CORTICOSTERONE BINDING BY MOUSE SERA DURING FOETAL AND POST-NATAL DEVELOPMENT

1977 ◽  
Vol 84 (1) ◽  
pp. 177-190 ◽  
Author(s):  
Lia Savu ◽  
Emmanuel Nunez ◽  
Max-Fernand Jayle
Keyword(s):  
Amniotic Fluid ◽  
Binding Sites ◽  
Binding Proteins ◽  
Serum Proteins ◽  
Equilibrium Dialysis ◽  
Normal Adult ◽  
Scatchard Analysis ◽  
Mammalian Development ◽  
Single Class ◽  
Mean Values

ABSTRACT The binding properties of corticosterone binding globulin (CBG) of mouse sera have been studied by equilibrium dialysis and electrophoretic techniques, at different stages of foetal and post-natal development. Scatchard analysis has demonstrated in all cases a single class of high affinity saturable binding sites for corticosterone. Remarkable increases of the binding capacities were observed in the foetal and pregnant sera, as compared to normal adult and immature levels. The mean values of n1M1 × g−1 of serum proteins (concentration of binding sites, n1 × moles of binding proteins M1) were 21 10−8 in 14–19 day pregnant females, 17 10−8 in the amniotic fluid, 4.2 10−8 in 14–19 day embryos, and only 0.8 10−8 in the normal adult female. Neonatal mice, aged 0–6 days exhibited no CBG activities. The association constants showed values of 2.5–4.1 108 m−1 when measured with foetal sera, and of 1.2–2.1 108 m−1 with pregnant or control adult sera and with the amniotic fluid, at 25°C. Comparative electrophoretic, thermal denaturation and competition studies with foetal and pregnant plasma CBG's are also reported. The results are discussed in relation to the origin of CBG in the foetal serum, and also with respect to similar studies in the rat, guinea pig and man. The possible biological implications of serum steroid binding proteins in mammalian development are briefly outlined.

Insulin-like growth factor receptors in normal and tumorous adult human adrenocortical glands

1997 ◽  
Vol 136 (3) ◽  
pp. 296-303 ◽  
Author(s):  
Matthias M Weber ◽  
Christoph J Auernhammer ◽  
Wieland Kiess ◽  
Dieter Engelhardt
Keyword(s):  
Binding Sites ◽  
Normal Adult ◽  
Scatchard Analysis ◽  
Single Class ◽  
Α Subunit ◽  
High Affinity ◽  
Adult Human ◽  
Igf I ◽  
Receptor Concentration ◽  
Adrenocortical Carcinomas

Abstract We have identified and characterized insulin-like growth factor (IGF)-I and IGF-II/mannose-6phosphate (IGF-II/M6P) receptors in normal adult human adrenocortical tissue. Furthermore, we investigated the IGF-I receptor concentration and binding characteristics in benign and carcinomatous adrenocortical tumors. Membrane preparations of 14 normal adrenocortical glands showed a mean specific 125I-IGF-I binding (SB) of 5·0±0·5% and a competition by unlabeled ligands which is characteristic of the IGF-I receptor. The Scatchard analysis revealed a single class of high affinity binding sites with a dissociation constant (Kd) of 0·16±0·03 nmol/l, and a receptor concentration (RC) of 19·2±2·5 nmol/kg protein. Affinity cross-linking experiments with normal and tumorous adrenocortical tissue displayed a band at an apparent molecular mass of 135 kDa, corresponding to the size of the normal α-subunit of the IGF-I receptor. In agreement, 125I-IGF-II binding to normal adult human adrenocortical membranes was characteristic for the IGF-II/M6P receptor, and the Scatchard analysis revealed the presence of a single class of high affinity binding sites (SB 7·5±0·5, RC 1137±265 nmol/kg protein, Kd 2·20±0·46 nmol/l, n=6). The identity of the IGF-II/M6P receptor in adrenocortical tissue was further confirmed by Western blotting showing a specific band at 220 kDa. When 125I-IGF-I binding in adrenocortical hyperplasias (SB 4·1±0·4% RC 19·6± 2·0 nmol/kg protein, Kd 0·19±0·04 nmol/l, n=4) and adenomas (SB 4·0±1·1% RC 17·5± 3·1 nmol/kg protein, Kd 0·21±0·nmol/l,04 n=4) was compared with the 125I-IGF-I binding in normal adrenocortical tissue, similar IGF-I receptor concentration and binding kinetics were found. In contrast, three out of four hormonally active adrenocortical carcinomas showed a strongly elevated specific 125I-IGF-I binding with a 3- to 4-fold increase in IGF-I receptor concentration, as compared with normal adrenocortical tissue. This resulted in a significantly higher mean specific binding and receptor concentration in adrenocortical carcinomas, while the binding kinetics and the size of the α-subunit of the IGF-I receptor remained unaltered (n =4, SB 13·8±4·2%, RC 72·2 ± 21·3 nmol/kg protein, Kd 0·17±0·02 nmol/l). In summary, we show that intact IGF-I and IGF-II receptors are present in normal adult human adrenocortical tissue. While the abundance of the IGF-I receptor in adrenocortical hyperplasias and adenomas was similar to normal tissue, a strong overexpression of the intact IGF-I receptor was found in three out of four adrenocortical carcinomas. European Journal of Endocrinology 136 296–303

Characterization of Thromboxane A2/Prostaglandin H2 Receptors in Porcine Coronary Artery -The Inhibitory Effect of a Novel Dibenzoxepin Derivative, KW-3635

1992 ◽  
Vol 67 (05) ◽  
pp. 582-584 ◽  
Author(s):  
Ichiro Miki ◽  
Akio Ishii
Keyword(s):  
Coronary Artery ◽  
Binding Sites ◽  
Thromboxane A2 ◽  
Inhibitory Effect ◽  
Scatchard Analysis ◽  
Single Class ◽  
Porcine Coronary Artery ◽  
Equilibrium Binding ◽  
H2 Receptors

SummaryWe characterized the thromboxane A2/prostaglandin H2 receptors in porcine coronary artery. The binding of [3H]SQ 29,548, a thromboxane A2 antagonist, to coronary arterial membranes was saturable and displaceable. Scatchard analysis of equilibrium binding showed a single class of high affinity binding sites with a dissociation constant of 18.5 ±1.0 nM and the maximum binding of 80.7 ± 5.2 fmol/mg protein. [3H]SQ 29,548 binding was concentration-dependently inhibited by thromboxane A2 antagonists such as SQ 29,548, BM13505 and BM13177 or the thromboxane A2 agonists such as U46619 and U44069. KW-3635, a novel dibenzoxepin derivative, concentration-dependently inhibited the [3H]SQ 29,548 binding to thromboxane A2/prosta-glandin H2 receptors in coronary artery with an inhibition constant of 6.0 ± 0.69 nM (mean ± S.E.M.).

scholarly journals Identification and analysis of human erythropoietin receptors on a factor-dependent cell line, TF-1

Blood ◽  
1989 ◽  
Vol 73 (2) ◽  
pp. 375-380 ◽  
Author(s):  
T Kitamura ◽  
A Tojo ◽  
T Kuwaki ◽  
S Chiba ◽  
K Miyazono ◽  
...  
Keyword(s):  
Cell Line ◽  
Binding Sites ◽  
Bone Marrow Cells ◽  
Scatchard Analysis ◽  
Hematopoietic Growth Factors ◽  
Single Class ◽  
Gm Csf ◽  
Molecular Weights ◽  
Human Erythropoietin ◽  
Macrophage Colony

Abstract We have recently established a novel cell line, TF-1, from bone marrow cells of a patient with erythroleukemia, that showed an absolute growth dependency on each of three hematopoietic growth factors: erythropoietin (EPO) granulocyte-macrophage colony-stimulating factor (GM-CSF), and interleukin 3 (IL-3). EPO stimulated the proliferation of TF-1 cells even at the physiologic concentration (0.03 U/mL). We performed binding experiments on TF-1 cells using radioiodinated EPO. The binding of radioiodinated EPO to TF-1 was specific, time- and temperature-dependent, and saturable. Scatchard analysis of the saturation binding data suggested the existence of a single class of binding sites (kd = 0.40 nmol/L; number of binding sites = 1,630 per cell). TF-1 cells were usually maintained in RPMI 1640 containing 10% fetal bovine serum and 5 ng/mL GM-CSF. The kd and the number of the EPO receptors were not changed by incubating the cells with IL-3, although culturing the cells in the presence of EPO resulted in down-modulation of EPO receptors. The chemical cross-linking study demonstrated that two molecules with apparent molecular weights of 105 kilodalton (Kd) and 90 Kd were the binding components of EPO. Present data suggest that human EPO receptors are very similar to the previously reported murine EPO receptors.

Binding of cadmium(II) and zinc(II) to human and dog serum albumins. An equilibrium dialysis and 113Cd-NMR study

1991 ◽  
Vol 69 (12) ◽  
pp. 809-820 ◽  
Author(s):  
William Goumakos ◽  
Jean-Pierre Laussac ◽  
Bibudhendra Sarkar
Keyword(s):  
Serum Albumin ◽  
Human Serum ◽  
Binding Sites ◽  
Metal Ion ◽  
Equilibrium Dialysis ◽  
Scatchard Analysis ◽  
Serum Albumins ◽  
High Affinity ◽  
Nmr Study ◽  
Nmr Techniques

The binding of Cd(II) and Zn(II) to human serum albumin (HSA) and dog serum albumin (DSA) has been studied by equilibrium dialysis and 113Cd(II)-NMR techniques at physiological pH. Scatchard analysis of the equilibrium dialysis data indicate the presence of at least two classes of binding sites for Cd(II) and Zn(II). On analysis of the high-affinity class of sites, HSA is shown to bind 2.08 ± 0.09 (log K = 5.3 ± 0.6) and 1.07 ± 0.12 (log K = 6.4 ± 0.8) moles of Cd(II) and Zn(II) per mole of protein, respectively. DSA bound 2.02 ± 0.19 (log K = 5.1 ± 0.8), and 1.06 ± 0.15 (log K = 6.0 ± 0.2) moles of Cd(II) and Zn(II) per mole of protein, respectively. Competition studies indicate the presence of one high-affinity Cd(II) site on both HSA and DSA that is not affected by Zn(II) or Cu(II), and one high-affinity Zn(II) site on both HSA and DSA that is not affected by Cd(II) or Cu(II). 113Cadmium-HSA spectra display three resonances corresponding to three different sites of complexation. In site I, Cd(II) is most probably coordinated to two or three histidyl residues, site II to one histidyl residue and three oxygen ligands (carboxylate), while for the most upfield site III, four oxygens are likely to be involved in the binding of the metal ion. The 113Cd(II)-DSA spectra display only two resonances corresponding to two different sites of complexation. The environment around Cd(II) at sites I and II on DSA is similar to sites I and II, respectively, on HSA. No additional resonances are observed in any of these experiments and in particular in the low field region where sulfur coordination occurs. Overall, our results are consistent with the proposal that the physiologically important high-affinity Zn(II) and Cd(II) binding sites of albumins are located not at the Cu(II)-specific NH2-terminal site, but at internal sites, involving mostly nitrogen and oxygen ligands and no sulphur ligand.Key words: albumin, human serum, dog serum, cadmium, zinc, copper, NMR, equilibrium dialysis, binding.

Involvement of estrogen receptors α and β in the regulation of cervical permeability

2000 ◽  
Vol 278 (4) ◽  
pp. C689-C696 ◽  
Author(s):  
George I. Gorodeski ◽  
Dipika Pal
Keyword(s):  
Estrogen Receptor ◽  
Binding Sites ◽  
Estrogen Receptor Α ◽  
Binding Activity ◽  
Scatchard Analysis ◽  
Single Class ◽  
Estrogen Receptor Modulators ◽  
Transcription Inhibitor ◽  
Estrogen Binding ◽  
In Cells

Estrogen increases the permeability of cultured human cervical epithelia (Gorodeski, GI. Am J Physiol Cell Physiol 275: C888–C899, 1998), and the effect is blocked by the estrogen receptor modulators ICI-182780 and tamoxifen. The objective of the study was to determine involvement of estrogen receptor(s) in mediating the effects on permeability. In cultured human cervical epithelial cells estradiol binds to high-affinity, low-capacity sites, in a specific and saturable manner. Scatchard analysis revealed a single class of binding sites with a dissociation constant of 1.3 nM and binding activity of ∼0.5 pmol/mg DNA. Estradiol increased the density of estrogen-binding sites in a time- and dose-related manner (half time ≈ 4 h, and EC50≈ 1 nM). RT-PCR assays revealed the expression of mRNA for the estrogen receptor α (αER) and estrogen receptor β (βER). Removal of estrogen from the culture medium decreased and treatment with estrogen increased the expression of αER and βER mRNA. In cells not treated with estrogen, ICI-182780 and tamoxifen increased βER mRNA. In cells treated with estrogen, neither ICI-182780 nor tamoxifen had modulated significantly the increase in αER or βER mRNA. The transcription inhibitor actinomycin D blocked the estrogen-induced increase in permeability, and it abrogated the estradiol-induced increase in estrogen binding sites. These results suggest that the estrogen-dependent increase in cervical permeability is mediated by an αER-dependent increase in transcription.

scholarly journals Identification of serum GH-binding proteins in the goldfish (Carassius auratus) and comparison with mammalian GH-binding proteins

1999 ◽  
Vol 161 (2) ◽  
pp. 255-262 ◽  
Author(s):  
Y Zhang ◽  
TA Marchant
Keyword(s):  
Binding Sites ◽  
Carassius Auratus ◽  
Binding Proteins ◽  
Binding Capacity ◽  
Sea Bream ◽  
Single Class ◽  
Binding Characteristics ◽  
High Affinity ◽  
Goldfish Carassius Auratus ◽  
Reducing Conditions

The present study constitutes the characterization of a specific, high-affinity GH-binding protein (GHBP) in the serum of a teleost, the goldfish (Carassius auratus). GH-binding assay and ligand blotting techniques were employed to identify GHBPs in goldfish serum and hepatocyte culture medium. The binding characteristics and apparent molecular weights (Mr) of goldfish GHBPs were also compared with those of rabbit and rat. LIGAND analysis identified a single class of high-affinity and low-capacity binding sites for iodinated recombinant carp GH (rcGH) in the goldfish serum, with an association constant (Ka) of 20.1x10(9) M-1 and a maximum binding capacity (Bmax) of 161 fmol ml-1 serum. A single class of binding sites for iodinated recombinant sea bream GH and bovine GH (bGH) was also found in goldfish serum, but with a much lower affinity than that of rcGH. The binding affinity for iodinated bGH in rabbit and rat sera was found to be similar to that reported previously. Ligand blotting revealed multiple forms of GHBPs in sera of goldfish, rabbit and rat with Mr ranging from 70 kDa to 400 kDa and 27 kDa to 240 kDa under non-reducing and reducing conditions respectively. A prominent band with Mr of 66 kDa and a minor band with Mr of 27 kDa were observed to occur in sera from all three species under reducing conditions. Iodoacetamide promoted the shedding of three GHBPs with Mr of 25, 40 and 45 kDa from the cultured goldfish hepatocytes. The appearance of all bands was completely inhibited by the presence of excess unlabeled rcGH. Our results provide clear evidence that a GHBP exists in the goldfish and indicate that more information on teleost GHBPs is needed if the physiology of growth in teleosts is to be fully understood.

scholarly journals Fluorescence-polarization studies on binding of 4-methylumbelliferyl β-d-galactopyranoside to Ricinus communis (castor-bean) agglutinin

1980 ◽  
Vol 191 (2) ◽  
pp. 395-400 ◽  
Author(s):  
M I Khan ◽  
M K Mathew ◽  
P Balaram ◽  
A Surolia
Keyword(s):  
Binding Sites ◽  
Fluorescence Polarization ◽  
Castor Bean ◽  
Ricinus Communis ◽  
Equilibrium Dialysis ◽  
Scatchard Analysis ◽  
Polarization Data ◽  
Appreciable Change ◽  
Number Of Binding Sites ◽  
Polarization Studies

The binding of Ricinus communis (castor-bean) agglutinin 1 to saccharides was studied by equilibrium dialysis and fluorescence polarization by using the fluorescently labelled sugar 4-methylumbelliferyl beta-D-galactopyranoside. No appreciable change in ligand fluorescence of 4-methylumbelliferyl beta-D-galactopyranoside was considerably polarized on its binding to the lectin. The association constants obtained by Scatchard analysis of equilibrium-dialysis and fluorescence-polarization data do not differ much from each other, and at 25 degrees C, Ka = 2.4 (+/- 0.2) X 10(4)M-1. These values agree reasonably well with that reported in the literature for Ricinus agglutinin 1. The number of binding sites obtained by the different experimental procedures is 1.94 +/- 0.1 per molecule of 120 000 daltons and is equal to the reported value of 2. The consistency in the values of Ka and number of binding sites indicate the absence of additional subsites on Ricinus agglutinin 1 for its specific sugars. In addition, the excellent agreement between the binding parameters obtained by equilibrium dialysis and fluorescence polarization indicate the potential of ligand-fluorescence-polarization measurements in the investigation of lectin-sugar interactions.

Characteristics and developmental changes of corticotrophin-releasing hormone-binding sites in the fetal sheep anterior pituitary gland

1991 ◽  
Vol 130 (2) ◽  
pp. 223-229 ◽  
Author(s):  
F. Lü ◽  
K. Yang ◽  
J. R. G. Challis
Keyword(s):  
Binding Sites ◽  
Anterior Pituitary ◽  
Time Course ◽  
Scatchard Analysis ◽  
Maximum Output ◽  
Similar Time ◽  
Single Class ◽  
Fetal Sheep ◽  
Releasing Hormone ◽  
Receptor Number

ABSTRACT The responses of the fetal sheep pituitary to corticotrophin-releasing hormone (CRH) change during gestation with maximum output of ACTH around days 120–130, and decreased ACTH output near term. However, there is no information available concerning the extent to which these responses may be modulated by alterations in the number of CRH receptors. Therefore we measured specific CRH-binding sites, and changes in binding characteristics in membrane preparations from fetal sheep anterior pituitaries collected at days 65–70, 85–88, 100–110, 125–130 and at term (approximately 145 days). Binding assays were carried out using 125I-labelled Tyr-ovine CRH (125I-Tyr-oCRH), incubated with crude membrane fractions for 90 min at 22 °C. Binding was time- and temperature-dependent, linear with protein concentration, saturable and specific for oCRH. Scatchard analysis of binding data for individual tissues revealed a single class of CRH-binding sites with high affinity (Kd ≃1 nmol/l) that did not change significantly with gestational age. However, the number of CRH-binding sites increased progressively from days 65–70 to a maximum at days 125–130, then decreased at term. These results demonstrate the presence of specific CRH-binding sites in the fetal sheep anterior pituitary. Furthermore, the change in CRH receptor number with advancing pregnancy follows a similar time-course to the changes reported previously in responsiveness of the fetal sheep anterior pituitary to exogenous CRH stimulation in vivo. These results suggest that alterations in CRH receptor number may contribute to changes in responsiveness of the fetal sheep anterior pituitary to CRH during gestation. Journal of Endocrinology (1991) 130, 223–229

BINDING OF CALCIUM TO HUMAN AND BOVINE FACTOR X

1987 ◽  
Author(s):  
D M Monroe ◽  
D W Deerfield ◽  
D L Olson ◽  
T N Stewart ◽  
H R Roberts ◽  
...  
Keyword(s):  
Binding Site ◽  
Binding Sites ◽  
Calcium Binding ◽  
Human Factor ◽  
Equilibrium Dialysis ◽  
Equilibrium Constants ◽  
Scatchard Analysis ◽  
Factor X ◽  
Amino Terminal ◽  
Bovine Factor

Human and bovine factor X contain 11 and 12 glutamyl residues respectively within the first forty amino terminal residues that are posttranslationally modified to y-carboxyglutamyl (Gla) residues. Calcium binding to these Gla residues and at other sites is critical for activity in factor X. We have measured calcium binding to human factor X by equilibrium dialysis for the first time. We have also re-examined calcium binding to bovine factor X in order to compare the two species. Factor X (10 μM) was incubated with 45Ca in 20 mM Tris (pH 7.5), 100 mM NaCl in a half cell separated by a 12-14000 molecular weight fast-equilib-rium disk membrane at 25°C for 24 hours. Four aliquots (100 μL each) were removed from each side of the cell and counted. Data were analyzed with a variety of models that allow for more than one class of binding site and for cooperativity among binding sites. Calcium binding to bovine factor X was best simulated by a model that assumes 1 very tight site, 3 cooperative tight sites, and 18 equivalent, non-interacting sites. Based on data from des(Gla)factor X, the first site is probably a high affinity non-Gla binding site. Our results differ from two previously published reports that indicated either 1 tight and 39 loose noncooperative sites (R.H. Yue & M.M. Gertler (1978) Thrombos. Haemostas. (Stuttg.) 40, 350) or 20 calcium binding sites with the first 4 being cooperative (M.J. Lindhout & H.C. Hemker (1978) Biochimica Biophysica Acta 533, 318). Our data on human factor X fit the same model as used for bovine factor X; however, coop-erativity is less in the 3 cooperative sites. Shown below are the first six thermodynamic equilibrium constants derived from a Scatchard analysis of binding data (values are M−1).Both proteins demonstrate the same total number of binding sites and essentially the same value for the first, tight binding site. Bovine factor X exhibits cooperativity, whereas human factor X has reduced cooperativity.

scholarly journals A Direct Free Thyroxine (T4) Immunoassay with the Characteristics of a Total T4 Immunoassay

2007 ◽  
Vol 53 (5) ◽  
pp. 911-915 ◽  
Author(s):  
Kristofer S Fritz ◽  
R Bruce Wilcox ◽  
Jerald C Nelson
Keyword(s):  
Serum Protein ◽  
Binding Proteins ◽  
Serum Proteins ◽  
Equilibrium Dialysis ◽  
Free Thyroxine ◽  
Free T4 ◽  
Serum T4 ◽  
Presence And Absence

Abstract Background: Direct free thyroxine (T4) measurements have been linked to both T4-binding serum protein concentrations and protein-bound T4 concentrations. Whether this is evidence of a relationship to total T4 concentrations has not been reported. Methods: We compared an analog-based direct free T4 immunoassay and a total T4 immunoassay. Each assay was applied to the fractions of serum T4 obtained by ultrafiltration and equilibrium dialysis. Both were applied to serum-based solutions in which free T4, T4-binding proteins, protein-bound T4, and total T4 were systematically varied, held constant, or excluded. Results: Neither the free T4 assay nor the total T4 assay detected dialyzable or ultrafilterable serum T4. Both assays detected and reported the T4 retained with serum proteins. Both free and total T4 results were related to the same total T4 concentrations in the presence and absence of T4-binding proteins. Both results were similarly related to total T4 concentrations when free T4 was held constant while total T4 was varied. Both were similarly related to a total T4 concentration that was held constant while free T4 progressively replaced protein-bound T4. These free T4 results, like total T4 results, were unresponsive to a 500-fold variation in dialyzable T4 concentrations. Conclusion: New experiments extend the characterization of a longstanding and incompletely characterized analog-based free T4 immunoassay. These free T4 measurements relate to total T4 concentrations in the same way that total T4 measurements do.

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