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1.
The binding of estradiol-17 beta (E2), diethylstilbestrol (DES), and polyene fatty acids, in particular arachidonate (C20:4), to alpha 1-fetoprotein (alpha-FP) and albumin purified from mouse embryo sera was studied using equilibrium dialysis and electrophoretic techniques. E2, arachidonate, and DES all bind to alpha-FP, but with decreasing strength. E2 is a high affinity, low capacity ligand (Ka approximately 0.8 X 10(8) M-1 and approximately 0.3 sites/mol of alpha-FP at 25 degrees C); arachidonate is a weaker ligand disposing of more sites (Ka approximately 0.3 X 10(7) M-1 and 4-5 sites/mol of alpha-FP); the binding of DES is of comparatively low affinity and capacity (Ka approximately 0.2 X 10(7) M-1 and n approximately 0.7/mol of alpha-FP). In spite of different structures and equilibrium parameters, E2, DES, and arachidonate are able to compete with each other for binding to the fetoprotein. The C22:4 and C22:6 fatty acids are also efficient concentration-dependent inhibitors of E2 or DES binding. Albumin binds the fatty acids and DES, but equilibrium parameters are different from those of alpha-FP. In particular, arachidonate is a better ligand for albumin, where it interacts with at least two classes of apparent sites (Ka1 approximately 0.3 X 10(8) M-1 and n1 approximately 1; Ka2 approximately 0.2 X 10(7) M-1 and n2 approximately 30). In contrast to alpha-FP, albumin virtually does not bind E2. Also, no competition could be demonstrated between DES and fatty acid ligands for binding to albumin. None of the studied interactions, with either albumin or alpha-FP, was modified even by high doses of bilirubin. The possible functions of the various binding activities present in fetal sera in the process of growth are discussed.  相似文献   

2.
The interaction of fatty acids with rat alpha-fetoprotein and albumin was measured using a partition equilibrium method. alpha-Fetoprotein (AFP) displays one high-affinity binding site for fatty acids and albumin near two binding sites. The AFP association constants for most fatty acids were similar to those of albumin (in the 10(7) M-1 range) whereas for docosahexaenoic acid it was 9.7 x 10(8) M-1, about 50-fold higher than that corresponding to albumin. This difference justifies docosahexaenoic acid in fetal or neonatal serum being mainly bound to AFP and can indicate a highly specific role of AFP in the transport of this fatty acid.  相似文献   

3.
The optimal conditions of the binding of human lactotransferrin to human alveolar macrophages have been determined and the necessity to measure the binding in absence of bovine serum albumin was demonstrated. In these conditions, diferric lactotransferrin and iron-free lactotransferrin are reversibly bound with the following parameters: association constant Ka = 2 and 5 X 10(6) M-1, respectively, and the number of binding sites N = 1.2 and 1 X 10(7), respectively. The binding of the two forms of lactotransferrins was inhibited by various neoglycoproteins, the highest inhibition being obtained with L-fucosyl, then, in the following decreasing order: D-mannosyl greater than N-acetyl-D-glucosaminyl greater than D-galactosyl. In the same conditions, the binding of serotransferrin (Ka = 2 X 10(7) M-1 and 1.6 X 10(7) M-1; N = 5 X 10(4) and 8 X 10(4) for diferric and iron-free protein, respectively) was not inhibited. These results suggest that the recognition of lactotransferrin is mediated by one or several membrane lectins, the fucose being one of the sugar playing an important role in the association. On the contrary, the binding of serotransferrin does not depend on a membrane lectin system.  相似文献   

4.
We use gel equilibration and electrophoretic techniques to compare the binding properties of thyroxine binding globulin and thyroxine binding prealbumin in rat sera. The evidence indicates that TBG bears the serum lowest capacity highest affinity sites for thyroxine (T4) and triiodothyronine (T3) (Ka1 greater than or equal to 10(9) M-1) as well as weaker saturable T3 sites (Ka2 approximately 10(8) M-1). TBPA bears for T4 only Ka2 approximately 10(8) M-1 sites and for T3 only Ka approximately 10(6) M-1 sites. Consistent with these parameters are the specific responses of TBG and TBPA binding activities to varying serum concentrations of T4, T3, oleic acid, the drugs diphenylhydantoin or salicylate. The primary attack of these compounds is aimed at TBG. Small T4, oleate or DPH doses chase the TBG-bound T4 to TBPA, high doses of T4 or oleate but not of DPH inhibiting the T4 binding to both proteins. In the T3-serum interactions, all tested compounds displace the TBG-bound hormone without chasing it to TBPA. The high reactivity of TBG sites designates the protein as crucially involved in modulating the free vs bound serum levels of T4 and T3 against physiological or pathological variations of binding competitors.  相似文献   

5.
The change of transferrin receptors in regenerating rat liver cells after partial hepatectomy was demonstrated. The binding of 125I-labeled transferrin to the cells began to increase after 24 h of partial hepatectomy and reached about double that of the non-operated normal rat liver cells. A Scatchard analysis of binding parameters showed 1.62 X 10(5) (Ka: 1.04 X 10(7) M-1) in normal cells and 3.36 X 10(5) (Ka: 1.23 X 10(7) M-1) in regenerating cells. This reflected on increase of transferrin receptor number and little change occurred in the binding affinity between transferrin and its surface receptor.  相似文献   

6.
The specific binding of IgG to jejunal brush borders was greatest at acidic pH, at neutral pH no specific binding occurred. Specific binding declined with age-no specific binding occurred in borders from 20-and 24-day-old animals. There was no specific binding of IgG to borders from ileal enterocytes. Human transferrin and bovine serum albumin did not bind specifically to borders. The affinity of binding (-Ka) and the receptors site numbers per border estimated for rat IgG were 18.64 X 10(6) M-1 to 3.53 X 10(6) sites; for human IgG, 25.06 X 10(6) M-1 to 3.30 X 10(6) sites; for bovine IgG, 10.48 X 10(6) M-1 to 2.11 X 10(6) sites and for sheep IgG, 7.26 X 10(6) M-1 to 2.34 X 10(6) sites.  相似文献   

7.
Using the antigens testosterone-17 beta-hemisuccinate and testosterone-3-(o-carboxymethyl) oxime, each coupled to bovine serum albumin, we have produced 44 monoclonal antibodies to testosterone. Of the 17 monoclonal antibodies raised against the 17 beta-linked antigen 8 showed extremely low affinity for testosterone (Ka less than or equal to 8 X 10(7) M-1) and none had an affinity greater than 5 X 10(9) M-1. Of the 27 monoclonal antibodies raised against the 3-linked antigen 2 had affinities less than 8 X 10(7) M, 7 had affinities greater than 5 X 10(9) M-1 and one had an affinity (Ka = 9 X 10(10) M-1) greater than that of a high affinity rabbit antiserum (Ka = 6 X 10(10) M-1). The affinity constant (Ka = 5 X 10(9) M-1) measured in the serum of the mouse whose spleen gave rise to the greatest number of high affinity antibodies, was significantly higher than those measured in the sera of the remaining mice (Ka = 0.7 - 3 X 10(8) M-1). The cross-reactions of the monoclonal antibodies varied widely but none showed an overall improvement in specificity when compared with the corresponding rabbit antisera. Results suggest that as well as the structure of the steroid antigen careful selection of the spleen donor facilitates the development of monoclonal antibodies with good binding characteristics.  相似文献   

8.
S M Ho  P Ofner 《Steroids》1986,47(1):21-34
The goal of the present research was characterization of the interaction of 5 alpha-androstane-3 beta, 17 beta-diol (3 beta-diol) with prostatic estradiol-17 beta(E2) binding sites to address the role of this 5 alpha-dihydrotestosterone(DHT)a metabolite in prostatic regulation. Using dextran-charcoal assay we demonstrated specific 3 beta-diol and E2 binding sites in rat ventral prostate cytosol (RVPC) and dog prostate cytosol (DPC). In both cytosols, E2 binding is of high affinity (Ka congruent to 10(9) M-1; RVPC:68 fmol/mg protein), DPC:170 fmol/mg protein), and 3 beta-diol binding is of moderate affinity (Ka congruent to 10(8) M-1; RVPC:62 fmol/mg protein, DPC:165 fmol/mg protein). Unlabeled 3 beta-diol competes effectively for cytosolic 3H-E2 binding sites, whereas unlabeled DHT, 5 alpha-androstane-3 alpha, 17 beta-diol (3 alpha-diol) and testosterone (T) are poor competitors for 3H-E2 binding sites. Using DNA-cellulose column chromatography, we separated prostatic androgen and estrogen binding activities. The E2 binding activity which adhered to DNA-cellulose was displaced by 100-fold excess 3 beta-diol but not by DHT. Thus data from two assay procedures show competition of 3 beta-diol for 3H-E2 binding sites in rat and dog prostate.  相似文献   

9.
The binding of triton X-100 to bovine serum albumin has been shown to exhibit positive cooperativity. Subsequent equilibrium dialysis studies indicate that the binding of Triton X-100 to sheep serum albumin likewise shows positive cooperativity, the first two stepwise equilibrium constants being K1 = 1.24 X 10(4) M-1 and K2 = 1.62 X 10(4) M-1. However, the mechanism for Triton X-100 binding to human serum albumin differs in that the binding isotherm indicates the binding sites are independent and identical. In the latter case the binding is described by the Scatchard model with an equilibrium constant of K = 7.2 X 10(3) M-1. The studies were conducted at 16 degrees C in pH 7.0, I = 0.05 phosphate buffer.  相似文献   

10.
The binding of Cu(II) to native human, porcine, bovine and ovine ceruloplasmin (Cp) and to bovine serum albumin (bSA) has been studied at pH 7.4, 30 mM barbital buffer. The results were analyzed for the strength and the number of binding sites using Scatchard plots. Evidence for additional copper binding sites in Cp and bSA was obtained suggesting a role for copper ion in the homeostatic regulation of Cu(II) and other metal ions in the serum. In the binding studies the Cp was freed of exogenous Cu(II) by passing it over a Chelex-100 column. Two flow rates were used, 4 ml/hr and 40 ml/hr, which removed Cu(II) of different affinities. Cp passed at the slower flow rate (Cp4) only contained the prosthetic copper atoms. Cp passed at the faster flow rate (Cp40) contained one additional copper atom with a Ka approximately 10(7) M-1. Another 2-6 Cu(II) ion could be added to the Cp40 with an average affinity of about Ka approximately 10(5) M-1. The Cu(II) ions found in Cp provide two distinguishable classes: (1) the prosthetic copper atoms and (2) the exogenous copper atoms that can be removed by Chelex-100. For bSA one copper atom was bound strongly with a Ka value approaching 10(12) - 10(13) M-1 and was not removed by Chelex-100 at any flow rate. A second copper atom was found with a Ka = 5.2 x 10(6) M-1 and was removed by Chelex-100 at 4 ml/hr. Three additional copper atoms were bound with a Ka = 1.6 x 10(5) M-1; they were readily removed by Chelex-100 at 40 ml/hr but were nondialysable.  相似文献   

11.
The interaction of tubulin with chromatin has been studied using a radiolabeled tubulin binding assay and velocity sedimentation analysis on isokinetic sucrose gradients. Soluble chromatin was prepared by mild micrococcal nuclease digestion of rat liver nuclei and tubulin was purified from rat brain by temperature-dependent assembly-disassembly and phosphocellulose chromatography. The tubulin-binding assay is based on the ability of chromatin to precipitate quantitatively at physiological ionic strength allowing separation of free tubulin from chromatin-bound tubulin. The binding of tubulin to unfractionated soluble chromatin was rapid, reversible and saturable. Saturation of binding sites was obtained using tubulin concentrations ranging from 0.5 to 400 micrograms/ml, in the presence of a high concentration (2.5 mg/ml) of another acidic protein, bovine serum albumin. The Scatchard and Hill plots showed that tubulin bound to a single class of non-interacting sites and yielded values of (0.5-0.6) X 10(7) M-1 for an apparent Ka and a maximal binding capacity of 0.8 nmol tubulin/mg DNA, i.e. about 1 molecule of tubulin/10 nucleosomes. Similar binding parameters were obtained when binding experiments were performed with insoluble chromatin in 0.15 M NaCl. Velocity sedimentation analysis of tubulin-chromatin complexes revealed that tubulin bound to all classes of chromatin oligomers, irrespective of the length of the nucleosomal chain. Tubulin-trinucleosome complexes formed from isolated trinucleosome in the presence of an excess of tubulin were separated from free reactants. It was found that 10-15% of the starting oligonucleosomal species reacted with tubulin, in a stoichiometry of about 0.8 molecule of tubulin/nucleosome. Given the characteristics of the binding and the expected cellular free tubulin concentration, the tubulin-chromatin interaction could possibly take place in vivo, when the nuclear membrane breaks down during the first steps of mitosis.  相似文献   

12.
Diethylstilbestrol (DES) and certain chemically structural derivatives and analogs, indenestrol A (IA), indenestrol B (IB), indanestrol (IN), and pseudo-DES (PD), have been used as probes to examine various estrogenic responses previously considered interrelated and obligatory to the stimulation of uterine growth. All the analogs had poor uterotropic activity in vivo which ranged from 10-200 times less than that of estradiol or DES. The poor uterotropic activity was not due to poor binding affinity for the receptor. All compounds except IN interacted with the mouse uterine estrogen receptor with high affinity (approximately Ka 1.5-2.2 X 10(10) M-1). In addition, the compounds were able to translocate similar levels of receptor to the nucleus in vivo. Nuclear retention and occupancy of the estrogen receptor by the compounds was comparable to the patterns produced by DES or estradiol. The activity of uterine tissue responses was investigated during treatment with the compounds. Only IA stimulated uterine glucose-6-phosphate dehydrogenase to significant levels similar to DES or estradiol. Uterine progesterone receptor was induced to varying degrees by all compounds; the indenestrol isomers (IA and IB) were the most active. Uterine DNA synthesis was marginally stimulated by the derivatives and analogs except for IB which showed a response increase comparable to DES or estradiol. Because of the differential stimulation, these data suggest that in uterine tissue estrogen receptor stimulates certain biochemical responses independently and not in concert. The ability of a particular response to be increased may depend on the chemical nature of the ligand receptor complex and its interaction at genomic sites.  相似文献   

13.
Two rabbits (TG-1, TG-2) were immunized with human thyroglobulin (HTg) and bled serially. Antisera were obtained at different times after the first immunization and kept separately and studied. In both rabbits production of anti-HTg, and anti-thyroid hormone antibodies such as anti-thyroxine (T4) and anti-triiodothyronine (T3) antibodies was observed. Binding parameters of anti-HTg antibodies with HTg, T4, and T3 were calculated in two selected antisera (70-day and 249-day). The Scatchard's plots of these antibodies were all curve-linear and were analyzed in two components: one, higher binding constant (Ka1) and smaller binding capacity (Cap1) and the other, lower binding constant (Ka2) and larger binding capacity (Cap2). Ka1 values of anti-HTg, anti-T4, and anti-T3 antibodies in sera from TG-1 obtained from 70-day and 249-day bleeding were 1.1 X 10(10) M-1, 6.0 X 10(9) M-1. 7.9 X 10(8) M-1 and 1.7 X 10(10) M-1, 6.5 X 10(9) M-1, 1.0 X 10(9) M-1, respectively. Those from TG-2 were 1.7 X 10(10) M-1, 1.8 X 10(9) M-1, 6.4 X 10(8) M-1 and 2.0 X 10(10) M-1, 3.1 X 10(9) M-1, 1.6 X 10(9) M-1, respectively. The significance of the production of anti-HTg and anti-thyroid hormone antibodies in rabbits immunized with HTg in relation to the antigenic structure of HTg molecule was discussed.  相似文献   

14.
The transport of small molecules in the blood, normally assured by serum albumin in the adult, is not well known in the fetus since the albumin concentration is low in fetal serum and inversely related to the alpha 1-fetoprotein concentration. In order to investigate whether rat alpha 1-fetoprotein might be a fetal counterpart to albumin, the binding properties of these two proteins have been compared with respect to a series of molecules of biological importance, especially during fetal development: thyroid hormones and indole analogues. Though high-affinity binding of thyroxine was found with both rat alpha 1-fetoprotein and albumin, a significant difference in the number of binding sites for this hormone was found with the two proteins. Further, while rat serum albumin strongly bound L-tryptophan and indolyl-3-acetic acid (Ka approximately equal to 10(5) M-1), rat alpha 1-fetoprotein did not bind any of the indoles tested. These results are discussed with respect to the physiological and pharmacological significance of the transport role of these proteins.  相似文献   

15.
The interaction of bovine serum albumin with dihydrotestosterone bearing a spin label at C-3 was studied using electron spin resonance (ESR) spectroscopy. Quantitative binding parameters (Ka approximately 10(5) M-1; maximum binding capacity; two sites/mol albumin) obtained by ESR were in good agreement with those given by equilibrium dialysis. ESR study at various temperatures allowed the calculation of the thermodynamic parameters of the steroid-protein interaction: deltaG=-6.8 kcal/mol; deltaH=-7.9 kcal/mol; deltaS=-3.2 cal/mol per degree and confirmed a transition temperature of about 65 degrees C for albumin. Na, Liland Ca salts had a generally favorable effect on the interaction whereas other ions (e.g. Hg, Cu) impaired the binding process. Study of the width of the ESR spectra of the protein-bound spin-labelled steroid and extrapolation of a 2 T value to infinite viscosity (Azz coupling constant) indicated a non-polar binding site, which became increasingly hydrophobic as the temperature was raised. Since this methodology can give both pertinent quantitative and qualitative data, ESR spectroscopy should be of value in the study of steroid-protein interactions of biological significance.  相似文献   

16.
Squash seeds proteinase inhibitors form stoichiometric complexes with bovine trypsinogen. In terms of association constants (Ka), the interaction is weak. The inhibitors bind to the zymogen with Ka values of approx. 10(4)M-1 i.e. 2 X 10(7) times weaker than to bovine beta-trypsin. Squash inhibitor with Lys at the P1 position binds to trypsinogen with a Ka value 2.1-fold higher than the inhibitor with Arg at P1. The Ile-Val binding cleft and the Ca2+ binding site of trypsinogen are cooperatively linked to the inhibitor binding site. Although these three sites are spatially separated, either binding of calcium ion or Ile-Val dipeptide to trypsinogen increase the Ka values 3-fold and more than 100-fold, respectively. In the presence of Ile-Val trypsinogen resynthetizes extremely slowly (about 10(4) times slower than beta-trypsin) the reactive site peptide bond in squash inhibitors.  相似文献   

17.
Antisera were prepared against two types of estradiol-3-sulfate-bovine serum albumin (BSA) conjugates. The haptens were coupled to BSA through the C-6 position in the steroid molecule by the glutaraldehyde (A) or the carbodiimide method (B). In comparison the antiserum produced by method A had a high affinity for estradiol-3-sulfate (Ka = 5.64 X 10(8) M-1); that produced by method B had an even higher affinity (Ka = 2.62 X 10(9) M-1). Furthermore the latter had no significant cross-reaction with other estrogen sulfates (less than 3.83%), and no cross-reaction with other steroids (less than 0.03%). The former revealed a little cross-reactivity with some of related steroids.  相似文献   

18.
The binding of estrone-3-sulfate (E1-3-S) and estradiol-3-sulfate (E2-3-S) to adult stallion plasma was determined and compared with the binding to equine serum albumin (ESA). On the ESA molecule, two binding sites for E1-3-S with an association constant of 1.3 x 10(5) M-1 and several sites of weaker affinity were found; the data for E2-3-S showed the existence of four binding sites of moderate affinity (1 x 10(5) M-1) and several sites of weaker affinity. The removal of albumin from the stallion plasma resulted in the absence of binding of E1-3-S or E2-3-S, whereas the removal of glycoproteins resulted in binding parameters similar to those obtained with whole plasma. These results indicate that ESA is the only estrogen sulfate binder in horse plasma. Under physiological conditions, 95% of E1-3-S was bound to ESA.  相似文献   

19.
A comparative study of the hormonal specificity of the affinity, the equilibrium association constant (Ka) and the kinetics of [3H]-estradiol (3H-E2) interaction with high molecular weight specifically binding E2 proteins from liver cytosol of male and female rats and with uterine estrogen receptors was carried out. The hormonal specificity of the affinity for the E2-binding proteins from the three sources was found to be similar, i.e. only the compounds possessing the estrogen activity competed with 3H-E2 for the binding sites. The values of the apparent equilibrium constants (Ka) for the proteins from male rat liver and female rat liver and uterus were equal to (6,6 +/- 1,2) . 10(9) M-1, (7,4 +/- 0,9) . 10(9) M-1 and (11,2 +/- 2,3) . 10(9) M-1, respectively. The dissociation kinetics of the 3H-E2--protein complexes from the three tissues at 0--4 degrees were two-phase: during the first 8--12 hours the dissociation processes were characterized by the dissociation rate constants (k-1) equal to (4--5) . 10(-5) S-1; then the k-1 values were decreased approximately by one order of magnitude. The kinetics of 3H-E2 association with the three types of proteins are presumably two-phase as well. During the first 10--15 min the association process can be characterized by association rate constants equal to (8--27). .10(5 M-1 S-1; then these values decreased about 4-fold. The data obtained suggest that the high molecular weight estrogen--binding proteins from different tissues are similar in their E2-binding properties on the one hand, and may be interpreted as evidence for the heterogeneity of the populations of E2-binding proteins in various tissues, on the other.  相似文献   

20.
Human alpha-fetoprotein (AFP) is able to bind arachidonic acid with high affinity Ka = 10(7)M-1 and a limited number of binding sites NS = 3. Thirty different fatty acids were tested by competition using labelled arachidonic acid as tracer. This experiment demonstrated a great specificity for the fatty acid-AFP interaction. Only polyunsaturated long chain fatty acids were able to bind to AFP with high affinity. The metabolic products of arachidonic acid i.e. prostaglandins presented no affinity for the AFP molecule.  相似文献   

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