scholarly journals Characterization of human somatotropin binding to detergent-solubilized lactogenic receptors from rat liver

1981 ◽  
Vol 194 (2) ◽  
pp. 385-394 ◽  
Author(s):  
J S Bonifacino ◽  
S H Sánchez ◽  
A C Paladini
Keyword(s):  
Rat Liver ◽  
Rate Constant ◽  
Binding Capacity ◽  
Inhibitory Effect ◽  
Binding Activity ◽  
Dissociation Rate ◽  
Scatchard Analysis ◽  
Single Class ◽  
Solubilized Form ◽  
Triton X

Lactogenic receptors from rat liver microsomal fraction (‘microsomes’) were extracted by treatment with 1% (w/v) Triton X-100. Triton X-100 exerts an inhibitory effect on both the binding reaction and the separation of the free hormone from the complex. The association and dissociation of 125I-labelled human somatotropin are time- and temperature-dependent processes. The association rate constant, k1, is 6.7 × 10(6) mol . litre-1 . min-1 at 25 decrees C, and the dissociation rate constant, k-1, is 1.1 × 10(-3) min-1 at 25 degrees C. Scatchard analysis of saturation data reveals the existence of a single class of receptors and that solubilization leads to a slight decrease in affinity and a sharp increase in binding capacity. The dissociation constant, Kd, of the solubilized preparation is 0.22 nM and the binding capacity 2900 fmol/mg of protein. Similar results were obtained from competition experiments. Binding of 125I-labelled human somatotropin to the solubilized receptors is specifically inhibited by hormones with lactogenic activity. Incubation of the solubilized preparation with trypsin resulted in an 80% decrease in binding activity. The solubilized form of the receptor has a slightly increased sensitivity to the inactivation by trypsin, heat and extremes of pH, with respect to the membrane-bound form.

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)

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.).

Expression of insulin-like growth factor receptors in primary human thyroid neoplasms

1989 ◽  
Vol 121 (1) ◽  
pp. 112-120 ◽  
Author(s):  
Tohru Yashiro ◽  
Yoshito Ohba ◽  
Hitomi Murakami ◽  
Takao Obara ◽  
Toshio Tsushima ◽  
...  
Keyword(s):  
Thyroid Cancer ◽  
Binding Capacity ◽  
Specific Binding ◽  
Human Thyroid ◽  
Scatchard Analysis ◽  
Type I ◽  
Single Class ◽  
Normal Tissues ◽  
Igf I ◽  
Cancer Tissues

Abstract. The presence of IGF-I receptors was demonstrated in normal and neoplastic tissues of human thyroid. Binding of [125I]IGF-I to thyroid membranes was dependent on time and temperature of incubation, and maximal binding was achieved at 4°C and 18 h of incubation. [125I] IGF-I binding was dose-dependently displaced by unlabelled IGF-I; half-maximal inhibition occurred at concentrations of 10–20 μg/l. IGF-II and insulin had relative potencies of 5 and 1% compared with IGF-I. Scatchard analysis of binding data revealed a single class of IGF-I receptors with high affinity (Ka: 1.2–8.6 × 109 1/mol) in normal thyroid tissues. Affinity cross-linking and autoradiography demonstrated the type I IGF receptors. Specific binding of [125I] IGF-I in thyroid cancer tissues (9.69 ± 2.07% per 200 μg protein; mean ± sem, N = 8) was significantly (p <0.05) higher than that in the surrounding normal tissues (3.03 ± 0.35%, N = 8). In contrast, there was no difference in the binding between adenoma tissues (4.19 ± 0.53%, N = 5) and the adjacent normal tissues (2.94 ± 0.24%, N = 5). The higher IGF-I binding in cancer tissues was due to an increase in the binding capacity without any change in the affinity. The presence of IGF-I receptors suggests a possible role of IGF-I and its receptors in the growth of thyroid cancer cells.

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.

Partial purification and properties of the TSH receptors from human thyroid plasma membranes

1983 ◽  
Vol 103 (2) ◽  
pp. 198-204 ◽  
Author(s):  
Yasuhiro Iida ◽  
Junji Konishi ◽  
Kanji Kasagi ◽  
Keigo Endo ◽  
Takashi Misaki ◽  
...  
Keyword(s):  
Sodium Acetate ◽  
Plasma Membranes ◽  
Binding Capacity ◽  
Human Thyroid ◽  
Partial Purification ◽  
Affinity Column ◽  
Scatchard Analysis ◽  
Dependent Manner ◽  
Con A ◽  
Triton X

Abstract. Human thyroid plasma membranes were solubilized with 0.5% Triton X-100 and TSH receptors were purified by using DEAE-Sephadex, Con A and TSH affinity chromatography. A TSH binding activity was bound to DEAE-Sephadex equilibrated with 0.05 m sodium acetate, pH 6.3, 0.2% Triton X-100 and was eluted by a linear gradient of 0.1 m to 1.0 m ammonium acetate, pH 6.3. Eighty-five per cent of the activity was absorbed to Con A Sepharose and was eluted with 0.5 m α-methyl-D-mannoside, 0.05 m sodium acetate, pH 6.0. Seventy-five per cent of the TSH binding capacity could be absorbed to TSH-affinity column and was eluted with 0.1 m glycine-HCl, pH 3.0. By sequential application of the above procedures, more than 100-fold purification of the receptor activity was attainable. [125I]TSH binding of this fraction was inhibited by addition of unlabelled TSH in a dose-dependent manner. Scatchard analysis gave a curvilinear plot with a high affinity association constant of 0.72 × 109 m−1. By using Ultrogel AcA 34 gel filtration, the molecular size of the hormonereceptor complex was estimated to be 180 000.

scholarly journals Evidence that non-covalent forces, thiol and disulphide groups affect the structure and binding properties of the prolactin receptor on hepatocytes from pregnant rats

1985 ◽  
Vol 228 (2) ◽  
pp. 383-390 ◽  
Author(s):  
K Yamada ◽  
D B Donner
Keyword(s):  
Growth Hormone ◽  
Binding Capacity ◽  
Prolactin Receptor ◽  
Sodium Dodecyl ◽  
Specific Effect ◽  
Scatchard Analysis ◽  
Pregnant Rats ◽  
Receptor Complexes ◽  
Affinity Labelling ◽  
Triton X

Incubation of hepatocytes from pregnant rats with dithiothreitol decreased specific 125I-prolactin (125I-prl) binding to such cells by about 20% relative to control. This was not due to a non-specific effect of dithiothreitol on the cell membrane, since reduction also altered the binding of prl to solubilized partially purified receptor. Exposure of hepatocytes to N-ethylmaleimide (6 mM) for periods as brief as 1 min decreased the subsequent specific binding of 125I-prl by more than 50%. N-Ethylmaleimide was less effective as an inhibitor of binding when applied after hepatocytes had been exposed to 125I-prl, binding being decreased by about 15%. Scatchard analysis demonstrated that the effect of N-ethylmaleimide resulted from loss of receptor-binding capacity without any substantial effect on the affinity of the prl receptor for hormone. Dithiothreitol diminished the affinity of lactogenic sites for prolactin without altering cellular binding capacity. These observations suggest that thiol and disulphide groups are present in the prl receptor and that these functional moieties regulate the formation and properties of prl receptor complexes. The species to which 125I-prl had bound were identified by affinity labelling. 125I-prl was covalently coupled into saturable complexes of Mr 65000 and 50000. 125I-human growth hormone (125I-hGH) was covalently incorporated into complexes of Mr 300 000, 220 000, 130 000, 65 000 and 50 000. Bovine growth hormone (bGH), but not prl, competed for 125I-hGH uptake into the 300 000-, 220 000- and 130 000-Mr complexes, indicating that these species were somatogenic. Prl, but not bGH, inhibited 125I-hGH uptake into 65 000- and 50 000-Mr complexes. This demonstrated that 125I-hGH in the presence of bGH could affinity-label lactogenic receptors. 125I-prl aggregates in Triton X-100, whereas 125I-hGH does not. Therefore lactogenic complexes to which 125I-hGH was bound in the presence of excess bGH were solubilized in Triton X-100 and characterized sequentially by gel filtration and affinity labelling. Prl receptors were eluted from columns of Sepharose 6B as a species of Mr380 000. Fractionation of the 380 000-Mr species on sodium dodecyl sulphate polyacrylamide gels resulted in the isolation of complexes of Mr 65 000 and 50 000. Thus non-covalent forces stabilize aggregates of the monomeric prolactin receptor.

Receptors for angiotensin I1 on platelets from man

1984 ◽  
Vol 66 (6) ◽  
pp. 725-731 ◽  
Author(s):  
Yuan Ding ◽  
Christopher J. Kenyon ◽  
Peter F. Semple
Keyword(s):  
Angiotensin Ii ◽  
Binding Capacity ◽  
Specific Binding ◽  
Venous Blood ◽  
Kinetic Studies ◽  
Free Fluid ◽  
Scatchard Analysis ◽  
Ang Ii ◽  
Temperature Dependent ◽  
Single Class

1. Platelets were prepared from peripheral venous blood on iso-osmotic density gradients of Percoll, resulting in a good recovery of cells (50–80%) which were relatively free of contaminating blood cells (erythrocyte <0.1%, leucocyte <0.1%). 2. At 22°C, specific binding of 125labelled angiotensin II (300 pmol/l) was time and temperature dependent, saturable, reversible and linear with cell concentration. 3. Scatchard analysis of saturation curves revealed a single class of binding sites with Kd 1.5 ± 0.4 × 10−10 mol/l and total binding capacity 6.3 ± 1.2 receptorslplatelet. Similar values (Kd 2.4 ± 0.7 × 10−10 mol/l and binding capacity 6.5 ± 1.0 receptors/cell) were obtained by displacement analysis. From kinetic studies the forward and reverse rate constants were 3.1 × 108 mol min−1 1−1 and 3.6 × 10−2/min giving a Kd of 1.2 × 10−10mol/l. 4. The relative binding potencies for angiotensin I1 and analogues were: [Sar1, Thr8]ANC II > ANG II > ANG III > [Sar1, Ala8]ANG II > ANG I. 5. Incubation with an extracellular marker (51Cr-labelled EDTA) demonstrated that binding of angiotensin II to platelets was not due to free fluid endocytosis.

scholarly journals Studies on membrane proteins involved in ribosome binding on the rough endoplasmic reticulum. Ribophorins have no ribosome-binding activity

1987 ◽  
Vol 245 (3) ◽  
pp. 811-819 ◽  
Author(s):  
H Yoshida ◽  
N Tondokoro ◽  
Y Asano ◽  
K Mizusawa ◽  
R Yamagishi ◽  
...  
Keyword(s):  
Membrane Protein ◽  
Rat Liver ◽  
Concanavalin A ◽  
Protein Fraction ◽  
Proteolytic Enzymes ◽  
Binding Capacity ◽  
Liver Microsomes ◽  
Binding Activity ◽  
Rat Liver Microsomes ◽  
Ribosome Binding

A membrane protein fraction showing affinity for ribosomes was isolated from rat liver microsomes (microsomal fractions) in association with ribosomes by treatment of the microsomes with Emulgen 913 and then solubilized from the ribosomes with sodium deoxycholate. This protein fraction was separated into two fractions, glycoproteins, including ribophorins I and II, and non-glycoproteins, virtually free from ribophorins I and II, on concanavalin A-Sepharose columns. The two fractions were each reconstituted into liposomes to determine their ribosome-binding activities. The specific binding activity of the non-glycoprotein fraction was approx. 2.3-fold higher than that of the glycoprotein fraction. The recovery of ribosome-binding capacity of the two fractions was about 85% of the total binding capacity of the material applied to a concanavalin A-Sepharose column, and about 90% of it was found in the non-glycoprotein fraction. The affinity constants of the ribosomes for the reconstituted liposomes were somewhat higher than those for stripped rough microsomes. The mode of ribosome binding to the reconstituted liposomes was very similar to that to the stripped rough microsomes, in its sensitivity to proteolytic enzymes and its strong inhibition by increasing KCl concentration. These results support the idea that ribosome binding to rat liver microsomes is not directly mediated by ribophorins I and II, but that another unidentified membrane protein(s) plays a role in ribosome binding.

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).

Human orbital fibroblasts in culture bind and respond to endothelin

1993 ◽  
Vol 265 (1) ◽  
pp. C138-C142 ◽  
Author(s):  
T. J. Smith ◽  
R. J. Kottke ◽  
H. Lum ◽  
T. T. Andersen
Keyword(s):  
Binding Capacity ◽  
Human Fibroblasts ◽  
Cellular Protein ◽  
Binding Activity ◽  
Dermal Fibroblasts ◽  
Acetoxymethyl Ester ◽  
Single Class ◽  
Free Concentration ◽  
Amino Acid Peptide ◽  
Orbital Fibroblasts

Human fibroblasts in primary cell culture were studied for their ability to bind to endothelin (ET), a 21-amino acid peptide with profound vasoconstricting properties. When 125I-labeled ET-1 was incubated with confluent orbital fibroblasts in the presence of increasing concentrations of unlabeled ligand, a single class of binding site was defined with a dissociation constant of 1.42 x 10(-8) M and a maximal binding capacity of 9.1 x 10(-10) mol/micrograms protein. ET-3 was a substantially less potent competitor for 125I-ET-1 binding sites than was unlabeled ET-1. Dermal fibroblasts demonstrated approximately 75% less ET-1 saturation binding activity, on a cellular protein basis, than did those from the orbit. Orbital fibroblasts responded to ET-1 (10(-9) M) with a rapid and transient increase in the free concentration of intracellular Ca2+ ([Ca2+]i) as assessed by monitoring acetoxymethyl ester of fura 2 fluorescence intensity. Rechallenge with the peptide elicited a substantially attenuated response than that seen after the initial treatment. There was no consistent effect of ET-1 on [Ca2+]i in dermal cultures. ET-3 failed to influence [Ca2+]i in either type of fibroblast. It would appear that orbital fibroblasts bind and respond to ET in a manner distinct from that observed in dermal fibroblasts, raising the possibility that the peptide may have site-specific actions in orbital connective tissue.

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