RESUMEN
BACKGROUND: Previous CSF studies in Rett syndrome suggest reduced turnover of the biogenic monoamines serotonin and dopamine. Because diminished turnover may result from CNS folate depletion, the authors studied transport of folate across the blood-brain barrier. METHODS: In four patients with Rett syndrome, the authors measured CSF values of 5-methyltetrahydrofolate (5MTHF), biogenic monoamine end-metabolites, and pterins together with serum and red blood cell folate. In CSF, the overall folate binding capacity by the two soluble folate-binding proteins FBP1 and FBP2 (sFBP) was measured using a radioligand binding method for H3-labeled folate. A specific immunoreactive test (ELISA) detected sFBP1, which normally contributes to 30 to 35% of the total folate binding capacity. Genetic analysis included DNA sequencing of the MECP2, FBP1, and FBP2 genes. Empirical treatment with oral folinic acid was evaluated. RESULTS: Two patients without and two with mutations of the MECP2 gene had normal values for red blood cell folate, serum folate, homocysteine, and methionine. In CSF, all patients had low values for 5MTHF, neopterin, and the serotonin end-metabolite 5-hydroxyindoleacetic acid (5-HIAA). Genetic analysis of FBP1 and FBP2 genes had normal results. Compared to controls, patients with Rett syndrome had normal immunoreactive sFBP1 in CSF, whereas the total folate binding capacity was disproportionately lowered. Empirical treatment with oral folinic acid normalized 5-MHTF and 5-HIAA levels in CSF, and led to partial clinical improvement. CONCLUSION: Irrespective of the MECP2 genotype, 5MTHF transfer to the CNS is reduced in Rett syndrome. Folinic acid supplementation restores 5MTHF levels and serotoninergic turnover. The lowered folate binding capacity of FBP is not explained by a defect of the FBP1 or FBP2 gene, but most likely occurs as a secondary phenomenon in Rett syndrome.
Asunto(s)
Sistema Nervioso Central/metabolismo , Ácido Fólico/metabolismo , Receptores de Superficie Celular , Síndrome de Rett/metabolismo , Monoaminas Biogénicas/metabolismo , Biomarcadores , Barrera Hematoencefálica , Proteínas Portadoras/análisis , Proteínas Portadoras/genética , Preescolar , Femenino , Receptores de Folato Anclados a GPI , Humanos , Leucovorina/uso terapéutico , Isoformas de Proteínas/análisis , Isoformas de Proteínas/genética , Pterinas/análisis , Síndrome de Rett/tratamiento farmacológico , Análisis de Secuencia de ADN , Tetrahidrofolatos/líquido cefalorraquídeoRESUMEN
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.
Asunto(s)
Proteínas Portadoras/química , Proteínas Portadoras/aislamiento & purificación , Fluorescencia , Interacciones Hidrofóbicas e Hidrofílicas , Iones/química , Leche/química , Receptores de Superficie Celular , Triptófano/química , Animales , Bovinos , Cromatografía , Femenino , Receptores de Folato Anclados a GPI , Ácido Fólico/análogos & derivados , Ácido Fólico/química , Concentración de Iones de Hidrógeno , Focalización Isoeléctrica , Punto Isoeléctrico , Ligandos , Nefelometría y Turbidimetría , Unión Proteica/fisiología , Ensayo de Unión RadioliganteRESUMEN
Binding of folate (pteroylglutamate) and 5-methyltetrahydrofolate, the major endogenous form of folate, to folate binding protein purified from cow's milk was studied at 7 degrees C to avoid degradation of 5-methyltetrahydrofolate. Both folates dissociate rapidly from the protein at pH 3.5, but extremely slowly at pH 7.4, most likely due to drastic changes in protein conformation occurring after folate binding. Dissociation of 5-methyltetrahydrofolate showed no increase at 37 degrees C suggesting that protein-bound-5-methyltetrahydrofolate is protected against degradation. Binding displayed two characteristics, positive cooperativity and a binding affinity that increased with decreasing concentrations of the protein. The binding affinity of folate was somewhat greater than that of 5-methyl tetrahydrofolate, in particular at pH 5.0. Ligand-bound protein exhibited concentration-dependent polymerization (8-mers formed at 13 microM) at pH 7.4. At pH 5.0, only folate-bound forms showed noticeable polymerization. The fact that folate at pH 5.0 surpasses 5-methyltetrahydrofolate both with regard to binding affinity and ability to induce polymerization suggests that ligand binding is associated with conformational changes of the protein which favor polymerization.
Asunto(s)
Radioisótopos de Carbono/metabolismo , Proteínas Portadoras/metabolismo , Ácido Fólico/metabolismo , Leche/química , Tetrahidrofolatos/metabolismo , Animales , Ácido Ascórbico/metabolismo , Receptores de Folato Anclados a GPI , Ácido Fólico/análogos & derivados , Concentración de Iones de Hidrógeno , Polímeros/metabolismo , Ensayo de Unión Radioligante , Receptores de Superficie Celular/metabolismoRESUMEN
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).
Asunto(s)
Proteínas Portadoras/metabolismo , Leche/química , Polímeros/metabolismo , Receptores de Superficie Celular , Animales , Tampones (Química) , Radioisótopos de Carbono/metabolismo , Receptores de Folato Anclados a GPI , Concentración de Iones de Hidrógeno , Ligandos , Unión ProteicaRESUMEN
The folate receptor (FR) in HeLa cells was characterized as to ligand binding mechanism, antigenic properties and membrane anchor in order to obtain information to be used for the design of biological agents targeting FR in malignant tumors. The receptor displayed the following binding characteristics in equilibrium dialysis experiments (37 degrees C, pH 7.4) with [3H] folate: a high-affinity type of binding that exhibited positive cooperativity with a Hill coefficient > 1.0 and an upward convex Scatchard plot, a slow radioligand dissociation at pH 7.4 becoming rapid at pH 3.5 and inhibition in the presence of other folates. The molecular size of the receptor was 100 kDa on gel filtration with Triton X-100, or similar to that of high molecular weight human milk folate binding protein (FBP). The latter protein represents a 25 kDa molecule which equipped with a hydrophobic glycosylphosphatidyl inositol (GPI) membrane anchor susceptible to cleavage by phosphatidylinositol specific phospholipase C (PI-PLC) forms micelles of 1kDa size with Triton X-100. The HeLa cell FR immunoreacted with antibodies against purified human milk FBP in ELISA, and in a fluorescence activated cell sorting system, where HeLa cells exposed to increasing concentrations of antibody showed a dose-dependent response. Exposure to PI-PLC decreased the fraction of immunolabeled cells indicating a linkage of FR to cell membranes by a GPI anchor. HeLa cells incubated with radiofolate showed a continuous uptake with time, however, with a complete suppression of uptake in the presence of an excess of cold folate. Prewash of cells at acidic pH to remove endogenous folate increased the uptake. Binding and uptake of [3H] folate was increased in cells grown in a folate-deprived medium. The HeLa FR seems to be epitope related to human milk FBP.
Asunto(s)
Proteínas Portadoras/metabolismo , Leche Humana/metabolismo , Sitios de Unión , Ensayo de Inmunoadsorción Enzimática , Epítopos , Receptores de Folato Anclados a GPI , Células HeLa , Humanos , Ligandos , Fosfatidilinositoles , Receptores de Superficie Celular/metabolismoRESUMEN
The present study was performed to establish the antigenic identity and origin of the folate binding protein in human saliva. We identified a folate receptor in human parotid and submandibular gland which immunoreacted with antibodies against human milk folate binding protein, as evidenced by ELISA and immunostaining of ductal epithelium and secretory glandular material. The receptor concentration was 0.4-1.4 nmol 3H-folate bound/g protein. Ligand binding was of a high-affinity (K=10(10) M(-1)) type, exhibited positive cooperativity, a slow radioligand dissociation at pH 7.4, and inhibition by folate analogues. The concentration of immunoreactive folate binding protein in saliva as determined by ELISA with antibodies against human milk folate binding protein was several fold higher than that determined by radioligand binding (nil - 1 nM). This indicates that a major fraction of the immunoreactive material does not bind 3H-folate, and could represent a precursor form of the protein. In conclusion, the folate binding protein in human saliva seems to be a secretory product of the salivary glands. The protein is also epitope-related to folate binding proteins in other human mucosal secretions.
Asunto(s)
Proteínas Portadoras/metabolismo , Glándula Parótida/metabolismo , Receptores de Superficie Celular , Saliva/metabolismo , Animales , Cromatografía en Gel , Ensayo de Inmunoadsorción Enzimática/métodos , Epítopos de Linfocito B/metabolismo , Receptores de Folato Anclados a GPI , Humanos , Peso Molecular , Glándula Parótida/patología , Conejos , Ensayo de Unión RadioliganteRESUMEN
High-affinity folate receptors are expressed in normal ovaries and ovarian carcinomas. Binding of [3H]folate in human ovary, serous ovarian carcinoma tissue, and ascites is a complex process that has not been well characterized. This study shows changes in binding affinity and mechanism of binding with decreasing receptor concentration, inhibition by folate derivatives, and a slow radioligand dissociation at pH 7.4 becoming rapid and complete at pH 3.5. The receptor seems to be positively charged since it elutes in the front effluent of a DEAE-Sepharose CL-6B ion-exchange column at pH 6.3. The gel filtration profile of Triton X-100-solubilized tissue and ascites contained two peaks of radioligand-bound receptor (25 and 100 kDa). Exposure of ascites to cleavage by phosphatidylinositol-specific phospholipase C resulted in a partial conversion of the 100-kDa peak to a 25-kDa peak. This suggests that the receptor may be anchored to the membrane by a glycosylphosphatidyl residue that inserts into Triton X-100 micelles, resulting in a large molecular size on gel filtration. The receptor in ovarian carcinoma tissue immunoreacts with antibodies against purified human milk folate receptor protein as shown by enzyme-linked immunosorbent assay, immunoprecipitation, sodium dodecyl sulfate-polyacrylamide gel electrophoresis immunoblotting (a single band of 45 kDa), and immunohistochemistry. In only three of seven ovarian carcinomas did expression of radioligand-bound receptors exceed levels found in five normal ovaries. However, only receptors in ovarian carcinoma specimens showed a high degree of immunoreactivity. Hence, even without elevations of the total receptor level, a folate receptor isoform homologous to human milk folate receptor protein seemed to prevail in serous ovarian carcinomas.
Asunto(s)
Ascitis/metabolismo , Proteínas Portadoras/metabolismo , Cistadenocarcinoma Seroso/metabolismo , Ácido Fólico/metabolismo , Neoplasias Ováricas/metabolismo , Ovario/metabolismo , Receptores de Superficie Celular/metabolismo , Ascitis/patología , Sitios de Unión/efectos de los fármacos , Proteínas Portadoras/antagonistas & inhibidores , Cromatografía en Gel , Cromatografía por Intercambio Iónico , Cistadenocarcinoma Seroso/química , Cistadenocarcinoma Seroso/patología , Electroforesis en Gel de Poliacrilamida , Ensayo de Inmunoadsorción Enzimática , Femenino , Receptores de Folato Anclados a GPI , Ácido Fólico/análogos & derivados , Ácido Fólico/farmacología , Humanos , Immunoblotting , Iones , Peso Molecular , Neoplasias Ováricas/química , Neoplasias Ováricas/patología , Ovario/química , Fosfatidilinositol Diacilglicerol-Liasa , Fosfoinositido Fosfolipasa C , Estructura Terciaria de Proteína , Ensayo de Unión Radioligante , Receptores de Superficie Celular/antagonistas & inhibidores , Fosfolipasas de Tipo C/farmacologíaRESUMEN
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(10M-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.
Asunto(s)
Proteínas Portadoras/metabolismo , Receptores de Superficie Celular , Seminoma/metabolismo , Neoplasias Testiculares/metabolismo , Testículo/metabolismo , Anciano , Anciano de 80 o más Años , Proteínas Portadoras/química , Proteínas Portadoras/efectos de los fármacos , Cromatografía en Gel , Receptores de Folato Anclados a GPI , Ácido Fólico/metabolismo , Antagonistas del Ácido Fólico/farmacología , Humanos , Inmunohistoquímica , Leucovorina/farmacología , Masculino , Metotrexato/farmacología , Peso Molecular , Valores de Referencia , Seminoma/tratamiento farmacológico , Neoplasias Testiculares/tratamiento farmacológico , Testículo/efectos de los fármacos , TritioRESUMEN
The presence of a folate binding protein which immunoreacts with antibodies against human milk folate binding protein was demonstrated in ascitic fluids from seven patients with ovarian adenocarcinoma. Ascitic fluids collected from two patients with other malignancies contained non-immunoreactive FBP. Tumor tissue specimens from five patients with ovarian carcinoma contained immunoreactive FBP. By contrast to normal ovaries ovarian carcinoma tissue showed positive immunostaining on immunohistochemistry. Ascitic fluids from two patients with ovarian carcinoma exhibited single distinct bands on SDS-PAGE immunoblotting. The gel filtration profile of ovarian carcinoma tissue homogenate from two patients contained 25 and 1OOkDa peaks of radioligand-bound and immunoreactive folate binding protein, while ascitic fluid from one of the patients exhibited a large 100 kDa immunoreactive peak with no radioligand binding activity. The immunoreactive non-functional 100 kDa FBP could represent unprocessed precursor FBP. Future studies are necessary to evaluate whether determination of immunoreactive FBP in ovarian adenocarcinomatosis is of any diagnostic value.
Asunto(s)
Adenocarcinoma/química , Líquido Ascítico/química , Proteínas Portadoras/análisis , Ácido Fólico/metabolismo , Neoplasias Ováricas/química , Ovario/química , Receptores de Superficie Celular , Animales , Anticuerpos/inmunología , Proteínas Portadoras/química , Proteínas Portadoras/inmunología , Cromatografía en Gel , Ensayo de Inmunoadsorción Enzimática , Femenino , Receptores de Folato Anclados a GPI , Humanos , Immunoblotting , Inmunohistoquímica , Proteínas de la Leche/inmunología , Leche Humana , Peso Molecular , ConejosRESUMEN
The presence of a folate binding protein in fluid of benign cysts of human liver and mammary gland was demonstrated. Radioligand binding was of a high-affinity type (K approximately 10(10) M -1). The gel filtration profile of cystic fluid contained two peaks of radiolabelled folate, a large one of 25 kDa and a small one of 100 kDa. The concentration of radioligand bound protein in samples of cystic fluid ranged from nil to 6 nM. In most cases the protein immunoreacted with antibodies against human milk folate binding protein. The data suggest that fluid of human liver and mammary gland cysts contains a folate binding protein which appears to be homologous to human milk folate binding protein.
Asunto(s)
Proteínas Portadoras/análisis , Quistes/química , Enfermedad Fibroquística de la Mama/química , Ácido Fólico/metabolismo , Hepatopatías/metabolismo , Receptores de Superficie Celular , Unión Competitiva , Proteínas Portadoras/química , Proteínas Portadoras/metabolismo , Cromatografía en Gel , Ensayo de Inmunoadsorción Enzimática , Femenino , Receptores de Folato Anclados a GPI , Ácido Fólico/análisis , Humanos , Peso Molecular , Ensayo de Unión Radioligante , TritioRESUMEN
We have characterized the folate receptor in malignant and benign tissues of human female genital tract (Fallopian tube and benign and malignant tissues of uterus). Radioligand binding displayed characteristics similar to those of other folate binding proteins. Those include a high-affinity type of binding (K = 10(10)M-1), apparent positive cooperativity, a slow dissociation at pH 7.4 becoming rapid at pH 3.5, and inhibition of binding by folate analogues. The gel filtration profile of Triton X-100 solubilized tissue contained two large peaks of 3H-folate labelled protein (> = 130 and 100 kDa) as well as a 25 kDa peak. Only a single band of 70 kDa was seen on SDS-PAGE immunoblotting. The large molecular size forms on gel filtration appear to represent folate receptors having a hydrophobic membrane anchor inserted into Triton X-100 micelles. The folate receptor of female genital tract showed cross-reactivity in ELISA and positive immunostaining with rabbit antibodies against human milk folate binding protein. Variations in the ratio of immunoresponse to total high affinity folic acid binding suggests the presence of multiple isoforms of the receptor in different types of malignant and benign tissues.
Asunto(s)
Adenocarcinoma/química , Proteínas Portadoras/análisis , Genitales Femeninos/química , Quistes Ováricos/química , Neoplasias Ováricas/química , Receptores de Superficie Celular/análisis , Adenocarcinoma/patología , Animales , Proteínas Portadoras/química , Proteínas Portadoras/inmunología , Cromatografía en Gel , Reacciones Cruzadas , Electroforesis en Gel de Poliacrilamida , Trompas Uterinas/química , Femenino , Receptores de Folato Anclados a GPI , Genitales Femeninos/patología , Humanos , Immunoblotting , Técnicas para Inmunoenzimas , Peso Molecular , Quistes Ováricos/patología , Neoplasias Ováricas/patología , Conejos , Ensayo de Unión Radioligante , Receptores de Superficie Celular/inmunología , Útero/químicaRESUMEN
We have characterized a high-affinity folate receptor in human molar placenta tissue. Radioligand binding exhibited characteristics typical of other high-affinity folate binding proteins. Those included, positive cooperativity, a tendency to increased binding affinity with decreasing receptor concentration, a slow ligand dissociation at pH 7.4 becoming rapid at pH 3.5, and inhibition by folate analogues. The folate receptor cross-reacted with antibodies against human milk folate binding protein, e.g. the syncytothrophoblastic layer of molar placenta tissue sections showed strongly positive immunostaining. The gel filtration profile contained two radioligand-bound peaks (25 and 100 kDa), however, with considerable overlap. Only a single band of 70 kDa was seen on SDS-PAGE immunoblotting. The folate receptor in placental tissue may play a crucial role in the transfer of folate from maternal circulation to the fetus.
Asunto(s)
Proteínas Portadoras/química , Mola Hidatiforme/química , Placenta/química , Receptores de Superficie Celular/metabolismo , Neoplasias Uterinas/química , Antídotos/farmacología , Proteínas Portadoras/efectos de los fármacos , Proteínas Portadoras/metabolismo , Ensayo de Inmunoadsorción Enzimática , Femenino , Receptores de Folato Anclados a GPI , Antagonistas del Ácido Fólico/farmacología , Humanos , Immunoblotting , Inmunohistoquímica , Leucovorina/farmacología , Metotrexato/farmacología , Embarazo , Ensayo de Unión Radioligante , Receptores de Superficie Celular/química , Receptores de Superficie Celular/efectos de los fármacos , Trofoblastos/químicaRESUMEN
The presence of a soluble folate receptor in fluids of non-neoplastic ovarian cysts was demonstrated. Radioligand binding exhibited characteristics typical of high-affinity folate-binding proteins. These included positive cooperativity, a tendency to increased binding affinity with decreasing receptor concentration, a slow ligand dissociation at pH 7.4 and inhibition by folate analogues. The folate receptor was probably synthesized in the lining epithelial cells of the cysts which showed positive immunostaining with antibodies against human milk folate-binding protein. The gel filtration profile of cystic fluid contained two radioligand-bound peaks, 25 and 100 kDa, whereas a single band of 70 kDa was seen on SDS-PAGE immunoblotting. Treatment with the enzyme phosphatidylinositol-specific phospholipase C resulted in a partial conversion of the 100 kDa peak to the 25 kDa peak. This suggests that insertion of a hydrophobic glycosylphosphatidylinositol tail into Triton X-100 micelles could give rise to large molecular size forms of the receptor on gel filtration.
Asunto(s)
Proteínas Portadoras/análisis , Exudados y Transudados/metabolismo , Quistes Ováricos/metabolismo , Receptores de Superficie Celular , Proteínas Portadoras/química , Proteínas Portadoras/inmunología , Ensayo de Inmunoadsorción Enzimática , Femenino , Receptores de Folato Anclados a GPI , Humanos , Ensayo de Unión RadioliganteRESUMEN
Binding of 3H-folate in human ovarian adenocarcinoma tissue was of a high-affinity type (K approximately 10(10) M-1) and displayed apparent positive cooperatively. A high-affinity folate receptor was also present in ascitic fluid and pleural effusion. Radioligand dissociation was slow at pH 7.4, but rapid at pH 3.5. The folate analogues methotrexate and in particular 5-formyltetrahydrofolate acted as inhibitors of 3H-folate binding in ascitic fluid. Ovarian adenocarcinoma tissue showed immunostaining with rabbit antibodies against human milk folate-binding protein. The gel filtration diagram contained two peaks of radiolabelled folate (at 25 and 100 kDa). The 25 kDa peak was predominant in ascitic fluid and pleural effusion. A single band of 70 kDa was seen on SDS-PAGE immunoblotting of tissue and malignant effusions. The concentration of folate receptor in tissue and fluid specimens could be determined by an immunochemical method (ELISA) utilizing antibodies against human milk folate-binding protein.
Asunto(s)
Adenocarcinoma/química , Líquido Ascítico/química , Proteínas Portadoras/análisis , Neoplasias Ováricas/química , Derrame Pleural/química , Receptores de Superficie Celular , Ensayo de Inmunoadsorción Enzimática , Femenino , Receptores de Folato Anclados a GPI , Humanos , Immunoblotting , Inmunohistoquímica , Ensayo de Unión RadioliganteRESUMEN
The hypothesis that folate depletion is a risk factor for development of colonic neoplasia prompted us to study the presence of a putative folate receptor in human colon mucosa. Binding of 3H-folate to normal and malignant mucosa studied by equilibrium dialysis was of high-affinity type (K = 10(10) L/mol) and displayed apparent positive cooperativity. Radioligand dissociation was slow at pH 7.4, but rapid at pH 3.5. As compared to methotrexate, 5-formyltetrahydrofolate was a potent inhibitor of binding. Gel filtration revealed a 25 kDa and a 100 kDa peak of folate-binding activity. Immunoreactivity studies were performed with rabbit antibodies against human 25 kDa milk folate-binding protein. Immunoblotting showed a single band at 65 kDa, and tissue sections exhibited immunostaining of mucosal areas. The present folate receptor with characteristics similar to those of other high-affinity folate-binding proteins may serve as a regulator of intracellular folate concentration in colon mucosa.
Asunto(s)
Proteínas Portadoras/metabolismo , Colon/metabolismo , Neoplasias del Colon/metabolismo , Mucosa Intestinal/metabolismo , Receptores de Superficie Celular/metabolismo , Electroforesis en Gel de Poliacrilamida , Ensayo de Inmunoadsorción Enzimática , Receptores de Folato Anclados a GPI , Ácido Fólico/metabolismo , Humanos , Immunoblotting , Ensayo de Unión RadioliganteRESUMEN
Binding of 3H-folate to human mammary tumor homogenate was of a high-affinity type (K = 10(10) M-1) and displayed apparent positive cooperativity. Radioligand dissociation was slow at pH 7.4, but rapid at pH 3.5. As compared to methotrexate, 5-formyltetrahydrofolate acted as a strong inhibitor of radioligand binding. Gel chromatography of radioligand-labeled homogenate of tumor tissue revealed three peaks: a small > or = 110 kDa peak and two major peaks of folate-binding activity (M(r) approximately 25 kDa and M(r) approximately 100 kDa). Mammary tumor tissue showed immunostaining with rabbit antibodies against human milk folate binder. A parallel elevation in the concentrations of folate-binding protein and triglyceride in tumor tissue as compared to normal tissue adjacent to the tumor was compatible with the localization of folate-binding protein in the triglyceride-rich fraction of mammary gland homogenate.
Asunto(s)
Adenocarcinoma/química , Neoplasias de la Mama/química , Proteínas Portadoras/análisis , Receptores de Superficie Celular , Adenocarcinoma/metabolismo , Adenocarcinoma/ultraestructura , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/ultraestructura , Proteínas Portadoras/metabolismo , Cromatografía en Gel , Ensayo de Inmunoadsorción Enzimática , Femenino , Receptores de Folato Anclados a GPI , Humanos , Inmunohistoquímica , Peso Molecular , Ensayo de Unión Radioligante , Triglicéridos/análisis , Triglicéridos/metabolismo , TritioRESUMEN
An adult male patient of Dutch ancestry has a slowly progressive neurological disease characterised by a cerebellar syndrome, distal spinal muscular atrophy, pyramidal tract dysfunction, and perceptive hearing loss. A severe folate deficiency state was found in CSF in combination with a normal serum and red cell folate state. Two unknown abnormal metabolites were present in CSF. The concentration of immunoreactive folate binding protein in CSF was unusually low, whereas the concentration of the protein measured with radioligand (3H-folate) binding was unusually high. The transfer of folate over the choroid plexus seems to be disturbed, potentially reflecting a defect in the choroid plexus folate binder.
Asunto(s)
Proteínas Portadoras/líquido cefalorraquídeo , Sistema Nervioso Central/metabolismo , Deficiencia de Ácido Fólico/líquido cefalorraquídeo , Ácido Fólico/metabolismo , Enfermedades del Sistema Nervioso/líquido cefalorraquídeo , Receptores de Superficie Celular , Adolescente , Receptores de Folato Anclados a GPI , Deficiencia de Ácido Fólico/metabolismo , Humanos , Masculino , Vitamina B 12/líquido cefalorraquídeoRESUMEN
C.d. and fluorescence spectroscopy have been used to investigate the effect of ligand binding on the structure and stability of folate-binding protein (FBP) from cow's whey. The c.d. spectrum of unligated FBP predicts the following secondary structure: 22% helix, 25% antiparallel beta-strand, 5% parallel beta-strand, 17% turn and 31% random-coil structure. Folate binding to FBP results in significant changes in the c.d. spectrum. Analysis of the spectrum shows a 10% decrease in antiparallel beta-strand as a result of ligand binding. Folate binding also leads to strong quenching of FBP tryptophan fluorescence. The magnitude of the quench is proportional to ligand binding. The guanidinium chloride-induced unfolding of FBP is shown to be a multistate process. Detection by c.d. and fluorescence spectroscopy lead to non-identical transitions. Modelling studies are consistent with the existence of a stable folding intermediate. Ligand binding to FBP increases the apparent folding stability of the molecule. Simultaneous detection by c.d. and fluorescence indicate that the apparent increased folding stability is derived from ligand-induced aggregation of FBP.
Asunto(s)
Proteínas Portadoras/química , Proteínas Portadoras/metabolismo , Conformación Proteica , Estructura Secundaria de Proteína , Receptores de Superficie Celular , Animales , Proteínas Portadoras/efectos de los fármacos , Bovinos , Dicroismo Circular , Femenino , Receptores de Folato Anclados a GPI , Ácido Fólico/metabolismo , Cinética , Ligandos , Matemática , Leche/metabolismo , Modelos Teóricos , Pliegue de Proteína , Espectrometría de Fluorescencia , Triptófano/análisisRESUMEN
Binding of 3H-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 revealed two major folate binding proteins (M(r) approximately 100 and 25 kDa), but only one single band (M(r) approximately 65-70 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 maximum 3H-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).
Asunto(s)
Ácido Fólico/metabolismo , Próstata/metabolismo , Receptores de Superficie Celular , Proteínas Portadoras/química , Proteínas Portadoras/aislamiento & purificación , Proteínas Portadoras/metabolismo , Receptores de Folato Anclados a GPI , Humanos , Concentración de Iones de Hidrógeno , Técnicas In Vitro , Cinética , Masculino , Peso Molecular , Ensayo de Unión Radioligante , Semen/metabolismoRESUMEN
High-affinity 3H-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(r) approximately 100 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%.