte plasma membranes. Identification of 15 amino acids at the N-terminus region revealed strong sequence homology with two previously described membrane glycoproteins: CD36, a ubiquitous protein originally identified in platelets and PAS IV, a protein that is enriched in the apical membranes of lipidsecreting mammary cells during lactation. Antibody against PAS IV cross-reacted with the adipocyte protein. This, together with the N-terminal sequence homology, suggested that the adipocyte protein belongs to a family of related intrinsic membrane proteins which include CD36 and PAS IV.     

Variations in Sulfhydryl, Disulfide, and Protein Content during Synchronous and Asynchronous Growth of HeLa Cells

The cellular contents of protein-bound and nonprotein sulfhydry (—SH) and disulfide (—SS—) groups were measured in both asynchronous and synchronous HeLa S3 cultures. About 90% of these groups are associated with proteins, the majority in the —SH form. The content of protein-bound groups, and hence the total content of —SH and —SS— groups (28 × 10^-15 moles/cell, or 1.1 × 10^-6 moles/g protein on average), changes in parallel with the protein content (which varies between 2 and 4 × 10^-10 g/cell) as asynchronous populations pass from the lag through the exponential to the stationary phase of growth. The concentration of nonprotein —SH groups, in contrast, increases 10-fold during lag phase and decreases in stationary phase; it follows the protein concentration closely during the exponential phase, at a level of about 2.8 × 10^-15 moles/cell. In synchronous cultures the protein content doubles during the cell cycle, possibly in an exponential fashion. The total —SH and —SS— content also doubles, but the rate of increase appears to fluctuate. The concentrations of the protein-bound groups show 2- to 3-fold fluctuations per unit protein: protein-bound —SH groups and mixed —SS— linkages rise to maxima while protein-bound —SS— groups fall to a minimum at the G₁/S transition, and fluctuations in these groups occur again during G₂. In addition, the protein-bound —SH concentration falls continuously during the S phase. The nonprotein —SH concentration undergoes the largest (relative) fluctuations, dropping from 4 × 10^-15moles/cell in early G₁ to about 0.4 × 10^-15 moles/cell (of standard protein content) at the end of G₁, and then rising to 30 times this value by the end of S.