Comparison of the specific binding of cortisol in human amnion, amniotic fluid, and plasma

1987 ◽  
Vol 65 (1) ◽  
pp. 54-59 ◽  
Author(s):  
L. Riopel ◽  
W. Gibb
Keyword(s):  
Amniotic Fluid ◽  
Concanavalin A ◽  
Specific Binding ◽  
Binding Protein ◽  
Maternal Plasma ◽  
Binding Activity ◽  
Human Amnion ◽  
Corticosteroid Binding Globulin ◽  
Influence Of Ph ◽  
Contraceptive Treatment

The purpose of this study was to compare the specific cortisol-binding protein found associated with human amnion with specific cortisol binding in human amniotic fluid and plasma. The electrophoretic mobility on polyacrylamide gels of the specific cortisol binding in amnion, amniotic fluid, and maternal plasma was identical. The influence of pH on cortisol binding activity was similar in all tissues and the cortisol binding was immunoprecipitable by a polyclonal antibody raised against human corticosteroid-binding globulin. The interaction of the cortisol binding protein with concanavalin A was studied in preterm amniotic fluid, term amniotic fluid, term amnion, and plasma from pregnant women at term and women under oral contraceptive treatment. Binding to concanavalin A was similar in term amnion and term amniotic fluid but was less than that found with both preterm amniotic fluid and term plasma. These results indicate that the cortisol binding portein associated with human amnion has similar characteristics to plasma corticosteroid-binding globulin, but that its state of glycosylation appears to be more like that of the cortisol binding protein in term amniotic fluid rather than in plasma.

scholarly journals Characteristics of solubilized human-somatotropin-binding protein from the liver of pregnant rabbits

1980 ◽  
Vol 187 (2) ◽  
pp. 479-492 ◽  
Author(s):  
T Tsushima ◽  
N Sasaki ◽  
Y Imai ◽  
F Matsuzaki ◽  
H G Friesen
Keyword(s):  
Binding Site ◽  
Concanavalin A ◽  
Specific Binding ◽  
Binding Protein ◽  
Liver Homogenate ◽  
Deae Cellulose ◽  
Sedimentation Coefficient ◽  
Binding Activity ◽  
Affinity Constant ◽  
Triton X

A specific binding site for somatotropin was solubilized by 1% (v/v) Triton X-100 from a crude particulate membrane fraction of pregnant rabbit liver, partially purified and characterized. The solubilized binding site retained many of the characteristics observed in the original particulate fraction, indicating that extraction of the binding site with Triton X-100 does not cause any major changes in its properties. The binding of human 125I-labelled-somatotropin to the solubilized binding site is a saturable and reversible process, depending on temperature, incubation time, pH and ionic environment. Analysis of the kinetic data revealed a finite number of binding sites with an affinity constant of 0.32 × 10(10)M-1. The binding activity for human 125I-labelled-somatotropin was adsorbed to a concanavalin-A-Sepharose column and was dissociated from the column with alpha-methyl-D-glucoside, suggesting that the binding protein may be a glycoprotein. Using affinity chromatography on concanavalin-A-Sepharose, ion-exchange chromatography on DEAE-cellulose and gel filtration on Sepharose 6B, the binding protein was purified 1000-4000-fold from the original liver homogenate. When the partially purified preparation was chromatographed on Sepharose 6B, the binding protein eluted as a molecule with an apparent molecular weight of 200000, with a Stokes' radius of 4.9 nm. Sucrose-density-gradient centrifugation of the preparation showed that the sedimentation coefficient of the binding protein was 7.2S. Isoelectric focusing experiments revealed that a major part of the protein has an acidic pI (4.2-4.5). Exposure of the protein to trypsin decreased the binding activity for human 125I-labelled-somatotropin or bovine 125I-labelled-somatotropin, whereas ribonuclease, deoxyribonuclease, phospholipase C or neuraminidase had little or no effect.

scholarly journals Transcriptional and Posttranscriptional Regulation of Insulin-Like Growth Factor Binding Protein-3 by Cyclic Adenosine 3′,5′-Monophosphate: Messenger RNA Stabilization Is Accompanied by Decreased Binding of a 42-kDa Protein to a Uridine-Rich Domain in the 3′-Untranslated Region

1999 ◽  
Vol 13 (3) ◽  
pp. 495-504
Author(s):  
N. E. Erondu ◽  
J. Nwankwo ◽  
Y. Zhong ◽  
M. Boes ◽  
B. Dake ◽  
...  
Keyword(s):  
Growth Factor ◽  
Untranslated Region ◽  
Specific Binding ◽  
Binding Protein ◽  
Binding Activity ◽  
Insulin Like Growth Factor ◽  
Factor Binding ◽  
Mdbk Cells ◽  
Camp Induction ◽  
Igfbp 3

Abstract The Madin Darby bovine kidney (MDBK) cell line was used to investigate the mechanisms underlying the cAMP regulation of insulin-like growth factor binding protein-3 (IGFBP-3) gene expression. Treatment of confluent monolayers either with forskolin or cAMP produced a 60- to 75-fold induction of IGFBP-3 mRNA and protein levels. This effect did not require new protein synthesis as inhibition of translation by cycloheximide actually caused a 2-fold increase in the cAMP induction. The rates of IGFBP-3 gene transcription, assessed by nuclear run-on assays, increased approximately 15-fold in cells exposed to cAMP. In addition, the half-life of the IGFBP-3 mRNA transcript was increased ∼3-fold in the presence of cAMP. Gel mobility shift and competition experiments revealed the specific binding of an approximately 42-kDa cytoplasmic protein factor to the 3′-untranslated region (3′-UTR) of the IGFBP-3 mRNA. A 21-nucleotide uridine-rich segment that contained no AUUUA motif was sufficient for the specific binding. The binding activity of this protein was reduced after cAMP treatment but was increased by phosphatase treatment. In conclusion, the cAMP induction of IGFBP-3 mRNA in MDBK cells occurred at both the transcriptional and posttranscriptional levels. The IGFBP-3 mRNA stabilization in MDBK cells probably involved the phosphorylation of a member of the family of U-rich region mRNA-binding proteins and is the first reported member whose RNA-binding activity is reduced by cAMP.

scholarly journals Solubilization and purification of a membrane-associated 3,3′,5-tri-iodo-l-thyronine-binding protein from rat erythrocytes

1990 ◽  
Vol 270 (3) ◽  
pp. 577-582 ◽  
Author(s):  
R C Angel ◽  
J A Botta ◽  
R D Morero ◽  
R N Farias
Keyword(s):  
Hormone Receptors ◽  
Specific Binding ◽  
Binding Protein ◽  
Active Form ◽  
Binding Activity ◽  
Erythrocyte Membranes ◽  
Major Protein ◽  
Sds Page ◽  
Affinity Labelling ◽  
Zwitterionic Detergent

3,3′,5-Tri-iodo-L-thyronine (L-T3) binding sites from rat erythrocyte membranes were solubilized in an active form by using the zwitterionic detergent CHAPS or the anionic detergent lauroylsarcosine. The binding protein was successively purified by Sephadex G-200 and affinity chromatography. The purified material retained its binding activity and exhibited high affinity and specificity compared with those displayed in the original membrane. Yield was about 10% of the starting activity. The specific binding activity was enriched by approx. 100-fold, which represents a purity of only 0.1%. Analysis of the purified preparation on SDS/PAGE showed two major protein bands (Mr 64,000 and Mr 50,000), but these could not represent the binding protein since the purity obtained was low. However, affinity-labelling experiments with N-bromoacetyl-L-[125I]T3 in intact membranes showed that two proteins (also with Mr values of 64,000 and 50,000) bound the hormone specifically, suggesting a co-migration of hormone receptors and contaminants on gel electrophoresis.

scholarly journals ICBP90, a Novel Methyl K9 H3 Binding Protein Linking Protein Ubiquitination with Heterochromatin Formation

2007 ◽  
Vol 28 (2) ◽  
pp. 705-717 ◽  
Author(s):  
Panagiota Karagianni ◽  
Larbi Amazit ◽  
Jun Qin ◽  
Jiemin Wong
Keyword(s):  
Transcriptional Repression ◽  
Mammalian Cells ◽  
Specific Binding ◽  
Binding Protein ◽  
Biological Effects ◽  
Binding Activity ◽  
Effector Proteins ◽  
Heterochromatin Formation ◽  
Protein Ubiquitination

ABSTRACT Methylation of histone H3 on lysine 9 is critical for diverse biological processes including transcriptional repression, heterochromatin formation, and X inactivation. The biological effects of histone methylation are thought to be mediated by effector proteins that recognize and bind to specific patterns of methylation. Using an unbiased in vitro biochemical approach, we have identified ICBP90, a transcription and cell cycle regulator, as a novel methyl K9 H3-specific binding protein. ICBP90 and its murine homologue Np95 are enriched in pericentric heterochromatin of interphase nuclei, and this localization is dependent on H3K9 methylation. Specific binding of ICBP90 to methyl K9 H3 depends on two functional domains, a PHD (plant homeodomain) finger that defines the binding specificity and an SRA (SET- and RING-associated) domain that promotes binding activity. Furthermore, we present evidence that ICBP90 is required for proper heterochromatin formation in mammalian cells.

Characterization of insulin-like growth factor-binding protein in ovine amniotic fluid

1990 ◽  
Vol 127 (2) ◽  
pp. 325-333 ◽  
Author(s):  
J.-F. Wang ◽  
L. J. Fraher ◽  
D. J. Hill
Keyword(s):  
Growth Factor ◽  
Amniotic Fluid ◽  
Binding Protein ◽  
Binding Activity ◽  
Scatchard Analysis ◽  
Insulin Like Growth Factor ◽  
Fetal Plasma ◽  
Igf I ◽  
Igf Binding ◽  
Igf Binding Protein

ABSTRACT We have characterized an insulin-like growth factor (IGF)-binding protein present in ovine amniotic fluid. Using an activated charcoal-binding assay, whole amniotic fluid specifically bound approximately 20–30% of 125I-labelled human (h) IGF-II added, while the binding of 125I-labelled hIGF-I was minimal. Radioimmunoassay for IGF-I or -II in ovine biological fluids showed that values in amniotic fluid were 9- to 13-fold less than in fetal plasma, while gel filtration of amniotic fluid on Sephadex G-50 eluted with 1 mol acetic acid/l revealed no additional binding activity which had been complexed to IGFs at neutral pH. Together, these observations suggest that the binding activity in amniotic fluid is largely unsaturated. Competition studies for the displacement of 125I-labelled IGF-II binding to amniotic fluid by increasing amounts of unlabelled IGF-I or -II, using the charcoal assay, showed that IGF-II was 30-fold more potent than IGF-I. Scatchard analysis revealed a single class of binding site for IGF-II, with a binding affinity of 0·68 ±0·18 litres/nmol (mean ± s.d., n = 3). Ligand blot analysis of amniotic fluid by separation on 8% SDS-PAGE, transfer to nitrocellulose membranes, incubation with 125I-labelled IGF-II and autoradiography revealed a single band of IGF-binding protein with approximate molecular size of 38 kDa. Additional IGF-binding species of 20, 28, 48 and > 180 kDa were present in ovine fetal plasma. Separation of amniotic fluid on Concanavalin A–Sepharose revealed that it had little carbohydrate content. These results show that ovine amniotic fluid contains an unsaturated, non-glycosylated IGF-binding protein with high affinity for IGF-II. These characteristics differ from those of the IGF-binding proteins purified from human amniotic fluid. Journal of Endocrinology (1990) 127, 325–333

Enhanced corticosterone-binding capacity in plasma after stimulation of the rat adrenal cortex: the possibility of a simultaneous release of protein and corticosterone

1987 ◽  
Vol 112 (1) ◽  
pp. 33-41 ◽  
Author(s):  
J. R. Bassett
Keyword(s):  
Binding Capacity ◽  
Specific Binding ◽  
Binding Protein ◽  
Plasma Concentrations ◽  
Plasma Corticosterone ◽  
Initial Increase ◽  
Scatchard Analysis ◽  
Corticosterone Concentration ◽  
Handling Stress ◽  
Corticosteroid Binding Globulin

ABSTRACT Exposure of rats to either footshock or handling stress produced a significant increase in both plasma corticosterone concentration and specific binding capacity. Non-specific binding was eliminated using the synthetic glucocorticoid, dexamethasone. The increase in both plasma corticosterone and specific binding capacity was biphasic following exposure to footshock. Adrenalectomy and pretreatment with betamethasone abolished both phases of the enhanced binding capacity and plasma steroid concentration. Intraperitoneal injection of ACTH (1–24) in animals pretreated with betamethasone resulted in a biphasic rise in plasma concentrations of corticosterone but only the initial increase in binding capacity. Dissociation constant (Kd) values, determined by Scatchard analysis, for adrenalectomized and betamethasone-pretreated animals were 546 and 556 pmol/l respectively. These values were significantly different from the Kd in animals with functional adrenals (631 pmol/l). The results are discussed in the light of a possible specific corticosteroid-binding globulin (CBG)-like binding protein of adrenal origin released in conjunction with corticosterone. This binding protein has a lower affinity for corticosterone and a shorter half-life than CBG. J. Endocr. (1987) 112, 33–41

Onchocerca retinol- and ivermectin-binding protein activity

Parasitology ◽  
1996 ◽  
Vol 112 (2) ◽  
pp. 221-225 ◽  
Author(s):  
P. G. Lal ◽  
E. R. James
Keyword(s):  
Adult Worm ◽  
Specific Binding ◽  
Binding Protein ◽  
Binding Activity ◽  
Retinol Binding Protein ◽  
Size Exclusion ◽  
Protein Activity ◽  
Molecular Weights ◽  
Molar Excess ◽  
Exclusion Chromatography

SummaryThe presence of retinol-binding protein (RBP) activity in Onchocerca cervicalis adult worms and interaction with ivermectin has been studied using high pressure size exclusion chromatography (HPSEC). Four distinct peaks of [3H]-retinol incorporation were obtained corresponding to approximate molecular weights of 150, 67, 19·7 and 4–6 kDa, the 2 smaller Mr peaks accounting for most of the binding activity. Competition for binding using non-labelled retinol at 200-fold molar excess indicated that specific binding of retinol occurred only to the 19–7 kDa fraction. Competition by ivermectin also inhibited binding of [3H]-retinol to the third peak. Following incubation with [3H]-ivermectin & peaks of similar molecular weights were also detected by HPSEC in soluble adult worm homogenate, However, in this case the 150 kDa fraction was most prominent. Both non-labelled ivermectin and non-labelled retinol at 200-fold molar excess reduced binding of [3H]-ivermectin to all & fractions. These data indicate that the putative Onchocerca RBP has an approximate molecular weight of 19·7 kDa, that retinol also binds to 3 additional fractions non-specifically, that the pattern of binding of ivermectin to adult worm material is quantitatively and qualitatively different from the binding exhibited by retinol, and that ivermectin interferes with the binding of retinol to the 19·7 kDa Onchocerca protein.

Free and bound androgens in the female guinea pig during gestation and after parturition and in the offsprings during the perinatal period

1980 ◽  
Vol 95 (1) ◽  
pp. 101-109 ◽  
Author(s):  
N. Rigaudière ◽  
P. Pradier ◽  
P. Delost
Keyword(s):  
Binding Capacity ◽  
Specific Binding ◽  
Equilibrium Dialysis ◽  
Plasma Concentrations ◽  
Perinatal Period ◽  
Maternal Plasma ◽  
Binding Activity ◽  
Low Concentrations ◽  
High Binding Affinity ◽  
Newborn Animals

Abstract. Total amounts of testosterone (T) and dihydrotestosterone (DHT) and their distribution between non-protein-bound (free), albumin-bound and PBG-(progesterone binding globulin)-bound fractions were determined by radioimmunoassay and equilibrium dialysis from plasma samples. The samples were taken from male guinea pigs during the perinatal period and from pregnant females during gestation and after parturition. Plasma proteins of the foetus and newborn animals appeared to have no high binding affinity for androgens. On the other hand albumin having a binding capacity of 56% for testosterone and 75% for DTH irrespective of the total androgen concentration may be considered to be an important low affinity binding protein. Maternal plasma developed a specific binding activity with the appearance of PBG in early pregnancy. Alterations in the binding capacity of PBG for T or DHT paralleled changes in plasma concentrations of both androgens, the highest values being observed on day 48 of pregnancy, with a prompt return to normal after parturition. Irrespective of the total androgen concentration, it was evident that PBG was capable of maintaining the free T and DHT at low concentrations (about 0.20 and 0.17 ng/10 ml, respectively). Besides the specific binding due to PBG a non-specific binding due to albumin was observed. The competition which exists between these two binding systems for T and DHT was evident when the quantity of bound androgens was expressed as a percentage. Neither the sex, nor the number of the foetuses, nor the interaction between the two, was found to have any significant effect in the maternal androgens, whether total or free hormone was considered.

scholarly journals A high-affinity oestrogen-binding protein in cockerel liver cytosol

1979 ◽  
Vol 180 (2) ◽  
pp. 347-353 ◽  
Author(s):  
C B Lazier ◽  
A J Haggarty
Keyword(s):  
Binding Sites ◽  
Proteinase Inhibitors ◽  
Specific Binding ◽  
Binding Protein ◽  
Gel Filtration ◽  
Binding Activity ◽  
Single Class ◽  
High Affinity ◽  
Significant Concentration

In contrast with several earlier reports, cytosol from cockerel liver contains a significant concentration of a protein that binds oestradiol with high affinity. To demonstrate the activity, certain alterations in the conventional method of preparation of cytosol must be made. Homogenization in sucrose-containing buffer at pH 8.4 in the presence of proteinase inhibitors and rapid fractionation of the cytosol with (NH4)2SO4 enables demonstration of a single class of oestradiol-binding sites with a Kd of about 1 nM and specificity only for oestrogens. The concentration is about 300 sites per cell in liver from 2-week-old cockerels. Oestradiol treatment in vivo decreases the number of exchangeable cytosol oestradiol-binding sites by about 80% for 1–4h, after which time it is gradually restored. Gel filtration of the cytosol preparation in the presence of high salt concentrations reveals that most of the oestradiol-binding activity is in high-molecular-weight aggregates, but a mild trypsin treatment generates a specific binding protein with an approximate mol.wt. of 40 000. This protein may be an oestrogen receptor.

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