RESUMEN
A delipidation procedure based on treatment with charcoal at pH 3 has been applied to highly purified rat alpha 1-foetoprotein preparations. The oestrogen binding properties of the delipidated proteins have been studied with an equilibrium dialysis technique, and compared with the properties of the untreated foetal protein, as well as those of preparations reconstituted from the defatted alpha 1-foetoprotein and the removed lipids. An important increase has been evidenced for the binding levels of oestrone, oestradiol-17 beta and diethylstilboestrol by the delipidated alpha 1-foetoprotein. A reversal of this effect has been obtained by incubating the delipidated protein either with the lipids extracted from the purified alpha 1-foetoprotein or with a potent competitor of the rat alpha 1-foetoprotein-oestrogen interaction, designated as 'L', previously demonstrated and isolated from whole rat sera, and tentatively characterized as a mixture of fatty acids. Scatchard analysis of the oestrone and oestradiol-17 beta binding parameters show that the enhanced fixation of the hormones after defatting is primarily due to a two-fold increase of the apparent number of binding sites/mol alpha 1-foetoprotein. The results are interpreted in terms of the probable, at least partial, identity between the lipids closely associated with the pure alpha 1-foetoprotein and the fatty acid mixture 'L' isolated from whole sera. The possible biological role of complex interplay between oestrophilic alpha 1-foetoproteins, phenolsteroids and fatty acids in the control of oestrogen levels during development is discussed briefly.
Asunto(s)
Estrógenos , Ácidos Grasos , alfa-Fetoproteínas , Animales , Dietilestilbestrol , Embrión de Mamíferos , Estradiol , Estrona , Femenino , Humanos , Unión Proteica , Radioinmunoensayo , Ratas , alfa-Fetoproteínas/metabolismoRESUMEN
Binding, spectral and immunological studies were performed to demonstrate the conformational changes in rodent and human alpha-fetoprotein (AFP) induced by a free fatty acid environment. Scatchard analysis of estradiol (E2) binding to purified rat AFP indicated that unsaturated fatty acids changed the number of binding E2 sites and the apparent E2 equilibrium dissociation constant which varied non-linearly with docosahexaenoic acid concentration. UV spectral analysis of rodent and human AFPs showed that the absorbance minimum of AFP incubated with unsaturated fatty acid (L-AFP) was red-shifted, broadened and less pronounced than that of purified native AFP (N-AFP). Immunochemical studies with specific polyclonal antibodies to purified rodent and human AFPs (N-AFP antibodies) showed that these proteins lost immunoreactivity after incubation with unsaturated fatty acid. N-AFP antibodies recognized fewer epitopes on L-AFP than on N-AFP, whatever the species. Specific anti-rat L-AFP antibodies were used to demonstrate specific epitopes on rat L-AFP. Rat L-AFP antibodies did not recognize rat N-AFP. Saturated fatty acids were without effect on the binding, spectral and immunological properties of rodent and human AFPs. RIA or ELISA values for human AFP from fetal serum, hepatoma serum, and cord serum, were reduced 80, 50 and 5%, respectively, by unsaturated fatty acids. This decrease correlated with the relative percentage of polyunsaturated fatty acid in each biological fluid. Such results indicate that an unsaturated fatty acid environment induces conformational changes in AFP which may modulate the endocrine and immune functions of this protein.
Asunto(s)
Ácidos Grasos no Esterificados/farmacología , Ácidos Grasos Insaturados/farmacología , alfa-Fetoproteínas/metabolismo , Animales , Intoxicación por Tetracloruro de Carbono/sangre , Cromatografía de Afinidad , Estradiol/metabolismo , Sangre Fetal/análisis , Feto , Humanos , Cinética , Conformación Proteica , Ratas , Ratas Endogámicas , Especificidad de la Especie , alfa-Fetoproteínas/aislamiento & purificación , alfa-Fetoproteínas/ultraestructuraRESUMEN
In polyacrylamide gels, highly purified rat alpha1-fetoprotein shows a molecular heterogeneity, i.e. a "slow" and a "fast" moving fraction. We have isolated by electrophoretic fractionation and subsequent elution these two forms of alpha1-fetoprotein, and we have studied comparatively the binding parameters for estradiol-17beta of whole alpha1-fetoprotein preparations and of the isolated forms. We have shown that the number of binding sites per molecule of whole alpha1-fetoprotein is always, in our experimental conditions, a fractional number, inferior to unity (0.3). Furthermore, the analysis of the binding parameters of the "two forms" of alpha1-fetoprotein allows discrimination between different classes of binding sites. For the "slow" fraction, the number of predominant binding sites per molecule of protein is close to unity (0.7-0.9), whereas for the "fast" fraction, a very low fractional value is found (0.1). The corresponding association constants are reproducibly different for the two fractions: Ka = 0.1.10(8) M-1 for the "slow" alpha1-fetoprotein, and Ka = 0.7.10(8) M-1 for the "fast" alpha1-fetoprotein. Traces of a very high affinity (10(9) M-1) minor class of binding sites are demonstrated in the "slow" fraction. These results point to the existence of a molecular population of alpha1-fetoprotein, some forms of which have a strong or very strong affinity, and some a negligible affinity, for estrogens.
Asunto(s)
Estradiol , Proteínas Fetales , alfa-Fetoproteínas , Animales , Sitios de Unión , Carcinoma Hepatocelular/sangre , Femenino , Neoplasias Hepáticas/sangre , Masculino , Unión Proteica , Conformación Proteica , Ratas , alfa-Fetoproteínas/inmunología , alfa-Fetoproteínas/aislamiento & purificaciónRESUMEN
We report detailed gas chromatography analyses of the non-esterified fatty acids in the sera of female rats during post-natal maturation. A marked age-dependent decrease of concentration is demonstrated for all classes of compounds. Total levels fall from about 0.8 mM at birth to about 0.25 mM, 60 days later. The decrease is most pronounced for the polyunsaturated acids, which represent 27 +/- 9% of total fatty acids at birth and 13 +/- 3% 60 days later. The effects of ovariectomy and adrenalectomy on the free fatty acid levels as a function of age are strikingly different before and after maturation. When ovariectomy is performed at 5, 9 and 15 days, the fatty acid levels respond by a significant (30-40%) decrease; when adrenalectomy is carried out at the same ages, a dramatic 3-5-fold increase of all classes of fatty acids is observed. By contrast, in older animals, both responses have virtually disappeared. Possible mechanisms underlying the age-dependent patterns and behaviour of the serum free fatty acids are briefly discussed.
Asunto(s)
Adrenalectomía , Castración , Ácidos Grasos no Esterificados/sangre , Factores de Edad , Animales , Cromatografía de Gases , Femenino , Ratas , Ratas EndogámicasRESUMEN
The binding of the lignans, enterolactone, enterodiol, nordihydroguaiaretic acid (NDGA), and the isoflavonic phytoestrogen equol, to human and rat alpha-fetoprotein (AFP) was studied. They had differential inhibitory effects (NDGA greater than equol greater than enterolactone greater than enterodiol) on the binding of estrone and estradiol to rat AFP and the binding of unsaturated fatty acid to both rat and human AFP. Inhibition was dose-dependent. The apparent dissociation constants (Kd) for phytoestrogens binding to AFP were: Kd NDGA = 5 +/- 1.2.10(-7) M, Kd equol = 6.7 +/- 0.8.10(-6) M, Kd enterolactone = 1.7 +/- 0.4.10(-5) M and Kd enterodiol = 2.2 +/- 0.6.10(-5) M. The Kd for estrone binding to rat AFP was increased by increasing concentrations of equol, but the number of esterone binding sites remained unchanged. This, plus the results of double-reciprocal plots, suggests that they compete for the same site(s). NDGA also competitively inhibited estrone binding at low NDGA concentrations (increased Kd), but high concentrations induced conformational changes in rat AFP, as both Kd and the number of binding sites (n) were altered. Both rat and human AFPs underwent changes in electrophoretic behaviour and loss of immunoreactivity with increasing NDGA, suggesting that NDGA binding induces conformational changes in the AFPs. However, equol did not alter the electrophoretic or immunological properties of either rat or human AFP, providing further evidence for qualitative differences in the effects of these diphenols. These findings indicate that phytoestrogens could play a role in AFP-dependent normal and pathological growth and development.
Asunto(s)
Estrógenos no Esteroides , Estrógenos/metabolismo , Isoflavonas , Lignanos , alfa-Fetoproteínas/metabolismo , 4-Butirolactona/análogos & derivados , 4-Butirolactona/metabolismo , Adulto , Animales , Ácido Araquidónico/metabolismo , Unión Competitiva , Butileno Glicoles/metabolismo , Cromanos/metabolismo , Equol , Humanos , Inmunoelectroforesis , Ligandos , Masoprocol/metabolismo , Fitoestrógenos , Preparaciones de Plantas , Ratas , Ratas Endogámicas , alfa-Fetoproteínas/aislamiento & purificaciónRESUMEN
Activity of the enzyme phosphatidylinositol 4,5-bisphosphate phospholipase C (PIP2-PLC) was demonstrated in MCF-7 human breast cancer cell homogenate. The addition of 10(-9) M 17 beta-estradiol to the culture medium elicited in the cells two types of responses depending on the period of exposure. Enzyme activity was rapidly activated at 15 s of incubation. After 5 min, PIP2-PLC activity was inhibited, and this effect continued at least until 24 h of exposure to the hormone. When 17 beta-estradiol was added in vitro to the total homogenate of untreated cells, enzyme activity was stimulated in a dose-dependent manner. These findings indicate that 17 beta-estradiol induces early and long-term modifications of the phosphoinositide signal pathway in intact MCF-7 cells as well as in vitro. The rapidity of the early effect suggests a non-genomic action of estradiol.
Asunto(s)
Estradiol/farmacología , Hidrolasas Diéster Fosfóricas/metabolismo , Transducción de Señal/efectos de los fármacos , División Celular , Activación Enzimática/efectos de los fármacos , Humanos , Fosfoinositido Fosfolipasa C , Factores de Tiempo , Células Tumorales Cultivadas/efectos de los fármacos , Células Tumorales Cultivadas/enzimologíaRESUMEN
Regulation by unsaturated fatty acids of glucocorticoid-sensitive gene transcription was studied in HeLa cells transiently transfected with a mouse mammary tumour virus-luciferase reporter gene. Arachidonic acid and docosahexaenoic acid by themselves had no effect on basal levels of luciferase expression. However, they were able to enhance dexamethasone-induced transcription by 1.4-2.3 times (25-42 times the control levels) in a dose-dependent manner (ED50: 18 and 8 microM) for arachidonic and docosahexaenoic acid, respectively. The glucocorticoid antagonist RU486 effectively antagonized the dexamethasone response as well as the synergistic effect observed in the presence of arachidonic and docosahexaenoic acids, suggesting that the glucocorticoid receptor was an intermediate in the fatty acid synergism of the dexamethasone response. These studies show that fatty acids may be playing a role in modulating the intracellular steroid hormone signalling pathway to co-regulate a glucocorticoid-sensitive promoter.
Asunto(s)
Ácido Araquidónico/farmacología , Dexametasona/farmacología , Ácidos Docosahexaenoicos/farmacología , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Glucocorticoides/farmacología , Luciferasas/biosíntesis , Animales , Dexametasona/antagonistas & inhibidores , Relación Dosis-Respuesta a Droga , Sinergismo Farmacológico , Glucocorticoides/antagonistas & inhibidores , Células HeLa/enzimología , Células HeLa/virología , Antagonistas de Hormonas/farmacología , Humanos , Virus del Tumor Mamario del Ratón/fisiología , Ratones , Mifepristona/farmacología , Receptores de Glucocorticoides/fisiología , Transcripción Genética/efectos de los fármacos , TransfecciónRESUMEN
Binding of the synthetic glucocorticoid dexamethasone to the rat liver cytosol glucocorticoid receptor was inhibited by physiological concentrations of nonesterified fatty acids as a function of increasing dose, degree of unsaturation, and chain length of the fatty acid. Polyunsaturated fatty acids were the most potent inhibitors. Scatchard analysis and Line-weaver-Burk plots of the binding data revealed that both the association constants and number of binding sites decreased and that polyunsaturated fatty acids inhibition was of a mixed non-competitive type. The dissociation rate constant of [3H]dexamethasone from glucocorticoid receptors was increased by up to 10 times in the presence of docosahexaenoic acid, whereas a competitive inhibitor like the glucocorticoid antagonist RU 38486 had no effect. Moreover, sucrose density gradient analysis showed that docosahexaenoic acid inhibited the binding of [3H] dexamethasone to both the 8.8S and 4S forms. The results strongly suggest that unsaturated fatty acids are interacting at a site on the receptor different from the hormone binding site and the heat shock protein and that by binding to a second site unsaturated fatty acids greatly change the conformation of the hormone binding site to reduce its affinity for the hormone, either partially or completely depending on the concentration and the class of the fatty acid.
Asunto(s)
Dexametasona/metabolismo , Ácidos Grasos no Esterificados/farmacología , Hígado/fisiología , Receptores de Glucocorticoides/metabolismo , Animales , Citosol/metabolismo , Cinética , Masculino , Peso Molecular , Ratas , Ratas Endogámicas , Receptores de Glucocorticoides/efectos de los fármacos , Receptores de Glucocorticoides/aislamiento & purificación , Relación Estructura-ActividadRESUMEN
Nonesterified fatty acids (NEFAs) have been recently shown in the rat to be involved in steroid hormone expression, having effects on plasma transport and intracellular activity. This study examines the influence of saturated and unsaturated NEFAs on estradiol (E2) binding to cytosol from human uterus, breast, and melanoma. Binding was analyzed after separation with dextran-coated charcoal or hydroxylapatite and by sucrose density gradient centrifugation. Unsaturated NEFAs induced a 2- to 10-fold increase (P less than 0.001) in E2 binding to cytosol from normal, fibromatous, and neoplastic uteri, while saturated NEFAs had a slight inhibitory effect (P less than 0.05). Similar effects were seen with cytosol from metastatic melanoma lymph nodes and neoplastic breast tissues. By contrast, unsaturated NEFAs did not increase E2 binding to serum from these patients. Density gradient centrifugation indicated that the increased binding was associated with the proteins present in the 2- to 4 S region. Analysis of E2 metabolites in the presence of unsaturated NEFAs showed the formation of water-soluble derivatives. Seventy percent of these E2 derivatives were trichloracetic acid precipitable, suggesting a covalent link between the steroid and a protein. The existence of such water-soluble metabolites could be erroneously interpreted as a true binding to soluble cytoplasmic receptors.
Asunto(s)
Estradiol/metabolismo , Ácidos Grasos no Esterificados/farmacología , Receptores de Estradiol/metabolismo , Receptores de Estrógenos/metabolismo , Mama/metabolismo , Neoplasias de la Mama/metabolismo , Centrifugación por Gradiente de Densidad , Citosol/metabolismo , Femenino , Humanos , Ganglios Linfáticos/metabolismo , Melanoma/metabolismo , Receptores de Estradiol/efectos de los fármacos , Neoplasias Uterinas/metabolismo , Útero/metabolismoRESUMEN
1. The present study was designed to investigate, in an in vitro model of the human intestinal barrier, the ability of epithelial cells to produce interleukin-1 (IL-1), the cellular mechanisms involved in IL-1 release, and the intracellular signalling pathways involved in IL-1 up-regulation during inflammatory stress. 2. This study was based on the human colonic epithelial cell line HT29-Cl.16E, maintained as polarized monolayers on filters mounted in culture chambers and treated with various proinflammatory cytokines (interferon gamma (IFN gamma), tumour necrosis factor alpha (TNF alpha), IL-1 beta) alone or in combination. 3. IL-1 production, restricted to IL-1 alpha, was induced by the combination of IFN gamma/TNF alpha. When IL-1 beta was added to IFN gamma/TNF alpha, it led to an additional production of IL-1 alpha. IL-1 alpha release was associated with cell damage, as shown by the correlation between lactate dehydrogenase (LDH) release and extracellular IL-1 production, and was not accounted for by a secretory mechanism. 4. Both IFN gamma/TNF alpha and IFN gamma/TNF alpha/IL-1 beta induced inducible nitric oxide synthase (iNOS) expression as shown by quantitation of NO2-/NO3- by use of the Griess reagent, quantitation of cells scoring positive with an anti-iNOS antibody and detection of mRNAs coding for iNOS by RT-PCR. The use of NG-monomethyl-L-arginine (L-NMMA), an inhibitor of NOS, led to the demonstration of two distinct signalling pathways in IL-1 production by HT29-Cl.16E cells, one dependent on NO (L-NMMA-sensitive) under treatment with IFN gamma/TNF alpha/IL-1 beta, and the other independent of NO (L-NMMA-insensitive) under treatment with IFN gamma/TNF alpha. 5. Moreover, we examined whether a redox-based mechanism could be responsible for the apparent discrepancy between NO production and NO implication in IL-1 production under IFN gamma/TNF alpha and IFN gamma/TNF alpha/IL-1 beta treatments. Experiments with cysteine, which acts as a powerful reductant, suggest that the nitrosonium character of NO is involved in the NO-dependent pathway in IL-1 production.
Asunto(s)
Colon/metabolismo , Interleucina-1/biosíntesis , Óxido Nítrico/biosíntesis , Estrés Fisiológico/metabolismo , Células Cultivadas/metabolismo , Epitelio/metabolismo , Humanos , Óxido Nítrico/fisiologíaRESUMEN
Liposoluble extracts of serum from healthy men and AIDS patients (stages IVC1 and IVD by CDC criteria) inhibited the incorporation of [3H]thymidine into isolated rat thymocytes, but AIDS extracts were less inhibitory, requiring 1.8 times more cortisol in the AIDS extracts than in the healthy extracts to inhibit [3H]thymidine incorporation by 50%. Although the total serum extracts from AIDS patients contained 1.7 times more cortisol than the extracts from healthy controls, the AIDS extracts decreased the binding affinity (Ka) of [3H]dexamethasone to rat thymus glucocorticoid receptors by 50% less than the healthy control extracts. The present study seems to indicate that a substance(s) can be extracted from the serum of AIDS patients that attenuates the inhibitory effect of cortisol on thymocyte proliferation and interferes with the binding of cortisol to the glucocorticoid receptor.
Asunto(s)
Síndrome de Inmunodeficiencia Adquirida/sangre , Dexametasona/farmacología , Timo/efectos de los fármacos , Animales , Células Cultivadas , Citosol/metabolismo , Dexametasona/antagonistas & inhibidores , Dexametasona/metabolismo , Ácidos Grasos no Esterificados/sangre , Humanos , Hidrocortisona/sangre , Masculino , Ratas , Ratas Sprague-Dawley , Receptores de Glucocorticoides/metabolismo , Solubilidad , Timidina , Timo/citologíaRESUMEN
The total liposoluble extract of sera from AIDS patients, IVC1 and IVD stages, containing cortisol and free fatty acids (FFA) inhibited [3H]dexamethasone binding to a lesser extent than did the same quantity of total liposoluble extract of sera from healthy men. FFA isolated from extracts of AIDS sera by Sephadex LH20 chromatography had less effect on [3H]dexamethasone binding to rat liver glucocorticoid receptor than those extracted from sera of healthy men. These results suggest the presence in sera of AIDS patients of a liposoluble substance which could be limiting the inhibitory effect of FFA on [3H]dexamethasone binding to glucocorticoid receptor by inducing a conformational change in glucocorticoid receptor that could alter the biological action of glucocorticoids. The pathological consequence could be the apparent contradiction of high cortisolemia and clinical symptoms of adrenal insufficiency that have been observed in AIDS patients.
Asunto(s)
Síndrome de Inmunodeficiencia Adquirida/sangre , Citosol/metabolismo , Hígado/metabolismo , Receptores de Glucocorticoides/metabolismo , Adulto , Animales , Dexametasona/metabolismo , Ácidos Grasos no Esterificados/metabolismo , Humanos , Hidrocortisona/metabolismo , Metabolismo de los Lípidos , Masculino , Ratas , Ratas Wistar , SolubilidadRESUMEN
We discuss the various experimental findings to date that indicate that AFP may be considered as a positive or negative modulator of estrogenic action. Moreover, we show that this protein, even when nonestrogenophilic, is able to bind other hydrophobic ligands, in particular nonesterified fatty acids. These fatty acids inhibit the binding of estrogens to murine AFP as well as to the cytosolic estrogen receptors. Thus, the AFPs of all species--whether or not estrogenophilic--might play an endocrinologic role through the intermediary of the unsaturated fatty acids to which they associate with high affinity.
Asunto(s)
Estrógenos/metabolismo , Ácidos Grasos Insaturados/metabolismo , alfa-Fetoproteínas/metabolismo , Animales , Estradiol/metabolismo , Estrona/metabolismo , Ácidos Grasos no Esterificados/metabolismo , Femenino , Semivida , Humanos , Intercambio Materno-Fetal , Modelos Biológicos , Ovario/metabolismo , Embarazo , Receptores de Estrógenos/metabolismo , Útero/metabolismoRESUMEN
We report the equilibrium binding parameters for the interactions of the estrogen analogue diethylstilbestrol (DES) with highly purified rat alpha 1-fetoprotein (AFP) and serum albumin preparations. At 25 degrees C and pH 7.4, an association constant (Ka) of about 1.5 X 10(6)M-1 and 2 sites/mole are measured with the DES-AFP system, whereas for the DES-albumin interaction, we find a Ka of approximately 2 X 10(5)M-1 and about 11 sites/mole of protein. The removal of fatty acids from pure AFP causes a reversible 3 fold increase of the number of DES binding sites; the same delipidation procedure applied to albumin slightly diminishes its DES binding parameters. We also demonstrate the capability of DES to displace competitively estradiol-17 beta (E2) from its high affinity sites on the estrophilic rat AFP. Finally, the binding behaviour of the two serum proteins towards the synthetic estrogen is compared to their interaction with the natural hormones. The physiological and pharmacological relevance of these data is discussed.
Asunto(s)
Dietilestilbestrol , Albúmina Sérica , alfa-Fetoproteínas , Animales , Unión Competitiva , Embrión de Mamíferos , Estradiol , Cinética , Ligandos , Unión Proteica , RatasRESUMEN
Stimulating lipase activity with heparin (200 IU/kg b.w.) increased the plasma free fatty acid (FFA) concentration of immature rats (15 days). The effect of this elevated FFA concentration on glucocorticoid binding to corticosteroid binding globulin (CBG), and liver cytosol glucocorticoid receptor (GR), was analyzed. The plasma FFA concentration increased 2-fold, 10 minutes (P < 0.001), 20 minutes (P < 0.01), and 60 minutes (P < 0.01) post-heparin. The corticosterone (B) and progesterone concentrations were unchanged 60 minutes post-injection. The binding activity of immature rat CBG for B dropped 50% (P < 0.001) 60 minutes post-heparin injection, decreased B binding and increased plasma FFA were correlated (r = -0.8). The decreased B binding resulted from a 2-fold decrease in the apparent number of CBG binding sites; the affinity constant (Ka) remained unchanged. The liver cytosol endogenous FFA content of immature rats was also increased 2-fold, 60 minutes after heparin-induced lipolysis. The increased cytosol FFA, with no significant change in glucocorticoid, was accompanied by a significant decrease in dexamethasone binding to liver cytosol glucocorticoid receptor. The decrease resulted from a significantly lower apparent Ka for dexamethasone and fewer receptor binding sites (n). There was a good inverse correlation between Ka (r = -0.93) and n (r = -0.90) and the increased liver cytosol FFA content. Thus the higher plasma FFA induced in vivo by lipase activation or a standard FFA mixture probably causes conformational changes in CBG and GR, reducing glucocorticoid binding to immature rat CBG and liver GR.
Asunto(s)
Ácidos Grasos no Esterificados/farmacología , Glucocorticoides/metabolismo , Hígado/metabolismo , Receptores de Glucocorticoides/metabolismo , Transcortina/metabolismo , Animales , Corticosterona/sangre , Citosol/metabolismo , Dexametasona/metabolismo , Ácidos Grasos no Esterificados/sangre , Ácidos Grasos no Esterificados/metabolismo , Heparina/farmacología , Cinética , Lipasa/sangre , Lípidos/sangre , Lipólisis/efectos de los fármacos , Masculino , Progesterona/sangre , Ratas , Ratas WistarRESUMEN
A highly active inhibitor of the binding of estrone and estradiol-17beta to rat alpha-fetoprotein is demonstrated for the first time in embryo, immature and adult rat sera as well as in fetal and adult human sera. The competitive character and the narrow specificity of this inhibition effect is shown. The major compound responsible for this activity is isolated by successive column Sephadex LH20 and thin layer chromatography: it is characterized as a nonpolar, nonphenolic, dialysable and thermostable substance, unreactive towards anti-estrone and anti-estradiol-17beta antibodies. The possible biological role of an endogenous non-estrogen ligand of rodent fetoproteins is discussed.
Asunto(s)
Fenómenos Fisiológicos Sanguíneos , Embrión de Mamíferos/fisiología , Antagonistas de Estrógenos/sangre , Estrógenos/sangre , alfa-Fetoproteínas/metabolismo , Adrenalectomía , Adulto , Animales , Animales Recién Nacidos/sangre , Especificidad de Anticuerpos , Sitios de Unión , Unión Competitiva , Castración , Cromatografía en Gel , Reacciones Cruzadas , Estradiol/sangre , Estrona/sangre , Femenino , Humanos , Hipofisectomía , Masculino , Unión Proteica , Radioinmunoensayo , RatasRESUMEN
The combination of polyacrylamide gel electrophoresis and Concanavalin-A-Sepharose affinity chromatography has permitted the isolation on a preparative scale, of four molecular forms of rat alpha 1-fetoprotein: a "slow" and a "fast" fraction, each separable into Concanavalin-A-adsorbed ("high carbohydrate", i.e. rich in accessible alphaD-Mannosyl and alphaD-Glu-cosyl residues) and a Concanavalin-A-non adsorbed ("low carbohydrate") fractions. These four iso-alpha 1-fetoproteins (iso-AFP) bind estradiol-17beta. However, they disclose differences in both their association constants and number of binding sites for this hormone. Very high affinity sites (10(9) are mainly located on the "slow-low carbohydrate" form. Low affinity, high capacity sites are preferentially located on the "high carbohydrate" form. These results confirm the molecular and functional heterogeneity of rat AFT and suggest that the carbohydrate moiety of the protein may have a role in estrogen-AFP interactions.
Asunto(s)
Estradiol , alfa-Fetoproteínas , Animales , Sitios de Unión , Cromatografía de Afinidad , Concanavalina A , Electroforesis en Gel de Poliacrilamida , Embrión de Mamíferos , Femenino , Inmunodifusión , Peso Molecular , Embarazo , Unión Proteica , Ratas , alfa-Fetoproteínas/aislamiento & purificaciónRESUMEN
The combination of polyacrylamide gel electrophoresis and Concanavalin-A-Sepharose affinity chromatography has permitted the isolation on a preparative scale, of four molecular forms of rat alpha1-fetoprotein: a "slow" and a "fast" fraction, each separable into Concanavalin-A-adorbed ("high carbohydrate", i.e. rich in accessible alphaD-Mannosyl and alphaD-Glucosyl residues) and a Concanavalin-A-non adsorbed ("low carbohydrate") fractions. These four iso-alpha-fetoproteins (iso-AFP) bind estradiol-17beta. However, they disclose differences in both their association constants and number of binding sites for this hormone. Very high affinity sites (10(9)) are mainly located on the "slow-low carbohydrate" form. Low affinity, high capacity sites are preferentially located on the "high carbohydrate" form. These results confirm the molecular and functional heterogeneity of rat AFP and suggest that the carbohydrate moiety of the protein may have a role in estrogen-AFP interactions.