scholarly journals An investigation of sites that bind human somatotropin (growth hormone) in the liver of the pregnant rabbit

1981 ◽  
Vol 198 (3) ◽  
pp. 605-614 ◽  
Author(s):  
H F Cadman ◽  
M Wallis
Keyword(s):  
Growth Hormone ◽  
Binding Sites ◽  
Specific Binding ◽  
Bovine Somatotropin ◽  
Scatchard Analysis ◽  
Single Class ◽  
Pregnant Rabbit ◽  
Membrane Bound ◽  
Ovine Prolactin ◽  
Triton X

The binding of 125I-labelled human somatotropin (growth hormone) to a crude membrane preparation from the liver of pregnant rabbit, and to receptors solubilized from this fraction by Triton X-100, was dependent on time, temperature and receptor concentration. At 4 degrees C a steady state was reached after 20 h, and maximum specific binding (as a percentage of total tracer added) was approx. 50% for both membrane-bound and solubilized receptors. Solubilization did not significantly affect the binding properties of the receptor at low concentrations of Triton X-100 (less than 0.05%, v/v, in the assay tube). However, at higher concentrations (approx. 0.1%, v/v), the detergent lowered the ability of some hormones, for example ovine prolactin, to displace 125I-labelled human somatotropin, but did not affect other hormones such as bovine somatotropin. Some somatogenic hormones, such as bovine somatotropin, and some lactogenic hormones, such as ovine prolactin, displaced 125I-labelled human somatotropin from membrane-bound and solubilized receptor preparations. Furthermore, 85% of 125I-labelled bovine somatotropin was displaced from membrane-bound receptors by ovine prolactin, and 125I-labelled ovine prolactin was almost completely displaced by bovine somatotropin. Scatchard analysis of the binding data for human somatotropin suggested a single class of binding sites in the membrane-bound receptor preparation, with an affinity (Ka) of 1.9 X 10(9) M-1 and a capacity of 1726 fmol/mg of protein; these values were slightly increased by solubilization (Ka = 3.2 X 10(9) M-1, capacity = 2103 fmol/mg of protein). Scatchard analysis of binding to membrane-bound receptors also indicated a single class of high-affinity binding sites for bovine somatotropin (Ka = 4.8 X 10(9) M-1, capacity = 769 fmol/mg) and for ovine prolactin (Ka = 6.1 X 10(9) M-1, capacity = 187 fmol/mg).

Growth hormone receptors in the liver and osmoregulatory organs of rainbow trout: characterization and dynamics during adaptation to seawater

1991 ◽  
Vol 130 (3) ◽  
pp. 425-433 ◽  
Author(s):  
T. Sakamoto ◽  
T. Hirano
Keyword(s):  
Growth Hormone ◽  
Rainbow Trout ◽  
Binding Sites ◽  
Chum Salmon ◽  
Hormone Receptors ◽  
Sea Water ◽  
Specific Binding ◽  
Head Kidney ◽  
Scatchard Analysis ◽  
Single Class

ABSTRACT Specific binding sites for chum salmon growth hormone (sGH) were identified in the membranes obtained from tissues of rainbow trout. Specific binding of 125I-labelled sGH (% per mg protein) was found in the liver (37%), ovary (6%), brain (6%), gill (4%), intestine (4%) and posterior body kidney (4%). Specific binding was not significant in head kidney, anterior body kidney, spleen, heart, skeletal muscle or skin. Scatchard analyses demonstrated the presence of a single class of high-affinity low-capacity receptors in the liver, gill, intestine and kidney. The association constants for the membranes from liver, gill, intestine and kidney were of the same order (1 litre/nmol). Chum salmon prolactin did not inhibit the binding of 125I-labelled sGH to receptors in the liver, gill, intestine and kidney. Transfer of rainbow trout from fresh water to 80% seawater evoked a rise in plasma concentration of GH and a significant decrease in the GH binding to the liver membranes after 1 day. Binding in the gill and kidney was not altered significantly. Membranes were treated with 4 mol MgCl2/l to remove bound GH from the receptors, and the results indicated that the reduction in binding in the liver after transfer to sea-water was probably due to receptor occupancy by increased endogenous GH. The occupancy of liver GH-binding sites was maximal 4 days after transfer. Total (MgCl2-treated) binding sites in the liver increased significantly 14 days after transfer. Scatchard analysis indicated that receptors were altered in capacity without changes in binding affinity. Although GH may also directly affect osmoregulatory organs through their GH receptors, the present results indicate the likelihood of at least partial mediation by the liver of the seawater-adapting action of GH in the rainbow trout. Journal of Endocrinology (1991) 130, 425–433

Specific binding sites for ovine prolactin in three amphibian cell lines

1985 ◽  
Vol 248 (1) ◽  
pp. C80-C87 ◽  
Author(s):  
M. Dunand ◽  
M. L. Aubert ◽  
J. P. Kraehenbuhl ◽  
B. C. Rossier
Keyword(s):  
Growth Hormone ◽  
Cell Lines ◽  
Binding Sites ◽  
Binding Capacity ◽  
Specific Binding ◽  
Human Growth ◽  
Functional Differentiation ◽  
Water Metabolism ◽  
Ovine Prolactin ◽  
Follicle Stimulating Hormones

Established cell lines (TB-6c and TB-M) obtained by continuous culture of epithelial cells from toad Bufo marinus urinary bladder, which, in culture, maintained a high degree of functional differentiation, exhibited a significant number of high-affinity (KA = 1-2 X 10(10) M-1) binding sites detected both with radioiodinated (125I) ovine prolactin (oPRL) and human growth hormone (hGH). Binding capacity was higher in the case of TB-6c cells (7,573 +/- 581 sites/cell) than with the TB-M cells (1,160 +/- 87). Similarly, binding sites for oPRL were characterized on Xenopus laevis kidney-derived cell line A6. With oPRL used both as tracer and standard, significant cross-reaction was observed with hGH, less with human or rat prolactin (PRL), and none with human chorionic somatomammotropin, bovine growth hormone, and rat luteinizing hormone or follicle-stimulating hormones. B. marinus pituitary extracts completely displaced the binding of 125I-oPRL to toad bladder binding sites. This finding of specific sites for PRL on amphibian bladder and kidney cells confirms that PRL exerts specific biological actions for the control of electrolyte and water metabolism in the amphibians.

scholarly journals Influence of divalent cations on the detection of somatogenic and lactogenic binding sites in mouse liver cells

1985 ◽  
Vol 228 (3) ◽  
pp. 761-764 ◽  
Author(s):  
G N Ciccia-Torres ◽  
J M Dellacha
Keyword(s):  
Binding Sites ◽  
Mouse Liver ◽  
Binding Capacity ◽  
Specific Binding ◽  
Divalent Cations ◽  
Isolated Hepatocytes ◽  
Liver Cells ◽  
Scatchard Analysis ◽  
Male Mice ◽  
Ovine Prolactin

Specific binding of 125I-labelled human somatotropin was demonstrated in isolated hepatocytes from male mice. In the presence of divalent cations (Ca2+ and Mg2+) the binding of 125I-labelled human somatotropin was competitive with ovine prolactin. Scatchard analysis of competition data indicated a KD of 1.4 +/- 0.2 nM and a binding capacity of 13 000 +/- 2000 sites/cell. In the absence of divalent cations and in the presence of EDTA, human and bovine somatotropins were found to be equally effective to displace bound 125I-labelled human somatotropin, while ovine prolactin showed a weak competition. In this case, the binding capacity was 8400 +/- 1500 sites/cell and the KD was 1.1 +/- 0.1 nM.

Comparison of growth hormone binding and metabolic response in rat adipocytes of epididymal, subcutaneous, and retroperitoneal origin

1985 ◽  
Vol 110 (1) ◽  
pp. 50-55 ◽  
Author(s):  
Stephen LaFranchi ◽  
Cheryl E. Hanna ◽  
Toni Torresani ◽  
Eugen Schoenle ◽  
Ruth Illig
Keyword(s):  
Growth Hormone ◽  
Binding Sites ◽  
Metabolic Response ◽  
Specific Binding ◽  
Human Growth ◽  
Scatchard Analysis ◽  
Rat Adipocytes ◽  
Significant Difference ◽  
Glucose Incorporation ◽  
Number Of Binding Sites

Abstract. We undertook a comparison of human growth hormone (hGH) binding and metabolic responses in rat adipocytes of epididymal, subcutaneous, and retroperitoneal origin to determine whether the site of fat depot biopsy might affect the response to hGH stimulation. The results showed highest specific binding in epididymal (3.6%), followed by subcutaneous (2.3%) and retroperitoneal adipocytes (1.5%); half-maximal binding was achieved at 14–18 ng/ml hGH for the three sites. Scatchard analysis of the binding data from each site was linear; there was no significant difference in binding affinities (2.1 to 3.3 × 109, m−1), but the number of binding sites was statiticially higher in epididymal (9.8 × 103) as compared to subcutaneous (7.5 × 103, P < 0.05) and retroperitoneal cells (3.3 × 103, P < 0.01). Stimulation with 5 to 2500 ng pituitary hGH produced a dose-related increase in glucose incorporation, with the largest increase in epididymal fat cells (31%, P <0.05) followed by subcutaneous cells (18%, P < 0.05); no significant increase was seen with retroperitoneal cells. Biosynthetic hGH produced a similar pattern of glucose incorporation in the three sites. Addition of hGH antibodies blocked the glucose incorporation in epididymal adipocytes using both pituitary-derived and biosynthetic hGH. It seems clear that this insulin-like effect is caused by hGH, not an insulin-like impurity. We conclude that the number of binding sites, perhaps related to adipose cell size, differs in adipose tissue from different locations and this influences the metabolic response to hGH stimulation.

THE PRESENCE OF LACTOGEN BUT NOT GROWTH HORMONE BINDING SITES IN THE ISOLATED RAT HEPATOCYTE

1977 ◽  
Vol 74 (2) ◽  
pp. 323-334 ◽  
Author(s):  
A. C. HERINGTON ◽  
N. M. VEITH
Keyword(s):  
Growth Hormone ◽  
Amino Acid ◽  
Binding Sites ◽  
Specific Binding ◽  
Female Rats ◽  
Scatchard Analysis ◽  
Hormone Binding ◽  
Binding Studies ◽  
Membrane Function ◽  
Liver Membranes

The binding of 125I-labelled human growth hormone (hGH) and bovine growth hormone (bGH) has been studied in hepatocytes isolated from female rats by perfusion with collagenase in situ. The cells appeared to retain normal membrane function, in that amino acid ([14C]α-aminoisobutyric acid) transport was both saturable and temperature-dependent. Amino acid ([14C]leucine) incorporation into protein was also linear over 3 h and was inhibited by cycloheximide. Binding of 125I-labelled hGH was dependent on time, temperature, hepatocyte concentration and hGH concentration. At 22 °C, binding reached a steady-state after 2·5 h and had a half-life of dissociation of 2–3 h. Hormone specificity studies indicated that binding was specific for hormones with prolactin-like activity (hGH, prolactins) and not for growth hormones themselves (bGH). Scatchard analysis revealed a single class of binding site with a binding capacity of 26·74 ± 3·73 fmol/106 cells and a binding affinity of 1·24 × 109 ± 0·17 × 109 (s.e.m.) l/mol (n = 10). There was a significant sex difference in binding (female > male) and binding was subject to marked regulation by oestrogens (stimulation of binding) and by androgens (inhibition). The lactogen-binding sites, therefore, were comparable in many respects to those previously reported in rat liver membranes. No distinct GH binding sites were demonstrable as shown by the lack of specific binding by 125I-labelled bGH, purified either by Sephadex chromatography or by binding to and elution from GH receptors in rabbit liver membranes. The value of receptor purification of tracer for use in hormone binding studies was indicated by a substantial lowering of non-specific binding.

Solubilization of active receptors for glucagon-like peptide-1(7-36)amide from rat lung membranes

1993 ◽  
Vol 264 (2) ◽  
pp. L146-L152
Author(s):  
R. Goke ◽  
F. Kolligs ◽  
G. Richter ◽  
B. Lankat-Buttgereit ◽  
B. Goke
Keyword(s):  
Divalent Cations ◽  
Binding Activity ◽  
Scatchard Analysis ◽  
Rat Lung ◽  
Single Class ◽  
Binding Data ◽  
Membrane Bound ◽  
Glucagon Like Peptide ◽  
Glucagon Like Peptide 1 ◽  
Triton X

We report on a protocol that allows the solubilization of active glucagon-like peptide (GLP)-1-(7–36)amide receptors from rat lung membranes. Digitonin-solubilized GLP-1(7–36)amide binding proteins from lung membranes most effectively, whereas (3-[(3-cholamidopropyl)- dimethylamino]-1-propane-sulfonate was less affective, and octyl-beta-glucoside, Triton X-100 and Lubrol PX were almost ineffective. Solubilization of binding activity was optimal at a digitonin concentration of 1%, a protein-to-detergent ratio of 1:10, and a pH between 7.0 and 8.0. Binding of GLP-1(7–36)amide to solubilized receptors was dependent on the concentration of solubilized protein. The presence of certain mono- and divalent cations was crucial for binding of GLP-1(7–36)amide to solubilized receptors. Scatchard analysis of the binding data revealed a single class of binding sites with dissociation and maximum binding constant values of 0.40 +/- 0.20 nM and 80.0 +/- 26.0 fmol/mg protein for membrane bound and 7.0 +/- 0.6 microM and 12.0 +/- 6.0 nmol/mg protein for solubilized receptors, respectively. In cross-linking experiments 125I-labeled GLP-1(7-36)amide was covalently attached to GLP-1(7–36)amide receptors on lung membranes. The apparent molecular mass of the solubilized receptor was 55,000 Da. This was proven in another experiment when receptor was consecutively cross-linked after solubilization. Nonhydrolyzable GTP analogues (GTP gamma S or GDP beta S) were unable to reduce GLP-1(7–36)amide-binding at solubilized receptors. This argues that the receptor is solubilized as a single protein and not as a receptor-G protein complex.(ABSTRACT TRUNCATED AT 250 WORDS)

FSH and LH up-regulate epidermal growth factor receptors in rat granulosa cells

1994 ◽  
Vol 140 (2) ◽  
pp. 171-177 ◽  
Author(s):  
H Fujinaga ◽  
M Yamoto ◽  
T Shikone ◽  
R Nakano
Keyword(s):  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Granulosa Cells ◽  
Binding Sites ◽  
Specific Binding ◽  
Scatchard Analysis ◽  
Dependent Manner ◽  
Single Class ◽  
Epidermal Growth ◽  
Dose Dependent

Abstract Epidermal growth factor (EGF) modulates ovarian folliculogenesis and steroidogenesis and its binding sites have been demonstrated in the ovary. We investigated the localization of EGF-binding sites in the rat ovary, and the effects of FSH and LH on EGF binding to cultured granulosa cells. Autoradiographic localization of 125I-labelled mouse EGF-binding sites was demonstrated in the granulosa and luteal cells. Displacement study and Scatchard analysis showed that a single class of specific binding sites for 125I-labelled mouse EGF was present in the granulosa cells, obtained from the ovaries of immature rats treated with diethylstilboesterol. The number of binding sites and the apparent dissociation constant were 4336 binding sites/cell and 3·42 pmol/l respectively. The granulosa cells were cultured for 48 h at 37 °C in medium alone or with increasing amounts of ovine FSH (oFSH; 1–1000 μg/l). FSH treatment increased 125I-labelled mouse EGF binding to the granulosa cells in a dose-dependent manner. After culture with oFSH (100 μg/l) for 48 h, the cells were cultured in medium alone or with increasing amounts of ovine LH (oLH; 1–1000 μg/l) for an additional 48 h. LH treatment also increased 125I-labelled mouse EGF binding in a dose-dependent manner, compared with the control. However, neither FSH nor LH altered receptor-binding affinity. Furthermore, after culture with oFSH (FSH-primed) or oFSH followed by oLH (LH-primed), tissue plasminogen activator (tPA) activities in the conditioned media were examined by fibrin autography. FSH-primed or LH-primed granulosa cells were more responsive to EGF action to induce an increase in tPA activity. In conclusion, it is suggested that functional receptors for EGF in rat granulosa cells are up-regulated by FSH and LH. Journal of Endocrinology (1994) 140, 171–177

Thromboxane binding and signal transduction in rat glomerular mesangial cells

1993 ◽  
Vol 264 (2) ◽  
pp. F292-F299 ◽  
Author(s):  
R. F. Spurney ◽  
J. J. Onorato ◽  
F. J. Albers ◽  
T. M. Coffman
Keyword(s):  
Signal Transduction ◽  
Dissociation Constant ◽  
Binding Sites ◽  
Mesangial Cells ◽  
Specific Binding ◽  
Maximal Response ◽  
Scatchard Analysis ◽  
Glomerular Mesangial Cells ◽  
Single Class ◽  
Alpha 7

Thromboxane A2 (TxA2) stimulates contraction of glomerular mesangial cells. However, mesangial cell TxA2 receptors have not been previously characterized. We therefore investigated TxA2 binding and TxA2-associated signal transduction pathways in rat glomerular mesangial cells using the specific thromboxane receptor agonist (1S-[1 alpha,2 beta(5Z),3 alpha-(1E,3S)4 alpha])-7-(3-[3-hydroxy-4-(p- iodophenoxy)-1-butenyl]7-oxabicyclo[2.2.1]hept-2-yl)-5-heptenoic acid (IBOP). In these cells, [125I]BOP binding was saturable, displaceable, and of high affinity. Scatchard analysis revealed a single class of binding sites with a dissociation constant (Kd) of 293 pM and a maximal density of binding sites (Bmax) of 33 fmol/mg protein. Specific binding was inhibited by the thromboxane agonist (15S)-hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5Z,13E-dienoic acid (U-46619) [inhibitor dissociation constant (Ki) = 297 nM] and the TxA2 receptor antagonists SQ 29548 (Ki = 1 nM) and (1R-[1 alpha(Z),2 beta,3 beta,5 alpha])-(+)-7-(5-[(1,1'-biphenyl)- 4-yl-methoxy]-3-hydroxy-2-(1-piperidinyl)cyclopentyl]-4-heptenoic acid (GR 32191) (Ki = 92 nM). Binding was also highly specific for thromboxane because prostaglandin E2 (Ki = 16 microM) and the inactive thromboxane metabolite, TxB2 (Ki = 41 microM), were approximately 1,000-fold less potent at inhibiting binding. IBOP stimulated phosphatidylinositol hydrolysis with an effective concentration of drug that produces 50% of the maximal response of 229 pM, which correlated well with the equilibrium Kd and enhanced phosphorylation of an acidic 80-kDa protein substrate for protein kinase C.(ABSTRACT TRUNCATED AT 250 WORDS)

scholarly journals Binding of androgens to a nuclear-envelope fraction from the rat ventral prostate

1980 ◽  
Vol 186 (3) ◽  
pp. 641-647 ◽  
Author(s):  
Y A Lefebvre ◽  
Z Novosad
Keyword(s):  
Nuclear Envelope ◽  
Binding Sites ◽  
Specific Binding ◽  
Ventral Prostate ◽  
Scatchard Analysis ◽  
Rat Ventral Prostate ◽  
Membrane Isolation ◽  
Triton X ◽  
Androgen Binding ◽  
Plasma Membrane Isolation

A nuclear-envelope fraction was isolated from the rat ventral prostate which is virtually free of DNA and contains little RNA or plasma membrane. Isolation of this nuclear-envelope fraction after incubation of purified nuclei with radioactive dihydrotestosterone results in labelling of the membrane. More binding of dihydrotestosterone is observed after incubations at 22 degrees C for 17 h than at 4 degrees C for 17 h or at 22 degrees C for 60 min. Scatchard analysis revealed a class of binding sites with KD 8.4 nM. Dihydrotesterone and testosterone were almost equally effective as competitors of labelled dihydrotestosterone binding on the purified nuclear-envelope fraction, whereas diethylstilboestrol was less effective and dexamethasone did not compete well. When the outer membrane of the nuclei was removed with Triton X-100, a 24% decrease in specific binding of androgens was observed. Castration 24 h before preparation of nuclei resulted in loss of the androgen binding to the membrane.

Direct effect of endothelin-1 on the granulosa cells of the porcine ovary

1992 ◽  
Vol 134 (1) ◽  
pp. 59-66 ◽  
Author(s):  
S. Kamada ◽  
T. Kubota ◽  
Y. Hirata ◽  
M. Taguchi ◽  
S. Eguchi ◽  
...  
Keyword(s):  
Granulosa Cells ◽  
Binding Sites ◽  
Inositol Phosphate ◽  
Binding Capacity ◽  
Specific Binding ◽  
Scatchard Analysis ◽  
Single Class ◽  
Apparent Dissociation Constant ◽  
Endothelin 1 ◽  
Porcine Granulosa Cells

ABSTRACT Specific binding sites for endothelin-1 (ET-1), a novel potent vasoconstrictor peptide, as well as the effects of ET-1 on cytosolic free Ca2+ concentration ([Ca2+]i), intracellular total inositol phosphate (IP) generation and steroidogenesis were studied in cultured porcine granulosa cells. Scatchard analysis of a binding study using 125I-labelled ET-1 indicated the presence of a single class of high-affinity binding sites with almost equal affinity for ET-1 and ET-3: the apparent dissociation constant was 0·59 nmol/l and the maximal binding capacity was 1·84 pmol/mg protein. Affinitylabelling of 125I-labelled ET-1 to the membranes using disuccinimidyl tartarate as a cross-linker revealed one major and one minor band with the apparent molecular weights of 32 kDa and 49 kDa respectively. ET-1 dose-dependently (1−100 nmol/l) induced rapid and transient increases in [Ca2+]i in fura-2-labelled cells. ET-1 also dose-dependently stimulated total IPs in cells prelabelled with myo-[3H]inositol. ET-1 had a slight stimulatory effect on the secretion of progesterone but not of oestradiol from porcine granulosa cells. The present data clearly demonstrate the presence of a non-selective ET receptor (ETB) in porcine granulosa cells coupled with phosphoinositide hydrolysis and [Ca2+]i mobilization, and suggest that ET-1 may play some role in the production of progesterone by porcine granulosa cells. Journal of Endocrinology (1992) 134, 59–66

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