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1.
G C Nicholson C S D''Santos T Evans J M Moseley B E Kemp V P Michelangeli T J Martin 《The Biochemical journal》1988,250(3):877-882
Receptors for the hypocalcaemic hormone, calcitonin (CT), have been identified in a membrane fraction prepared from term human placentae. Binding of 125I-labelled salmon CT (125I-sCT) to the membranes was time- and temperature-dependent, saturable (Bmax. 58 +/- 11 fmol/mg of protein), of high affinity (Kd 80 +/- 21 pM) and poorly reversible. Species-specific CTs and CT analogues competed for 125I-sCT binding with potencies proportional to their known biological potencies. Various unrelated peptide hormones did not compete, indicating that receptor binding was specific for CT. Photoaffinity labelling using a derivatized biologically active sCT analogue, [Arg11,18,3-nitrophenylazide-Lys14]sCT, identified a receptor component of Mr approximately 85,000, comparable with findings in osteoclasts and other target cells. The presence of CT receptors in the human placenta supports other evidence that CT may have a role in the regulation of placental function. 相似文献
2.
G C Nicholson C S D''Santos T Evans J M Moseley B E Kemp T J Martin 《The Biochemical journal》1988,253(2):505-510
Calcitonin (CT) binding activity has been extracted from a membrane fraction of human placenta using the zwitterionic detergent, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulphonic acid (Chaps). Approximately two-thirds of the available binding sites were extracted using 5 mM-Chaps. The binding characteristics of 125I-labelled salmon CT(125I-sCT) to the solubilized extract were similar to those obtained previously with placental membranes and other targets such as osteoclasts, renal cells and certain human cancer cell lines. 125I-sCT binding was saturable (Bmax. 75 +/- 6 fmol/mg of protein, n = 3) and Scatchard analysis revealed a single class of high-affinity binding sites (Kd 165 +/- 28 pM, n = 3). In competitive-binding studies, various species-specific CTs and CT analogues showed the same rank order of potencies as seen in CT bioassays and several unrelated peptides did not compete at high doses. A biologically active CT analogue, [Arg11,18, Lys14]sCT, derivatized with the photoreactive phenylazide cross-linking agent, N-hydroxysuccinimidyl-4-azidobenzoate, was used to identify receptor components of Mr approximately 88,000 and approximately 71,000 in both particulate placental membranes and the solubilized extract. Receptor components of Mr 85-90,000 have been identified in other CT target cells previously using chemical- and photoaffinity-labelling techniques. These results demonstrate the first successful solubilization of the CT receptor in a form which purification. 相似文献
3.
The binding of biologically active [125I]thyrotropin to purified plasma membranes prepared from bovine thyroid glands was studied. At 4°C, specific binding reached a maximum after 2 h of incubation and a plateau was maintained for up to 20 h. Degradation of [125I]thyrotropin was undetectable after 2 h of incubation and was only 10% of the total after 20 h.At pH 6.0, at which binding was maximal, a single class of binding sites, having a dissociation constant of approx. 25 nM, was evident. Dissociation studies revealed first order kinetics with a half-time of 2–3 min. At pH 7.5, binding curves were complex, suggesting two orders of binding sites with dissociation constants of approx. 200 nM and 80 pM. Further, at this pH, dissociation of the thyrotropin from its receptor was also complex, suggesting the presence of two first order reactions, one with a half-time similar to that seen at pH 6.0 and another with a half-time of 4 h. At both pH 6.0 and 7.5, insulin, glucagon, growth hormone, and prolactin were without effect on [125I]thyrotropin binding.Similar high affinity and low affinity binding sites were seen with porcine thyroid membranes, but only low affinity sites were seen with either rat liver membranes or human cultured lymphocytes. 相似文献
4.
The distribution of binding site for [125I]-eel-calcitonin (ECT) to rat central nervous system, studied by an autoradiographic technique, showed concentrations of binding in the diencephalon, the brain stem and the spinal cord. Large accumulations of grains were seen in the hypothalamus, the amygdala, in the fasciculus medialis prosencephali, in the fasciculus longitudinalis medialis, in the ventrolateral part of the periventricular gray matter, in the lemniscus medialis and in the raphe nuclei. The density of grains in the reticular formation and in the nucleus tractus spinalis nervi trigemini was more moderate. In the spinal cord, grains were scattered throughout the dorsal horns. Binding of the ligand was displaced equally by cold ECT and by salmon CT(sCT), indicating that both peptides bind to the same receptors. Human CT was much weaker than sCT in displacing [125I]-ECT binding. The administration of ECT into the brain ventricles of rats dose-dependently induced a significant and long-lasting enhancement of hot-plate latencies comparable with that obtained with sCT. The antinociceptive activity induced by ECT is compatible with the topographical distribution of binding sites for the peptide and is a further indication that fish CTs are active in the mammalian brain. 相似文献
5.
Calcitonin receptors of human osteoclastoma 总被引:2,自引:0,他引:2
G C Nicholson M A Horton P M Sexton C S D'Santos J M Moseley B E Kemp J A Pringle T J Martin 《Hormones et métabolisme》1987,19(11):585-589
Osteoclast-rich cultures were prepared by disaggregation of osteoclastomas (giant cell tumour of bone) and settlement onto glass or plastic surfaces. Autoradiography using [125I]-salmon calcitonin ([125I]-sCT) revealed specific binding only to multinucleate giant cells (osteoclasts) and a minor population of mononuclear cells. [125I]-sCT competitive binding studies indicated a Kd of 5 x 10(-10) M and receptor number of approximately 1 million sites/osteoclast. sCT treatment resulted in a dose-dependent rise in cAMP (EC50 10(-10) M). Homogenates of an osteoclastoma also demonstrated specific binding of [125I]-sCT. Chemical cross-linking of a labelled synthetic sCT derivative. [125I]-[Arg11,18,Lys14]-sCT, using disuccinimidyl suberate, resulted in labelling of a receptor component of approximate Mr 85-90,000. The multinucleate giant cells (osteoclasts) of human osteoclastomas possess large number of CT receptors which exhibit the same binding kinetics and apparent Mr as those of other CT target cells. 相似文献
6.
K Dang J Disa B Gout N Aiyar 《Journal of receptor and signal transduction research》1999,19(5):803-817
We have investigated the binding characteristics of rat [125I] adrenomedullin (AM) and human [125I] calcitonin gene-related peptide (CGRP) to membranes prepared from a number of porcine tissues including atrium, ventricle, lung, spleen, liver, renal cortex and medulla. These membranes displayed specific, high affinity binding for [125I] rat AM and [125I] human CGRP. Porcine lung displayed the highest density of binding sites for radiolabeled AM and CGRP followed by porcine renal cortex. Competition experiments performed with [125I] rat AM indicated that the rank order of potencies of various peptides for inhibiting [125I] rat AM binding to various tissues were rat AM > or = human AM > or = human AM(22-52) > h alpha CGRP > or = h alpha CGRP(8-37) > sCT except spleen, atrium, renal cortex and renal medulla where rAM and hAM were 20-300 fold more potent than hAM (22-52). When the same experiments were performed using [125I] h alpha CGRP as the radioligand, the rank order potencies for various peptides were rAM = hAM > h alpha CGRP > h alpha CGRP(8-37) in most of the tissues except in spleen and liver where h alpha CGRP was the most potent ligand. In lung, h alpha CGRP was almost as potent as rAM and hAM in displacing [125I] h alpha CGRP binding. These data suggest the existence of distinct CGRP and AM specific binding sites in contrast to previous reports that showed that both peptides interact differently in rat tissues. 相似文献
7.
Biologically potent analogues of salmon calcitonin which do not contain an N-terminal disulfide-bridged ring structure 总被引:3,自引:0,他引:3
The disulfide bridge formed between the cysteine residues at positions 1 and 7 of salmon calcitonin (sCT) is not required for biological activity. The analogues [Ala1,7]sCT,[AcmCys1,7]sCT and [AmcCys1,Ala7]sCT (AcmC = S-acetamido-methylcysteine) are linear sequences which retain full hypocalcemic activity in the intact rat and ability to activate adenylate cyclase of rat renal membranes. The secondary structure of these peptides in aqueous solution in the presence or absence of lipid is not greatly perturbed by the opening of the disulfide ring. In contrast with salmon calcitonin, substitution of Cys by AcmCys in human calcitonin results in greatly reduced hypocalcemic activity but no loss in the ability of the peptide to activate renal adenylate cyclase. Thus in vitro activation of adenylate cyclase by human calcitonin analogues is not always correlated with in vivo hypocalcemic potency. 相似文献
8.
Michael Mayer Shlomo Nir Richard J. Milholland F. Rosen 《Archives of biochemistry and biophysics》1976,176(1):28-36
The temperature dependence of uptake of [3H]dexamethasone by rat thymocytes in suspension and of the intracellular distribution of the bound hormone was studied as a function of time of incubation. The transport of [3H]dexamethasone was found to obey a simple solubility-diffusion mechanism. The permeability coefficient for glucocorticoid transport corresponded to values reported for other nonelectrolytes of a similar size through biological membranes. At temperatures ranging from 0 to 42 °C, the permeability coefficient increased with temperature and no maximum was observed. However, the maximum cellular uptake of the hormone varied depending on the temperature and time of incubation. Maximal uptake of [3H]dexamethasone was observed at 30 min when the reaction mixture was incubated at 30 °C; when incubated at 20 °C, maximum uptake of [3H]dexamethasone was observed at 3 h. These data were interpreted to mean that there was competition between two temperature-dependent processes, namely steroid transport and inactivation of intracellular binding sites. Intracellular hormone was observed to bind to specific sites as well as to nonspecific, presumably membranal sites. Two independent methods, one of which is based on a linear plot of uptake versus extracellular hormone concentration, gave similar values for the amount of specifically bound hormone, estimated to be 3300 molecules per cell. The binding results are in accord with the sequence of events previously proposed for the interaction of glucocorticoids with thymocytes. These events include nonspecific uptake, specific cytoplasmic binding, a highly temperature-dependent translocation into the nucleus, intranuclear binding, as well as receptor inactivation and regeneration. The amount of intracellular bound hormone and its distribution between the cytoplasmic and nuclear fractions showed no equilibrium or steady-state phenomenon throughout extended periods of incubation up to 28 h. The experiments verified kinetic equations which predicted maximum nuclear binding of the hormone at a given time, followed by an appreciable and progressive reduction in the binding of the hormone to cytoplasmic and nuclear fractions. 相似文献
9.
《Journal of receptor and signal transduction research》2013,33(5):803-817
AbstractWe have investigated the binding characteristics of rat [125I] adrenomedullin (AM) and human [125I] calcitonin gene-related peptide (CGRP) to membranes prepared from a number of porcine tissues including atrium, ventricle, lung, spleen, liver, renal cortex and medulla. These membranes displayed specific, high affinity binding for [125I] rat AM and [125I] human CGRP. Porcine lung displayed the highest density of binding sites for radiolabeled AM and CGRP followed by porcine renal cortex. Competition experiments performed with [125I] rat AM indicated that the rank order of potencies of various peptides for inhibiting [125I] rat AM binding to various tissues were rat AM ≥ human AM ≥ human AM(22–52) > hαCGRP ≥ hαCGRP(8–37) <<<< sCT except spleen, atrium, renal cortex and renal medulla where rAM and hAM were 20–300 fold more potent than hAM(22–52). When the same experiments were performed using [125I] hαCGRP as the radioligand, the rank order potencies for various peptides were rAM = hAM > hαCGRP > hαCGRP(8–37) in most of the tissues except in spleen and liver. where hαCGRP was the most potent ligand. In lung, hαCGRP was almost as potent as rAM and hAM in displacing [125I] hαCGRP binding. These data suggest the existence of distinct CGRP and AM specific binding sites in contrast to previous reports that showed that both peptides interact differently in rat tissues. 相似文献
10.
Calcitonin binding and degradation by two cultured human breast cancer cell lines (MCF 7 and T 47D). 总被引:3,自引:1,他引:2
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Two human breast cancer cell lines (MCF 7 and T 47D) possess calcitonin-responsive adenylate cyclase systems. Suspended cells of both lines specifically bound 125I-labelled salmon calcitonin with mean dissociation constants of 1.7 nM (MCF 7) and 1.4 nM (T 47D); mean receptor numbers were 5300 and 24400 per cell respectively. Measurement of specific binding to MCF 7 cells was obscured by rapid and substantial degradation of the labelled hormone. Degradation of 125I-labelled salmon calcitonin: (i) was of high capacity; (ii) lacked the specificity displayed by 125I-labelled salmon calcitonin binding to the same cells; and (iii) was not related to binding since cell incubation supernatants retained full degrading activity. The degrading activity was inhibited by corticotropin (1-24)-tetracosapeptide, insulin and bacitracin. Inclusion of bacitracin in the incubation resulted in apparently fewer numbers of lower affinity receptors on MCF 7 cells, whereas these parameters were identical to T 47D cells incubated in the presence or absence of bacitracin. Eel [2-aminosuberic acid 1,7]-calcitonin was resistant to proteolysis in the presence of either cell line. Analysis of hormone-receptor interactions with calcitonin-responsive cells should take account of potent calcitonin-degrading activities in some cell lines. 相似文献
11.
Some effects of calcitonin (CT) can also be produced by calcitonin gene-related peptide (CGRP), an alternative product of the calcitonin gene. This might be mediated by interaction of CGRP at the CT-receptor site. The human breast cancer cell line T47D possesses well characterized CT-receptors (KD = 2.3 x 10(-10) M for 125I salmon CT). 50% inhibition of 125I-sCT binding was achieved with 10(-9) M sCT, 5 x 10(-6) M rat CGRP and 10(-5) M human CGRP. Half maximal cAMP production in T47D cells was seen with 6 x 10(-10) M sCT, 5 x 10(-6) M rCGRP and 10(-5) M hCGRP. Binding and displacement capacity as well as the biological activity of CT and CGRP seems to correlate well. These findings suggest that CGRP in pharmacological doses acts via the CT-receptor. This could be explained by the homology and conformational similarities between CT and CGRP. 相似文献
12.
K W Cheng 《The Biochemical journal》1975,149(1):123-132
A simple method for preparing plasma membranes from bovine testes is described. Bovine testicular receptor has a high affinity and specificity for 125I-labelled human FSH (follicle-stimulating hormone). The specific binding of 125I-labelled human FSH to the plasma membranes is a saturable process with respect to the amounts of receptor protein and FSH added. The association and dissociation of 125I-labelled human FSH are time- and temperature-dependent, and the binding of labelled human FSH to bovine testicular receptor is strong and not readily reversible. Scatchard [Ann. N.Y. Acad. Sci. (1949) 51, 660-672] analysis indicates a dissociation constant, Kd, of 9.8 X10(-11)M, and 5.9 X 10(-14)mol of binding sites/mg of membrane protein. The testicular membrane receptor is heat-labile. Preheating at 40 degrees C for 15 min destroyed 30% of the binding activity. Specific binding is pH-dependent, with an optimum between pH 7.0 and 7.5. Brief exposure to extremes of pH caused irreversible damage to the receptors. The ionic strength of the incubation medium markedly affects the association of 125I-labelled human FSH with its testicular receptor. Various cations at concentrations of 0.1M inhibit almost completely the binding of 125I-labelled human FSH. Nuclectides and steroid hormones at concentrations of 1mM and 5mu/ml respectively have no effect on the binding of FSH to its receptor. Incubation of membranes with and chymotrypsin resulted in an almost complete loss of binding activity, suggesting that protein moieties are essential for the binding of 125I-labelled human FSH. Binding of 125I-labelled human FSH to bovine testicular receptor does not result in destruction or degradation of the hormone. 相似文献
13.
Photoaffinity labeling of rat liver nuclear extract with underivatized thyroid hormones was performed after incubation with 1 nM [3',5'-125I]thyroxine ([125I]T4) or [3'-125I]triiodothyronine [( 125I]T3) by irradiation with light above 300 nm. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the covalently photolabeled nuclear extract revealed four distinct hormone binding proteins of molecular masses 96, 56, 45, and 35 kilodaltons (kDa), respectively. Distribution of the hormone among these proteins was similar for T4 and T3. The 56- and 45-kDa proteins were the most prominently labeled. The specificity of the photoattachment of thyroid hormones to these nuclear proteins was verified by the irradiation of eight randomly chosen proteins and two proteins known to have thyroid hormone binding sites, human thyroxine binding globulin and bovine serum albumin. Only the latter two were photolabeled with [125I]T4. Competition studies performed by incubating nuclear extracts with [125I]T4 or [125I]T3 in the presence of increasing amounts of the corresponding unlabeled hormone (10-, 100-, and 1000-fold molar excess) demonstrated that (1) photoattachment of labeled T3 or T4 to the 56- and 45-kDa proteins was inhibited by 67-78% and 73-85%, respectively, after incubation with a 1000-fold molar excess of unlabeled hormone, (2) in the presence of lower molar excesses of the corresponding competitor (10- and 100-fold), photoattachment of labeled T3 or T4 to the 56- and 45-kDa receptors was gradually inhibited to a similar extent on both proteins, and (3) the 35- and 96-kDa proteins, although having thyroid hormone binding sites, display lower binding activities since the inhibition of photoattachment of labeled T3 or T4 by a 1000-fold molar excess of unlabeled hormone did not exceed 30-42% and 26-49%, respectively.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
14.
Nambi Aiyar Elayne Baker John Martin Arunbhai Patel Jeffrey M. Stadel Robert N. Willette Frank C. Barone 《Journal of neurochemistry》1995,65(3):1131-1138
Abstract: Calcitonin gene-related peptide (CGRP), a 37-amino-acid peptide, is a member of a small family of peptides including amylin or islet amyloid polypeptide and salmon calcitonin. These related peptides have been shown to display similar effects on in vitro and in vivo carbohydrate metabolism. The present study was initiated to identify and characterize the binding sites for these peptides in lung and nucleus accumbens membranes prepared from pig and guinea pig. Both tissues in either species displayed high-affinity (2-[125I]iodohistidyl10)humanCGRPα ([125I]hCGRPα) binding (IC50 = 0.4–7.7 nM), which was displaced by hCGRP8–37α with equally high affinity (IC50 = 0.4–7.3 nM). High-affinity binding for [125I]Bolton-Hunter human amylin ([125I]BH-h-amylin) was also observed in these tissues (IC50 = 0.2–6.0 nM). In membranes from the nucleus accumbens of both species, salmon calcitonin competed for amylin binding sites with high affinity (IC50 = 0.1 nM) but was poor in competing for amylin binding in lung membranes. Rat amylin8–37 competed for [125I]hCGRPα binding with higher affinity (IC50 = 5.4 nM) compared with [125I]BH-h-amylin binding (IC50 = 200 nM) in porcine nucleus accumbens, whereas in guinea pig nucleus accumbens, the IC50 values for rat amylin8–37 were 117 and 12 nM against [125I]hCGRPα and [125I]BH-h-amylin, respectively. Also, functional studies evaluating the activation of adenylate cyclase and generation of cyclic AMP in response to these agonists indicated that hCGRPα (EC50 = 0.3 nM), h-amylin (EC50 = 150 nM), and salmon calcitonin (EC50 = 1,000 nM) activated adenylate cyclase, resulting in increased cyclic AMP production in porcine lung membranes that was antagonized by hCGRP8–37α. The affinity of hCGRP8–37α was similar for all three peptides. The cyclic AMP responses to amylin and salmon calcitonin were significantly (p < 0.05) lower than that of hCGRPα and not additive, suggesting that they are acting as partial agonists at the same CGRP1-type receptor in porcine lung membranes. Similar observations were made for guinea pig lung membranes. However, human amylin and salmon calcitonin were weaker than hCGRPα in activating lung adenylate cyclase. None of these peptides activated adenylate cyclase in membranes prepared from the nucleus accumbens of both species. The data from these studies demonstrate both species and tissue differences in the existence of distinct CGRP and amylin binding sites and present a potential opportunity to study further CGRP and amylin receptor subtypes. 相似文献
15.
We have assessed the biological activity of salmon calcitonin I (sCT) using an in vivo biological assay of hypocalcemic activity in rats. The changes in biological activity observed are explained on the basis of changes in the conformational properties of the hormone analogues. Helical content in the presence and absence of lipids and detergents was assessed by using circular dichroism, and the section of the molecule that folds into a helix was predicted on the basis of the helix-coil transition theory of Mattice and co-workers. In the amino acid sequence of sCT, residue 8 is valine and residue 16 is leucine. The synthetic calcitonin derivatives [Gly8]sCT and [Ala16]sCT have higher biological activity than the native hormone although they have a lower helical content. The increased biological activity of these derivatives is ascribed to an increase in their conformational flexibility resulting from the substitution of amino acid residues with less bulky side chains and less tendency to form helical structures. The derivative [Met8]sCT has less substitution than sCT on the beta-carbon at position 8, but it has increased helix-forming potential in the region of residues 8-12. These two factors affect conformational flexibility in opposite ways, resulting in the biological activity of [Met8]sCT being slightly higher than that of sCT. However, increased conformational flexibility does not always increase biological activity. Substitution of the L-arginine at residue 24 for a D-arginine has little effect on the conformational properties or biological activity of sCT. However, [Gly8, D-Arg24]sCT is less active than sCT, [Gly8]sCT, or [D-Arg24]sCT.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
16.
Gonadotropin-releasing hormone (GnRH) binding sites have been characterized in the fully mature common carp ovary, using an analog of salmon GnRH ([D-Arg6,Trp7,Leu8,Pro9-NEt]-GnRH; sGnRH-A) as a labeled ligand. Binding of sGnRH-A to carp follicular membrane preparation was found to be time-, temperature-, and pH-dependent. Optimal binding was achieved after 40 min of incubation at 4 degrees C at pH 7.6; binding was found to be unstable at room temperature. Binding of radioligand was a function of tissue concentration, with a linear correlation over the range of 8.0-40.0 micrograms membrane protein per tube. Incubation of membrane preparations with increasing levels of [125I]sGnRH-A revealed saturable binding at radioligand concentrations greater than 400 nM. The binding of [125I]sGnRH-A to the carp ovary was also found to be reversible; addition of unlabeled sGnRH-A (10(-6) M) after reaching equilibrium resulted in complete dissociation of [125I]sGnRH-A within 30 min, and the log dissociation plot indicated the existence of a single class of binding sites. Addition of unlabeled sGnRH-A displaced the bound [125I]sGnRH-A in a dose-related manner. Hill plot as well as Scatchard analysis suggested the presence of one class of high affinity GnRH binding sites. Bound [125I]sGnRH-A was also found to be displaceable by other GnRH peptides, including sGnRH ([Trp7,Leu8]-GnRH), cGnRH-II ([His5,Trp7,Tyr8]-GnRH) and a GnRH antagonist ([D-pGlu1,D-Phe2,D-PTrp3,6]-GnRH; GnRH-ANT) in a parallel fashion, indicating that these peptides bind to the same class of binding sites.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
17.
Calcitonin and calcitonin gene-related peptide interact with the same receptor in cultured LLC-PK1 kidney cells 总被引:3,自引:0,他引:3
A Wohlwend K Malmstr?m H Henke H Murer J D Vassalli J A Fischer 《Biochemical and biophysical research communications》1985,131(2):537-542
Calcitonin and calcitonin gene-related peptide stimulate adenylate cyclase activity and plasminogen activator production in cultured renal tubular LLC-PK1 cells. Salmon [125I]calcitonin and human [125I]calcitonin gene-related peptide bound specifically to the cells. Salmon [125I]calcitonin binding was reduced at lower concentrations of non-radioactive salmon calcitonin than of human calcitonin gene-related peptide. For the stimulation of adenylate cyclase activity and plasminogen activator production, the potency of salmon calcitonin was higher than that of human calcitonin and calcitonin gene-related peptide. In a subclone of LLC-PK cells lacking salmon calcitonin binding sites, no specific binding of [125I]CGRP occurred, and adenylate cyclase activity and plasminogen activator production was not increased by the peptides. Thus, in LLC-PK1 cells the stimulation of adenylate cyclase activity and plasminogen activator production by calcitonin gene-related peptide is probably mediated by the calcitonin receptor. 相似文献
18.
Roberto Frairia Nicoletta Fortunati Laura Berta Annamaria Fazzari Federica Fissore Gianpiero Gaidano 《The Journal of steroid biochemistry and molecular biology》1991,40(4-6):805-812
Since the discovery of a specific membrane binding site for sex steroid binding protein (SBP) in human decidual endometrium and in hyperplastic prostate numerous speculations have been raised on the existence of an additional non-receptor-mediated system for steroid hormone action. In the present work SBP cell membrane binding was investigated in human estrogen target tissues other than those previously studied either in the absence of steroids or in the presence of varying amounts (10−10−10−6M) of estradiol, testosterone and dihydrotestosterone, respectively. Plasma membranes obtained by differential centrifugation from homogenized samples of pre-menopausal endometrium, endometrium adenocarcinoma, normal liver and post-menopausal breast showed a specific binding of highly purified [125I]SBP: a major displacement of labeled SBP was elicited by radioinert SBP, while no significant displacement occurred when other human plasma proteins were used as cold competitors (molar excess ranging 500–10,000-fold). A specific, time-dependent binding of [125I]SBP was also observed in MCF-7 and in Hep-G2 cell lines. The different patterns of specific binding, observed in membranes from different tissues when SBP was liganded with different sex steroid molecules, leads us to consider the tissue individuality of the receptor as a further entity in the membrane recognition system for SBP. 相似文献
19.
Comparison of Insulin Binding Proteins In Plasma and Nuclear Membranes of Obese and Lean Mouse Liver
《Journal of receptor and signal transduction research》2013,33(5-6):419-433
AbstractPlasma membranes obtained from obese (ob/ob) and lean (+/+ or +/ob) mouse livers were chemically crosslinked to [125I] -insulin and examined by electrophoresis and autoradiography. The pattern of crosslinked hormone was qualitatively similar in obese and lean plasma membranes. A major insulin binding protein of approximately M 120,000 was observed. Two additional bands were apparent, one which remained near the top of the gel and one about M 90,000. A minor band at approximately M 50,000 was also detected. For each of the insulin binding proteins a reduction in the amount of [125I]-insulin bound was observed with obese plasma membranes as compared with lean. For all proteins the insulin binding was specific as determined by competition with unlabeled hormone. In addition to plasma membrane receptors, insulin has also been reported to bind to nuclear membranes. The autoradiographic patterns of gels of [125]-insulin bound and crosslinked to nuclear membranes from obese and lean mouse livers indicated the presence of proteins of the same M as those described for plasma membranes. Nuclear membrane proteins bound less insulin than plasma membranes and, again, the obese was decreased relative to the lean. Contamination of the nuclear membrane fraction by plasma membranes was ruled out. Scatchard analyses of [125]-insul in bound to plasma and nuclear membranes indicated that the decrease in hormone binding in the obese mouse is a result of a reduction in the absolute number of receptors. The findings presented in this study provide additional support for this conclusion by demonstrating that membranes from obese mice are comprised of the same set of apparently unaltered insulin binding proteins. Further, the presence of similar insulin binding proteins in both nuclear and plasma membranes suggests a physiological relationship between these structures with respect to hormone binding and/or in the mechanism of action of insulin. 相似文献
20.
Nabil A. Mansour James J. Valdes Adil E. Shamoo Zoltan Annau 《Journal of biochemical and molecular toxicology》1987,2(1):25-42
Purified Torpedo nobiliana electric organ acetylcholine receptor (AChR) was reconstituted into membranes containing natural phospholipids supplemented with cholesterol (25% w/w). The reconstituted system facilitates the study of the effects of drugs on the regulation of the AChR channel complex under both resting and carbachol (carb)-stimulated conditions. Neostigmine (Neo) was the only carbamate to induce activation of [3-H]-phencyclidine ([3-H]-PCP) binding to the channel sites, acting as a weak agonist. The activation of [3-H]-PCP binding is dependent upon the nature of the reconstituted systems, with carb/Neo activation ratios of 8, 3, and 1 for the intact purified AChR vesicles fraction (PVF), the PVF reconstituted in phospholipid/cholesterol (CRPVF), and the PVF reconstituted in phospholipid (RPVF), respectively. The carbamates Neo, physostigmine (Physo), and pyridostigmine (Pyrido) inhibited carb-activated [3-H]-PCP binding with Ki values of 10, 20, and 1,600 μM, respectively. The inhibition was mixed competitivenoncompetitive in nature. The characteristic response of CRPVF to carb-stimulated [22-Na] influx was inhibited by the three carbamates, with IC-50 values of 6,50, and 1,000 μM for Neo, Physo, and Pyrido, respectively. The quaternary ammonium organophosphate ecothiophate (Eco) inhibited carb-stimulated [22-Na] influx with potency similar to that of Neo. Preincubation of AChR preparation with the carbamates and ecothiophate caused a reduction in the binding of [125-I]-α- bungarotoxin ([125-I]-α-BGT) with the following decreasing order of potency: Neo < Physo < Eco < Pyrido. Calcium has a direct modulatory role on the time-course inhibition of [125-I]-α-BGT binding by these drugs. While we observed a high potency of Neo and Physo in inhibiting [125-I]-α-BGT binding, it was undetectable for the carbamate insecticide 2-methyl-2-(methylthio)propionaldehyde-O-(methylcarbamoyl)oxime (aldicarb). These data suggest that the potent anticholinesterase carbamate agents interact differently with the AChR and its ionic channel. Their interactions with the nicotinic AChR channel system can be described as (a) weakly agonist, (b) directly acting on the open conformation of the channel, and (c) blocking the AChR-binding sites. 相似文献