Mediated uptake of folate by a high-affinity binding protein in sublines of L1210 cells adapted to nanomolar concentrations of folate

Summary An L1210 cell line (JT-1), which can grow in medium supplemented with 1nm folate, has been isolated. These cells exhibit a slower growth rate than folate-replete parental cells and have a lower ability to transport folate or methotrexate via the reduced folate transport system. Measurements at nanomolar concentrations of folate revealed that the adapted cells have acquired a high-affinity folate-binding protein. Binding to this component at 37°C was rapid and reached a maximum value after 30 min which corresponded in amount to 0.23±0.3 pmol/mg protein, and excess unlabeled folate added 30 min subsequent to the [³H]folate led to a rapid release of the bound substrate. Radioactivity bound to or released from the cells after 30 min at 37°C remained as unmetabolized folic acid. Binding was also rapid at 0°C but uptake at the plateau was only one-half the value obtained at 37°C. Half-maximal saturation of the binding component (K _D) occurred at a folate concentration of 0.065nm at pH 7.4, while the affinity for folate decreased 30-fold when the pH was reduced to 6.2 (K _D=2.0nm). 5-Methyltetrahydrofolate was also bound by this component (K _i=13nm at pH 7.4) but with a much lower affinity than for folate, while progressively weaker interactions were observed with 5-formyltetrahydrofolate (K _i=45nm) and methotrexate (K _i=325nm). When the same adaptation procedure was performed with limiting amounts of 5-formyltetrahydrofolate, two additional cell lines, JT-2 and JT-3, were isolated which expressed elevated levels of the folate-binding protein. The binding activity of the latter cells was 0.46 and 1.4 pmol/mg protein, respectively. When the level of binding protein was compared in cells grown at different concentrations of folate, an increase in medium folate from 1 to 500nm caused a sevenfold reduction in binding activity in the JT-3 cell line, while these same growth conditions had no effect on binding by the other cells. These results indicate that L1210 cells adapted to low concentrations of folate or 5-formyltetrahydrofolate contain elevated levels of a high-affinity binding protein and that this protein is able to mediate the intracellular accumulation of folate compounds. L1210 cells thus appear to have two potential uptake routes for folate compounds, the previously characterized anion-exchange system and a second route mediated by a high-affinity binding protein. An additional low-affinity, high-capacity transport system for folate that had been proposed previously was not observed under a variety of experimental conditions in either the adapted or parental cells.     

Effect of hydrogen ion concentration and buffer composition on ligand binding characteristics and polymerization of cow's milk folate binding protein

The ligand binding and aggregation behavior of cow's milk folate binding protein depends on hydrogen ion concentration and buffer composition. At pH 5.0, the protein polymerizes in Tris-HCl subsequent to ligand binding. No polymerization occurs in acetate, and binding is markedly weaker in acetate or citrate buffers as compared to Tris-HCl. Polymerization of ligand-bound protein was far more pronounced at pH 7.4 as compared to pH 5.0 regardless of buffer composition. Binding affinity increased with decreasing concentration of protein both at pH 7.4 and 5.0. At pH 5.0 this effect seemed to level off at a protein concentration of 10^–6 M which is 100–1000 fold higher than at pH 7.4. The data can be interpreted in terms of complex models for ligand binding systems polymerizing both in the absence or presence of ligand (pH 7.4) as well as only subsequent to ligand binding (pH 5.0).     

A high-affinity folate binding protein in human amniotic fluid. Radioligand binding characteristics,immunological properties and molecular size

The presence of a folate binding protein of high-affinity type (affinity constant 5 · 10⁹M^–1, maximum folate binding 3 nM) in human amniotic fluid was demonstrated in equilibrium dialysis experiments (37°C, pH 7.4) with the radioligand³H-folate. Dissociation of³H-folate from the binding protein was slow at pH 7.4 but rapid at pH 3.5. By use of rabbit antibodies against low molecular weight folate binding protein from human milk we determined the concentration of folate binding protein in 5 amniotic fluids (range 1.5–2.3 nM) in an Enzyme-Linked Immunosorbent Assay (ELISA). ultrogel AcA 44 chromatography of amniotic fluid showed that immunoreactive and radioligand bound folate binding protein coeluted in two peaks: a major one (M _r 25 000) and a minor one (M _r 100 000).     

A high-affinity folate binding protein in human semen

The presence of a folate binding protein of high-affinity type (affinity constant 3.10¹⁰M^–1, maximum folate binding 1.4 nM) in human semen was demonstrated in equilibrium dialysis experiments (37°C, pH 7.4) with the radioligand³H-folate. Radioligand dissociation from the binding protein was slow at pH 7.4, but rapid at pH 3.5. By use of rabbit antibodies against 25 kDa human milk folate binding protein we determined the concentration of folate binding protein in 16 speciments of human semen in an enzyme-linked immunosorbent assay. The concentration of immunoreactive folate binding protein was independent of the number of spermatozoa in individual specimens. Gel filtration showed that immunoreactive and radioligand bound folate binding protein coeluted in two peaks: a major one of 100 kDa and a minor one of 25 kDa.     

High-affinity folate binding in human mammary gland

High-affinity³H-folate binding in Triton X-100 solubilized human mammary gland tissue displayed characteristics, e.g. apparent positive cooperativity and increasing affinity with decreasing concentration of folate binding protein, shown to be typical of specific folate binding. Radioligand dissociation was slow at pH 7.4. A major fraction of the bound radioligand dissociated rapidly at pH 3.5, while a residual binding of 20% persisted even after prolonged dialysis at pH 3.5. Gel chromatography revealed two major folate binding proteins (M_r100 kDa and 25 kDa). However, only one single band was detectable on SDS-PAGE immunoblotting. The highest folate binding activity per g protein was associated with the upper triglyceride-containing layer of the 1000 g supernatant of the homogenate. The folate binding protein extracted from this layer had a low cross-reactivity (<5%) with rabbit antibodies against 25 kDa human milk folate binding protein. The folate binding protein in the 1000 g pellet and the aqueous phase of the 1000 g supernatant was present at a low concentration and had a cross-reactivity of 100%.     

Mediated uptake of folate by a high-affinity binding protein in sublines of L1210 cells adapted to nanomolar concentrations of folate

An L1210 cell line (JT-1), which can grow in medium supplemented with 1 nM folate, has been isolated. These cells exhibit a slower growth rate than folate-replete parental cells and have a lower ability to transport folate or methotrexate via the reduced folate transport system. Measurements at nanomolar concentrations of folate revealed that the adapted cells have acquired a high-affinity folate-binding protein. Binding to this component at 37 degrees C was rapid and reached a maximum value after 30 min which corresponded in amount to 0.23 +/- 0.3 pmol/mg protein, and excess unlabeled folate added 30 min subsequent to the [3H]folate led to a rapid release of the bound substrate. Radioactivity bound to or released from the cells after 30 min at 37 degrees C remained as unmetabolized folic acid. Binding was also rapid at 0 degrees C but uptake at the plateau was only one-half the value obtained at 37 degrees C. Half-maximal saturation of the binding component (KD) occurred at a folate concentration of 0.065 nM at pH 7.4, while the affinity for folate decreased 30-fold when the pH was reduced to 6.2 (KD = 2.0 nM). 5-Methyltetrahydrofolate was also bound by this component (Ki = 13 nM at pH 7.4) but with a much lower affinity than for folate, while progressively weaker interactions were observed with 5-formyltetrahydrofolate (Ki = 45 nM) and methotrexate (Ki = 325 nM). When the same adaptation procedure was performed with limiting amounts of 5-formyltetrahydrofolate, two additional cell lines, JT-2 and JT-3, were isolated which expressed elevated levels of the folate-binding protein. The binding activity of the latter cells was 0.46 and 1.4 pmol/mg protein, respectively. When the level of binding protein was compared in cells grown at different concentrations of folate, an increase in medium folate from 1 to 500 nM caused a sevenfold reduction in binding activity in the JT-3 cell line, while these same growth conditions had no effect on binding by the other cells. These results indicate that L1210 cells adapted to low concentrations of folate or 5-formyltetrahydrofolate contain elevated levels of a high-affinity binding protein and that this protein is able to mediate the intracellular accumulation of folate compounds. L1210 cells thus appear to have two potential uptake routes for folate compounds, the previously characterized anion-exchange system and a second route mediated by a high-affinity binding protein.(ABSTRACT TRUNCATED AT 400 WORDS)     

Ligand binding characteristics of two molecular forms,one equipped with a hydrophobic glycosyl phosphatidylinositol tail,of the folate binding protein purified from human milk

Two molecular forms of the folate binding protein were isolated and purified from human milk by a combination of cation exchange- and affinity chromatography. One protein (27 kDa) was a cleavage product of the other 100 kDa protein as evidenced by N-terminal amino acid sequence homology and a reduction in the molecular size of the latter protein to 27 kDa after cleavage of its hydrophobic glycosylphosphatidylinositol tail by phosphatidylinositol-specific phospholipase C. High-affinity binding of [³H]folate was characterized by upward convex Scatchard plots and increasing ligand binding affinity with decreasing concentrations of both proteins. Downward convex Scatchard plots and binding affinities showing no dependence on the protein concentration were, however, observed in highly diluted solutions of both proteins. Radioligand binding was inhibited by folate analogs, and dissociation of radioligand was slow at pH 7.4 but rapid and complete at pH 5.0 and 3.5. Ligand binding quenched the tryptophan fluorescence of the 27 kDa protein suggesting that tryptophan is present at the binding site and/or ligand binding induces a conformation change that affects tryptophan environment in the protein. The 27 kDa protein representing soluble folate binding protein exhibited a greater affinity for ligand binding than the 100 kDa protein which possesses a hydrophobic tail identical to the one that anchors the folate receptor to the cell membrane.     

High-affinity folate binding in human prostate

Binding of³H-folate in Triton X-100 solubilized human prostate homogenate was of a high-affinity type and displayed apparent positive cooperativity typical of specific folate binding. Radioligand dissociation was slow at pH 7.4, but rapid at pH 3.5. Gel chromatography reveled two major folate binding proteins (M_r100 and 25kDa), but only one single band (M_r65–70 kDa) was detectable on SDS-PAGE and immunoblotting with rabbit-anti human milk folate binding protein. Concentration of folate binding protein in prostate homogenate expressed as maximum³H-folate binding was 1.10 nmol/g protein, and the cross-reactivity with rabbit-anti human milk folate binding protein serum was 15% as determined by an enzyme-linked immunosorbent assay (median values; n=6).     

The interrelationship between ligand binding and thermal unfolding of the folate binding protein. The role of self-association and pH

The present study utilized a combination of DLS (dynamic light scattering) and DSC (differential scanning calorimetry) to address thermostability of high-affinity folate binding protein (FBP), a transport protein and cellular receptor for the vitamin folate. At pH 7.4 (pI = 7–8) ligand binding increased concentration-dependent self-association of FBP into stable multimers of holo-FBP. DSC of 3.3 μM holo-FBP showed Tm (76 °C) and molar enthalpy (146 kcal M^− 1) values increasing to 78 °C and 163 kcal M^− 1 at 10 μM holo-FBP, while those of apo-FBP were 55 °C and 105 kcal M^− 1. Besides ligand binding, intermolecular forces involved in concentration-dependent multimerization thus contribute to the thermostability of holo-FBP. Hence, thermal unfolding and dissociation of holo-FBP multimers occur simultaneously consistent with a gradual decrease from octameric to monomeric holo-FBP (10 μM) in DLS after a step-wise rise in temperature to 78 °C ≈ Tm. Stable holo-FBP multimers may protect naturally occurring labile folates against decomposition or bacterial utilization. DSC established an interrelationship between diminished folate binding at pH 5, especially in NaCl-free buffers, and low thermostability. Positively charged apo-FBP was almost completely unfolded and aggregated at pH 5 (Tm 38 °C) and holo-FBP, albeit more thermostable, was labile with aggregation tendency. Addition of 0.15 M NaCl increased thermostability of apo-FBP drastically, and even more so that of holo-FBP. Electrostatic forces thus seem to contribute to a diminished thermostability at low pH. Fluorescence spectroscopy after irreversible thermal unfolding of FBP revealed a weak-affinity folate binding.     

The preparation and properties of folate-binding protein from cow''s milk.

An improved affinity-chromatographic method for the preparation of folate-binding protein from cow's milk is described. Under dissociating conditions the protein appeared homogeneous in the ultracentrifuge, with a molecular weight of 35 000 +/- 1500, but it was heterogeneous on electrophoresis and ion-exchange chromatography and evidently consisted of several glycoproteins with similar molecular weights that all bound folic acid. Overall, the protein contained a high proportion of half-cystine (18 residues/molecule) and 10.3% of carbohydrate. At saturation it bound approx. 1 mol of folate/mol of protein at pH 7.2. Equilibrium-dialysis measurements of the binding of folic acid and 5-methyltetrahydrofolate to the purified protein gave non-linear Scatchard plots, the shapes of which depended on pH. The results were interpreted in terms of ligand binding to a polymerizing system in which the affinity of ligand for monomer was greater than its affinity for polymer. When the protein concentration was similar to that in cow's milk, dissociation constants (Kd) for folate and 5-methyltetrahydrofolate were 3 nM and 5 nM respectively at pH 7.2 and 37 degrees C, whereas Kd for the binding of folate to monomer was about 50 pM. The properties of the binding protein are discussed in relation to its possible role in folate absorption in the gut.     

A high-affinity folate binding protein in human urine. Radioligand binding characteristics,immunological properties and molecular size

High-affinity binding of [³H]folate in human urine displayed characteristics, e.g. apparent positive cooperativity, which are typical of specific folate binding. By means of a two-site enzyme-linked immunosorbent assay (ELISA) with rabbit antibodies against the low molecular weight folate binding protein from human milk, we measured folate binding protein concentrations in the range of 0.51 to 4.13 nM in urine samples from 16 apparently healthy individuals. Ultrogel AcA 44 chromatography of the urine showed that immunoreactive and radioligand bound folate binding protein coeluted in one large peak (M_r25,000).     

Ionic Charge, Hydrophobicity and Tryptophan Fluorescence of the Folate Binding Protein Isolated from Cow's Milk

A high-affinity folate binding protein was isolated and purified from cow's milk by a combination of cation exchange chromatography and methotrexate affinity chromatography. Chromatofocusing studies revealed that the protein possessed isoelectric points in the pH-interval 8–7. Polymers of the protein prevailing at pH values close to the isoelectric points seemed to be more hydrophobic than monomers present at pH 5.0 as evidenced by hydrophobic interaction chromatography and turbidity (absorbance at 340 nm) in aqueous buffer solutions (pH 5–8). Ligand binding seemed to induce a conformation change that decreased the hydrophobicity of the protein. In addition, Ligand binding quenched the tryptophan fluorescence of folate binding protein suggesting that tryptophan is present at the binding site and/or ligand binding induces a conformation change that affects tryptophan environment in the protein. There was a noticeable discordance between the ability of individual folate analogues to compete with folate for binding and the quenching effect.     

SYNTHESIS OF FOLATE RECEPTOR-TARGETED AND DOXORUBICIN-COUPLED CHEMOTHERAPEUTIC NANOCONJUGATE AND RESEARCH INTO ITS MEDICAL APPLICATIONS

Folate-targeted drug delivery has become an alternative therapy for the treatment of various cancers. Folate receptors are known to be responsible for cellular accumulation of folate and folate analogs with high binding affinity. The anthracycline antibiotic doxorubicin has a broad spectrum of antineoplastic action and a correspondingly widespread degree of clinical use. In this work, we aimed to prepare a folate receptor-targeted doxorubicin delivery system to achieve minimal effect of doxorubicin on healthy cells and more cytotoxicity of it on tumor cells. Folate–poly(ethylene glycol)–doxorubicin (FOL-PEG-DOX) nanoconjugate was synthesized through this aim and characterized with nuclear magnetic resonance (NMR), zetasizer, and atomic force microscopy (AFM). Doxorubicin release studies were also performed in vitro. The size of FOL-PEG-DOX was 78.84 nm. The results indicated that doxorubicin release rate from the conjugate was faster at pH 5.0 than pH 7.4 and the amide bond between DOX and PEG was more stable at pH 7.4 than pH 5.0. As a consequence, FOL-PEG-DOX nanoconjugate could be a potentially useful delivery system for folate receptor-positive cancer cells.     

Purification and characterization of folate binding proteins from rat placenta

Rat placenta contains virtually no unsaturated (i.e., apo-form) folate binding protein. However, by lowering the pH of a solubilized membrane preparation of this tissue to 3.5, the endogenous bound folate was dissociated from the protein and adsorbed to charcoal. The apo-form of the folate binding protein thus obtained was purified by affinity chromatography using pteroylglutamic acid covalently coupled to Sepharose 4B. A single protein band with an apparent M_r of 36 000 was observed by SDS-polyacrylamide gel electrophoresis of the eluate from the affinity matrix. Western blot of this preparation using a rabbit antiserum raised with the affinity eluate also identified a single 36 kDa protein band. However, peptide sequencing of the N-terminal region of the proteins in the affinity eluate established that it contained two homologous proteins. Computer alignment of the first 22 N-terminal amino acids of each rat placental protein with human, bovine milk and mouse folate binding proteins showed 50–64% identical homology and 27% homology when the eight proteins were aligned together. The affinity of both rat proteins is highest for pteroylglutamic acid (K_a = 1.6 − 109 l/mol) lower for N⁵-methyltetrahydrofolate and substantially lower for N⁵-formyltetrahydrofolate. In the dose-response range studied there was no apparent affinity for methotrexate. The folate binding proteins could be released from a preparation of placental membranes using phospholipase C indicating that these proteins belong to the class of proteins anchored to the plasma membrane by a glycosyl phosphatidylinositol adduct.     

Dependence of aggregation and ligand affinity on the concentration of the folate-binding protein from cow's milk

Ultracentrifugation and gel-filtration studies showed that the folate-binding protein from cow's milk possessed a remarkable aggregation tendency at pH 7.4. Aggregation was enhanced in the presence of folate which suggested an interrelationship between the mechanisms of ligand binding and polymerization. The degree of polymerization increased with increasing concentrations of binding protein. Thus, while the monomer prevailed at 1 nM, a polymer composed of more thn 32 monomers was formed at 130 microM. Two characteristics of folate binding, i.e., Scatchard plots that were convex upward and a ligand affinity that was inversely proportional to the concentration of binding protein, could be interpreted in terms of ligand binding to a polymerizing system in which the polymerization equilibria affect the ligand affinity.     

Characterization of folate uptake by choroid plexus epithelial cells in a rat primary culture model

Reduced derivatives of folic acid (folates) play a critical role in the development, function and repair of the CNS. However, the molecular systems regulating folate uptake and homeostasis in the central nervous system remain incompletely defined. Choroid plexus epithelial cells express high levels of folate receptor α (FRα) suggesting that the choroid plays an important role in CNS folate trafficking and maintenance of CSF folate levels. We have characterized 5-methyltetrahydrofolate (5-MTHF) uptake and metabolism by primary rat choroid plexus epithelial cells in vitro . Two distinct processes are apparent; one that is FRα dependent and one that is independent of the receptor. FRα binds 5-MTHF with high affinity and facilitates efficient uptake of 5-MTHF at low extracellular folate concentrations; a lower affinity FRα independent system accounts for increased folate uptake at higher concentrations. Cellular metabolism of 5-MTHF depends on the route of folate entry into the cell. 5-MTHF taken up via a non-FRα -mediated process is rapidly metabolized to folylpolyglutamates, whereas 5-MTHF that accumulates via FRα remains non-metabolized, supporting the hypothesis that FRα may be part of a pathway for transcellular movement of the vitamin. The proton-coupled folate transporter, proton-coupled folate transporter (PCFT), mRNA was also shown to be expressed in choroid plexus epithelial cells. This is consistent with the role we have proposed for proton-coupled folate transporter in FRα-mediated transport as the mechanism of export of folates from the endocytic compartment containing FRα.     

Ligand Binding Characteristics of a Glycosylphosphatidyl Inositol Membrane-Anchored HeLa Cell Folate Receptor Epitope-Related to Human Milk Folate Binding Protein

The folate receptor (FR) in HeLa cells was characterized as to ligandbinding mechanism, antigenic properties and membrane anchor in order toobtain information to be used for the design of biological agentstargeting FR in malignant tumors. The receptor displayed the followingbinding characteristics in equilibrium dialysis experiments(37°C, pH 7.4) with [³H] folate: a high-affinity type of bindingthat exhibited positive cooperativity with a Hill coefficient >1.0and an upward convex Scatchard plot, a slow radioligand dissociation atpH 7.4 becoming rapid at pH 3.5 and inhibition in the presence of otherfolates. The molecular size of the receptor was 100 kDa on gel filtrationwith Triton X-100, or similar to that of high molecular weight human milkfolate binding protein (FBP). The latter protein represents a 25 kDamolecule which equipped with a hydrophobic glycosylphosphatidylinositol (GPI) membrane anchor susceptible to cleavage byphosphatidylinositol specific phospholipase C (PI-PLC) formsmicelles of 100 kDa size with Triton X-100. The HeLa cell FRimmunoreacted with antibodies against purified human milk FBP inELISA, and in a fluorescence activated cell sorting system, whereHeLa cells exposed to increasing concentrations of antibody showed adose-dependent response. Exposure to PI-PLC decreased the fraction ofimmunolabeled cells indicating a linkage of FR to cell membranes by aGPI anchor. HeLa cells incubated with radiofolate showed a continuousuptake with time, however, with a complete suppression of uptake in thepresence of an excess of cold folate. Prewash of cells at acidic pH toremove endogenous folate increased the uptake. Binding and uptake of [³H]folate was increased in cells grown in a folate-deprived medium. The HeLaFR seems to be epitope related to human milk FBP.     

Anion specificity of the jejunal folate carrier: Effects of reduced folate analogues on folate uptake and efflux

Summary We previously reported that³H-folate uptake by rabbit jejunal brush-border membrane (BBM) vesicles was markedly stimulated by an outwardly directed OH^– gradient (pH_in 7.7, pH_out 5.5), inhibited by anion exchange inhibitors (DIDS, SITS, furosemide), and saturable (folateK _m=0.19 m) suggesting carrier-mediated folate/OH^– exchange (or H⁺/folate cotransport). In the present study, the anion specificity of this transport process was examined. Under conditions of an outwardly directed OH^– gradient, DIDS-sensitive folate uptake wascis inhibited (>90%) by reduced folate analogues: dihydrofolate (IC₅₀=0.40 m), folinic acid (IC₅₀=0.50 m), 5-methyltetrahydrofolate (IC₅₀=0.53 m), and (+)amethopterin (IC₅₀=0.93 M). In contrast, 10 m (–)amethopterin had only a modest effect on folate uptake (18% inhibition) suggesting stereospecificity of the folate/OH^– exchanger. The nonpteridine compounds which are transported by the folate carrier in L1210 leukemic cells (phthalate, thiamine pyrophosphate, and PO ₄ ^–3 ) did not inhibit jejunal folate uptake. Furthermore, folate uptake was not inhibited by SO ₄ ^–2 (4mm) or oxalate (4mm) thereby distinguishing this carrier from the previously described intestinal SO ₄ ^–2 /OH^– and oxalate/Cl^– exchangers. After BBM vesicles were loaded with³H-folate, the initial velocity of³H-folate efflux was stimulated by unlabeled folate in the efflux medium. The transstimulation of³H-folate efflux by unlabeled folate was furosemide (or DIDS) inhibitable and temperature sensitive. Half-maximal stimulation of furosemide-sensitive³H-folate efflux was observed with 0.25±0.05 m unlabeled folate, a concentration similar to theK _m for folate uptake. These data suggest that folate-stimulated³H-folate efflux is mediated by the folate/OH^– exchanger. With the exception of (–) amethopterin, reduced folate analogues also transstimulated furosemide-sensitive³H-folate efflux in a concentration-dependent manner suggesting stereospecific transport of these analogues by the folate/OH^– exchanger. In summary, folate transport by the jejunal folate/OH^– exchanger demonstrates bothcis inhibition and transstimulation by reduced folate analogues, but not by other inorganic or organic anions suggesting bidirectional transport of folate and a high degree of anion specificity.     

Characterization of a High-Affinity Folate Receptor in Normal and Malignant Human Testicular Tissue

We have characterized the folate receptor in normal and malignant tissue from male gonads. Radioligand binding displayed characteristics typical of other folate receptors. Those included a high-affinity type of binding (K = 10^{10 M–1}), apparent positive cooperativity changing into non-cooperativity at low receptor concentrations, a tendency to increased binding affinity with decreasing receptor concentrations, a slow dissociation at pH 7.4 becoming rapid at pH 3.5 and inhibition by folates, in particular oxidized forms. The gel filtration profile of Triton X-100 solubilized tissue contained a 25 and 100 kDa peak of radioligand-receptor. The latter peak could represent receptor equipped with a hydrophobic membrane anchor that inserts into Triton X-100 micelles. The concentration of radiolabelled receptor ranged from 0.41 nmol/g protein to 1.68 nmol/g protein in specimens of normal testicular tissue from patients with prostatic carcinomas and from 1.54 nmol/g protein to 3.82 nmol/g protein in testicular tissue from young individuals. Compared to normal testicular tissue the concentration of receptor in seminoma tissue was low (0.38–1.27 nmol/g protein) but showed a higher degree of immunoreactivity in the presence of antibodies against human milk folate binding protein as evidenced by ELISA and immunohistochemistry data. Hence a folate receptor isoform homologous to human milk folate binding protein is apparently expressed in seminomas where the total expression of receptor, however, seems to be lower than in normal testicles.     

Ligand binding characteristics and aggregation behavior of purified cow's milk folate binding protein depends on the presence of amphiphatic substances including cholesterol,phospholipids, and synthetic detergents

Folate binding protein was purified from cow's milk by a combination of cation exchange chromatography and methotrexate-AH-sepharose affinity chromatography. Dilution of the preparation to concentrations of protein less than 10 nM resulted in drastic changes of radioligand (folate) binding characteristics, i.e., a decrease in binding affinity with a change from upward to downward convex Scatchard plots and increased ligand dissociation combined with appearance of weak-affinity aggregated forms of the binding protein on gel filtration. These findings, consistent with a model predicting dimerization between unliganded and liganded monomers, were reversed in the presence of material eluted from the affinity column after adsorption of the protein(cofactor) or cholesterol, phospholipids, and synthetic detergents. The latter amphiphatic substances form micelles and lipid bilayers which could separate hydrophobic unliganded monomers from hydrophilic liganded monomers in the surrounding aqueous medium and thereby prevent association between these monomeric forms prevailing at low concentrations of the protein. Our data have some bearings on studies which show that cholesterol and phospholipids are necessary for the clustering of folate receptors in the cell membrane; a process required for optimum receptor function and internalization of folate.