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 Colchicine-binding protein of the liver. Its characterization and relation to microtubules.

1975 ◽  
Vol 66 (3) ◽  
pp. 609-620 ◽  
Author(s):  
C Patzelt ◽  
A Singh ◽  
Y L Marchand ◽  
L Orci ◽  
B Jeanrenaud
Keyword(s):  
High Speed ◽  
Specific Binding ◽  
Electrophoretic Analysis ◽  
Binding Activity ◽  
Low Concentrations ◽  
Electrophoretic Behavior ◽  
High Affinity Binding Site ◽  
High Concentrations

Colchicine-binding activity of mouse liver high-speed supernate has been investigated. It has been found to be time and temperature dependent. Two binding activities with different affinities for colchicine seem to be present in this high-speed supernate, of which only the high-affinity binding site (half maximal binding at 5 x 10(-6) M colchicine) can be attributed to microtubular protein by comparison with purified tubulin. Vinblastine interacted with this binding activity by precipitating it when used at high concentrations (2 x 10(-3) M), and by stabilizing it at low concentrations (10(-5) M). Lumicolchicine was found not to compete with colchicine. The colchicine-binding activity was purified from liver and compared with that of microtubular protein from brain. The specific binding activity of the resulting preparation, its electrophoretic behavior, and the electron microscope appearance of the paracrystals obtained upon its precipitation with vinblastine permitted its identification as microtubular protein (tubulin). Electrophoretic analysis of the proteins from liver supernate that were precipitated by vinblastine indicated that this drug was not specific for liver tubulin. Preincubation of liver supernate with 5 mM EGTA resulted in a time-dependent decrease of colchicine-binding activity, which was partly reversed by the addition of Ca++. However, an in vitro formation of microtubules upon lowering the Ca++ concentration could not be detected. Finally, a method was developed enabling that portion of microtubular protein which was present as free tubulin to be measured and to be compared with the total amount of this protein in the tissue. This procedure permitted demonstration of the fact that, under normal conditions, only about 40% of the tubulin of the liver was assemled as microtubules. It is suggested that, in the liver, rapid polymerization and depolymerization of microtubules occur and may be an important facet of the functional role of the microtubular system.

Inactivation of the β-adrenergic receptor in cardiac muscle by dithiols

1985 ◽  
Vol 63 (8) ◽  
pp. 932-936 ◽  
Author(s):  
Trevor I. Prior ◽  
Vandana Patel ◽  
G. I. Drummond
Keyword(s):  
Adrenergic Receptor ◽  
Reduced Glutathione ◽  
Binding Capacity ◽  
Specific Binding ◽  
Binding Activity ◽  
Ventricular Muscle ◽  
Adrenergic Antagonist ◽  
Equilibrium Dissociation ◽  
Rabbit Ventricular Muscle ◽  
Membrane Binding Sites

The effect of sulfhydryl reagents on binding of the β-adrenergic antagonist (−)-[3H]dihydroalprenolol hydrochloride ((−)-[3H]DHA) to a microsomal fraction of rabbit ventricular muscle was studied. Incubation with the disulfide reducing agents dithiothreitol (DTT), 2-mercaptocthanol, and reduced glutathione resulted in loss of (−)-[3H]DHA binding. At 500 μM DTT, less than 50% of specific binding activity remained; at 100 mM, binding was completely eliminated. 2-Mercaptoethanol and reduced glutathione were less effective than DTT at inhibiting binding activity. The total binding capacity (Bmax) decreased from 155.4 fmol mg−1 of protein, in the absence of DTT, to 92.4 and 77.5 fmol mg−1 at 0.25 and 0.7 mM DTT, respectively. The equilibrium dissociation constant (KD) increased from 7.6 nM, in the absence of DTT, to 10.3 nM at 0.25 mM DTT and to 20.8 nM at 0.7 mM DTT. Thus, DTT-induced decline in (−)-[3H]DHA binding results from a decrease in both the number and affinity of membrane binding sites for the tracer. Receptors could be protected from DTT inactivation by preincubation with β-adrenergic ligands. Oxidants could not reverse inactivation, with the exception of o-iodosobenzoate which was only partially effective. Thus, the β-adrenergic receptor of rabbit ventricular muscle contains essential disulfide moietie(s) which can be inactivated by reducing thiols.

A specific binding moiety for 1,25-dihydroxyvitamin D3 in basal lateral membranes of carp enterocytes

2000 ◽  
Vol 279 (3) ◽  
pp. E614-E621 ◽  
Author(s):  
Ilka Nemere ◽  
Dennis Larsson ◽  
Kristina Sundell
Keyword(s):  
Vitamin D ◽  
Gadus Morhua ◽  
Atlantic Cod ◽  
Binding Capacity ◽  
Specific Binding ◽  
Signal Transduction Pathway ◽  
Binding Activity ◽  
Protein Receptor ◽  
High Calcium ◽  
Plasma Membrane Receptor

Carp (Cyprinus carpio), a freshwater fish that lives in a low-calcium environment, and Atlantic cod (Gadus morhua), a seawater fish that lives in a high-calcium environment, were studied for the presence of a novel membrane binding protein (“receptor”) for the vitamin D metabolite, 1,25-dihydroxyvitamin D3[1,25(OH)2D3]. Basal lateral membranes from enterocytes of either species were prepared and analyzed for specific saturable binding. Membranes from carp revealed a dissociation constant of 1.23 nM with a maximal binding capacity of 212 fmol/mg protein. In comparison, membranes from Atlantic cod enterocytes revealed very low and nonsignificant levels of specific binding. The [3H]1,25(OH)2D3 binding activity in carp was further characterized for protein dependence, detergent extractability, and competition for binding with the metabolites 25(OH)D3 and 24 R,25(OH)2D3. Finally, introduction of 1,25(OH)2D3 to isolated carp enterocytes enhanced protein kinase C activity within 5 min, whereas intracellular Ca2+ concentrations were unaffected by a range of 1,25(OH)2D3 concentrations, as judged by fura 2 fluorescence. Thus the binding moiety may be a putative plasma membrane receptor for vitamin D, because it is functionally coupled to at least one signal transduction pathway.

scholarly journals Human somatotropin binding to rabbit kidney microsomal fraction

1981 ◽  
Vol 200 (2) ◽  
pp. 257-264 ◽  
Author(s):  
L P Roguin ◽  
S H Sánchez ◽  
J S Bonifacino ◽  
A C Paladini
Keyword(s):  
Binding Capacity ◽  
Specific Binding ◽  
Binding Activity ◽  
Rabbit Kidney ◽  
Scatchard Analysis ◽  
Temperature Dependent ◽  
Binding Reaction ◽  
Dissociation Equilibrium ◽  
Microsomal Membranes ◽  
Kidney Microsomes

Specific binding of 125I-labelled human somatotropin was demonstrated in microsomal membranes (microsomes) from rat and rabbit kidneys. Female rabbit kidney microsomes showed the highest binding activity and were used for further study. The association of 125I-labelled human somatotropin was time- and temperature-dependent and the binding reaction was reversible. Scatchard analysis of saturation data indicated a dissociation equilibrium constant, KD, of 56 pM and a binding capacity of 37 fmol per mg of protein. Similar results were obtained from competition experiments. Binding of 125I-labelled human somatotropin to the microsomes was specifically inhibited by hormones with lactogenic activity. The binding sites, as well as 125I-labelled human somatotropin, were not inactivated on incubation. Treatment of the microsomes with trypsin and chymotrypsin decreased the specific binding by over 90%. Preheating of the microsomes at 55 degrees C for 15 min abolished 50% of the specific binding activity.

PROGESTERONE-BINDING GLOBULIN AND TESTOSTERONE-BINDING ACTIVITY IN GUINEA PIG SERUM DURING PREGNANCY: RELATIONSHIP TO PROGESTERONE AND OESTROGENS

1976 ◽  
Vol 81 (2) ◽  
pp. 367-378 ◽  
Author(s):  
O. A. Lea ◽  
A. Bessesen ◽  
K. F. Støa
Keyword(s):  
Guinea Pig ◽  
Binding Sites ◽  
Binding Capacity ◽  
Post Partum ◽  
Equilibrium Dialysis ◽  
Binding Activity ◽  
Maximal Level ◽  
Sharp Drop ◽  
Endogenous Oestrogen ◽  
Competitive Protein Binding

ABSTRACT Progesterone levels in serum have been determined throughout pregnancy in guinea-pigs by a competitive protein-binding technique, using pregnant guinea pig plasma protein as binding agent. The concentrations of this protein (progesterone-binding globulin, PBG) as well as the testosteronebinding activity (TBA) have been quantitated by means of equilibrium dialysis. In order to correlate these parameters to the endogenous oestrogen production, the urinary oestrogen excretion was recorded. The concentration of progesterone, PBG and TBA showed a sharp rise on days 14–18 of gestation, reaching a maximal level on days 30–44. The progesterone level thereafter declined significantly towards parturition, followed by a sharp drop post partum. PBG and TBA followed a similar course, the decline towards parturition, however, being non significant. Neither progesterone, PBG nor TBA were significantly correlated to the urinary oestrogen excretion. The concentration of PGB was remarkably high, amounting to 1–2 × 10-5 m during most of the guinea pig pregnancy. This binding capacity generally exceeded the endogenous progesterone concentration by a factor of 20, as calculated on a molar basis. A mean of 64 % of the binding sites was available for testosterone binding, corresponding to a free binding capacity of 400 μg testosterone per 100 ml.

Androgen receptor in the Harderian gland of Rana esculenta

1991 ◽  
Vol 129 (2) ◽  
pp. 227-232 ◽  
Author(s):  
M. d'Istria ◽  
G. Chieffi-Baccari ◽  
L. Di Matteo ◽  
S. Minucci ◽  
B. Varriale ◽  
...  
Keyword(s):  
Androgen Receptor ◽  
Binding Capacity ◽  
Specific Binding ◽  
Secretory Activity ◽  
Harderian Gland ◽  
Nuclear Extract ◽  
Rana Esculenta ◽  
Binding Activity ◽  
Single Class ◽  
Nuclear Extracts

ABSTRACT An androgen receptor has been identified in the cytosolic and nuclear extracts of the Harderian gland of the frog, Rana esculenta. A single class of high-affinity binding sites was found: Kd = 1·9±1·3 (s.d.) nmol/l (n = 26) for the cytosolic extract and Kd = 0·9±0·8 nmol/l (n = 15) for the nuclear extract. The presence of binding activity in both nuclear and cytosolic extracts and the low rate of ligand-receptor dissociation are characteristics that distinguish this receptor from a steroid-binding protein. The Kd did not show any sex difference and did not exhibit any secretory activity-related change. Binding in both cytosolic and nuclear extracts was specific for androgens (testosterone = 5α-dihydrotestosterone); oestradiol-17β showed a 30% cross-reaction; moreover, specific binding of [3H]oestradiol-17β was not detectable. The binding capacity of the Harderian gland increased progressively in both fractions from October to December, reaching a peak in May, and decreased suddenly during July to August. The lack of any morphological sex-related difference in the Harderian gland of the green frog might be accounted for by the high amount of circulating androgens as well as a similar concentration of androgen receptor in both sexes. Journal of Endocrinology (1991) 129, 227–232

Identification of a set of calcium-binding proteins in reticuloplasm, the luminal content of the endoplasmic reticulum

1988 ◽  
Vol 91 (1) ◽  
pp. 61-70 ◽  
Author(s):  
D.R. Macer ◽  
G.L. Koch
Keyword(s):  
Endoplasmic Reticulum ◽  
Calcium Binding ◽  
Binding Proteins ◽  
Binding Capacity ◽  
Equilibrium Dialysis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Calcium Ionophore ◽  
Binding Activity ◽  
Calcium Binding Proteins ◽  
Molecular Weights

A procedure was developed for the isolation of reticuloplasm, the luminal material of the endoplasmic reticulum (ER). A reticuloplasm-rich extract was prepared from a murine plasmacytoma cell line that contains large amounts of ER, by first extracting the cytoplasmic contents using hypotonic lysis to yield ER-rich ‘shells’ followed by mechanical lysis to release the ER contents. The extract contains five major proteins with apparent molecular weights of 100, 75, 60, 58 and 55 (X 10(3] Mr by SDS-polyacrylamide gel electrophoresis. The 100, 75 and 58 (X 10(3] Mr species were identified as the known ER proteins endoplasmin, BiP and PD1, respectively. The ER association of the 60 and 55 (X 10(3] Mr proteins was confirmed by confocal fluorescence microscopy with affinity-purified antibodies. Equilibrium dialysis with isolated reticuloplasm gave a calcium-binding capacity of 300 nmoles calcium per mg protein with half-maximal binding at 3 mM-Ca2+. Purified endoplasmin bound 280 nmoles calcium per mg protein at a calcium concentration of 5 mM-Ca2+. A calcium overlay test revealed that, in addition to endoplasmin, reticuloplasm contained at least three other calcium-binding proteins: i.e. BiP, PDI and the 55 X 10(3) Mr protein, respectively, with endoplasmin and the 55 X 10(3) Mr protein (CRP55) accounting for the major proportion of the calcium-binding activity. Treatment of cells with calcium ionophore led to the specific over-expression of the major calcium-binding reticuloplasmins endoplasmin, BiP and CRP55. These studies show that the lumen of the ER contains a family of proteins with the capacity to bind significant amounts of calcium in the millimolar range and thereby to confer upon the ER the ability to perform a calcium storage function analogous to that of the sarcoplasmic reticulum in muscle cells.

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

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 Characterization of the Binding Specificity of K88ac and K88ad Fimbriae of Enterotoxigenic Escherichia coli by Constructing K88ac/K88ad Chimeric FaeG Major Subunits

2008 ◽  
Vol 77 (2) ◽  
pp. 699-706 ◽  
Author(s):  
Weiping Zhang ◽  
Ying Fang ◽  
David H. Francis
Keyword(s):  
Escherichia Coli ◽  
Amino Acids ◽  
Binding Capacity ◽  
Specific Binding ◽  
Binding Specificity ◽  
Binding Activity ◽  
Enterotoxigenic Escherichia Coli ◽  
Single Amino Acid ◽  
Fimbrial Subunit ◽  
K88 Fimbriae

ABSTRACT Enterotoxigenic Escherichia coli (ETEC) strains expressing K88 (F4) fimbriae are the major cause of diarrhea in young pigs. Three antigenic variants of K88 fimbriae (K88ab, K88ac, and K88ad) have been identified among porcine ETEC strains. Each K88 fimbrial variant shows a unique pattern in binding to different receptors on porcine enterocytes. Such variant specificity in fimbrial binding is believed to be controlled by the major subunit (FaeG) of the K88 fimbriae, because the genes coding for the only other fimbrial subunit are identical among the three variants. Uniqueness in binding to host receptors may be responsible for differences in the virulence levels of porcine diarrhea disease caused by K88 ETEC strains. To better understand the relationships between the structure of FaeG proteins and fimbrial binding function, and perhaps virulence in disease, we constructed and expressed various K88ac/K88ad faeG gene chimeras and characterized the binding activity of each K88 chimeric fimbria. After verifying biosynthesis of the chimeric fimbriae, we examined their binding specificities in bacterial adherence assays by using porcine brush border vesicles that are specific to either the K88ac or K88ad fimbria. Results showed that each fimbria switched binding specificity to that of the reciprocal type when a peptide comprising amino acids 125 to 163 was exchanged with that of its counterpart. Substitutions of a single amino acid within this region negatively affected the binding capacity of each fimbria. These data indicate that the peptide including amino acids 125 to 163 of the FaeG subunit is essential for K88 variant-specific binding.

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