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1.
Using everted sac technique we demonstrated the transfer of 125I-mEGF across the jejunal and ileal walls of suckling, weanling and adult rats. The transfer by the suckling rat jejunum and ileum was significantly inhibited by the presence of dinitrophenol and sodium azide or by the replacement of sodium with potassium or choline. RP-HPLC analysis detected carboxy-terminal processing of 125I-mEGF in suckling and adult rat jejunum and ileum. Suckling rat jejunum produced 125I-des(53)mEGF and 125I-des(49–53)mEGF, whereas 125I-des(48–53)mEGF was detected in suckling rat ileum or adult rat jejunum and ileum. All three forms of 125I-mEGF bound to anti-EGF antibody and EGF receptors. The receptor binding of 125I-des(53)mEGF was higher than that of 125I-mEGF, but those of 125-des(49–53)mEGF and 125I-des(48–53)mEGF were greatly diminished. Results indicate a carboxy-terminal processing of mouse EGF during uptake and transfer in the small intestine of developing and adult rats, and the resulting products showed altered receptor binding. An identical amino acid sequence of the C-terminal pentapeptide of EGF from mouse, human and possibly rat may suggest a biological significance of C-terminal processing of EGF in the small intestine. 相似文献
2.
Glucagon-like peptide-1 (7–36)amide (GLP-1 (7–36)amide) represents a physiologically important incretin in mammals including man. Receptors for GLP-1 (7–36)amide have been described in RINm5F cells. We have solubilized active GLP-1 (7–36)amide receptors from RINm5F cell membranes utilizing the detergents octyl-β-glucoside and CHAPS; Triton X-100 and Lubrol PX were ineffective. Binding of radiolabeled GLP-1(7–36)amide to the solubilized receptor was inhibited conentration-dependently by addition of unlabeled peptide. Scatchard analysis of binding data revealed a single class of binding sites with Ka= 0.26 ± 0.03 nM and Bmax= 65.4 ± 21.24 fmol/mg of protein for the membrane-bound receptor and Ka= 22.54 ± 4.42 μM and Bmax= 3.9 ± 0.79 pmol/mg of protein for the solubilized receptor. The binding of the radiolabel to the solubilized receptor was dependent both on the concentrations of mono- and divalent cations and the protein/detergent ratio in the incubation buffer. The membrane bound receptor is sensitive to guanine-nucleotides, however neither GTP-γ-S nor GDP-β-S affected binding or labeled peptide to solubilized receptor. These data indicate that the solubilized receptor may have lost association with its G-protein. In conclusion, the here presented protocol allows solubilization of the GLP-1(7–36)amide receptor in a functional state and opens up the possibility for further molecular characterization of the receptor protein. 相似文献
3.
It has been suggested that the F1-ATPase β-subunit is the enterostatin receptor. We investigated the binding activity of the purified protein with a labeled antagonist, β-casomorphin1–7, in the absence and presence of cold enterostatin. 125I-β-casomorphin1–7 weakly binds to the rat F1-ATPase β-subunit. Binding was promoted by low concentrations of cold enterostatin but displaced by higher concentrations. To study the relationship between binding activity and feeding behavior, we examined the ability of a number of enterostatin analogs to affect β-casomorphin1–7 binding to the F1-ATPase β-subunit. Peptides that suppressed food intake promoted β-casomorphin1–7 binding whereas peptides that stimulated food intake or did not affect the food intake displaced β-casomorphin1–7 binding. Surface plasmon resonance measurements show that the β-subunit of F1-ATPase binds immobilized enterostatin with a dissociation constant of 150 nM, where no binding could be detected for the assembled F1-ATPase complex. Western blot analysis showed the F1-ATPase β-subunit was present on plasma and mitochondrial membranes of rat liver and amygdala. The data provides evidence that the F1-ATPase β-subunit is the enterostatin receptor and suggests that enterostatin and β-casomorphin1–7 bind to distinct sites on the protein. 相似文献
4.
Parathyroid hormone (PTH) has been shown to have actions within the brain, suggesting the presence of central PTH receptors. This possibility was examined by determining the binding of 125I-labeled [Nle8,18,Tyr34]bovine PTH to the plasma membranes of rat and rabbit brains. Specific binding of the tracer to membranes of the whole brain was time and tissue dependent, and was greater with membranes from the hypothalamus than with membranes from the cerebellum, cerebrum, or brain stem. The binding of the tracer to rat hypothalamic membranes was saturable and competitively displaced by unlabeled PTH(1–34), PTH(3–34), [Nle8,18,Tyr34]PTH(1–34), and by PTH-related protein, indicating the presence of a single class of high-affinity (dissociation constant = 2–5 nM), low-capacity (maximum binding capacity, Bmax = 110–250 fmol/mg protein) binding site. The binding of radiolabeled PTH to these sites was not displaced by unrelated peptides of comparable molecular size (calcitonin, calcitonin-gene related peptide, adrenocorticotropin). The binding of PTH to these sites did not, however, appear to stimulate adenylate cyclase activity, as in peripheral PTH target sites. Thus, although these results indicate the presence of PTH receptors in the brain, these binding sites have a lower affinity than those in peripheral tissues and may utilize a different signal transduction system. 相似文献
5.
Harald Jüppner Abdul-Badi Abou-Samra Susumu Uneno Ernestina Schipani Henry T. Keutmann John T. Potts Jr. Gino V. Segre 《Peptides》1990,11(6):1139-1142
Biological properties of amino-terminal PTHrP analogues modified in the region 11–13 were examined using ROS 17/2.8 cells. [Leu11,D-Trp12,Arg13,Tyr36]PTHrP(1–36)amide had a 17-fold lower binding affinity for the receptor (apparent Kd: 5 × 10−8 M) than [Tyr36]PTHrP(1–36)amide or [Arg11,13,Tyr36]PTHrP(1–36)amide (apparent Kd for both: 2 × 10−9 M). Moreover, it is only a weak partial agonist despite completely inhibiting radioligand binding. [Leu11,D-Trp12,Arg13,Tyr36,Cys38]PTHrP(7–38) and PTHrP(7–34)amide had similar receptor affinities (apparent Kds: 5 × 10−8 M and 8 × 10−8 M), while that of [Nle8,18,Tyr34]bPTH(7–34)amide was more than 10-fold lower (apparent Kd: 2 × 10−6 M). These changes in biological properties suggest that high affinity receptor binding requires both amino- and carboxyl-terminal domains of the PTHrP(1–36) sequence and/or intramolecular interactions which are impaired by the D-Trp substitution for Gly12. 相似文献
6.
Bombesin (BN), gastrin-releasing peptide (GRP) and GRP(18–27) (neuromedin C) were equipotent and 30-fold more potent than neuromedin B (NMB) in inhibiting binding of 125I-GRP to and in stimulating amylase release from mouse pancreatic acini. In the present study we used 125I-GRP and chemical cross-linking techniques to characterized the mouse pancreatic BN receptor. After binding of 125I-GRP to membranes, and incubation with various chemical cross-linking agents, cross-linked radioactivity was analyzed by SDS-PAG electrophoresis and autoradiography. With each of 4 different chemical cross-linking agents, there was a single broad polypeptide band of Mr 80,000. Cross-linking did not occur in the absence of the cross-linking agent. Cross-linking was inhibited only by peptides that interact with the BN receptor such as GRP, NMB, GRP(18–27) or BN. Dose-inhibition curves for the ability of BN or NMB to inhibit binding of 125I-GRP to membranes or cross-linking to the 80,000 polypeptide demonstrated for both that BN was 15-fold more potent than NMB. The apparent molecular weight of the cross-linked polypeptide was unchanged by adding dithiothreitol. N-Glycanase treatment reduced the molecular weight of the cross-linked peptide to 40,000. The present results indicate that the BN receptor on mouse pancreatic acinar cell membranes resembles that recently described on various tumor cells in being a single glycoprotein with a molecular weight of 76,000. Because dithiothreitol had no effect, this glycoprotein is not a subunit of a larger disulfide-linked structure. 相似文献
7.
Specific receptors for adrenomedullin in cultured rat vascular smooth muscle cells 总被引:15,自引:0,他引:15
《FEBS letters》1994,340(3):226-230
The effects of synthetic rat adrenomedullin (rAM), a novel vasorelaxant peptide originally isolated from human pheochromocytoma, on receptor binding and cAMP generation were studied in cultured rat vascular smooth muscle cells (VSMC). A binding study using [125I]rAM revealed the presence of a single class of high-affinity (Kd1.3 × 10−8 M) binding sites for rAM in VSMC. The apparent Ki of rat calcitonin gene-related peptide (rCGRP) was 3 × 10−7 M. Affinity labeling of VSMC membranes with [125I]rAM revealed two distinct labeled bands with apparent molecular weights of 120 and 70 kDa, both of which were abolished by excess unlabeled rAM or rCGRP. rAM stimulated cAMP formation with an approximate EC50 of 10−8 M, the effect of which was additive with isoproterenol, but not with rCGRP. The rAM-induced cAMP response was unaffected by propranalol, indomethacin, or quinaerine, but inhibited by a CGRP receptor antagonist, human CGRP[8–37]. These data suggest that VSMC possesses specific AM receptors functionally coupled to adenylate cyclase with which CGRP interacts. 相似文献
8.
Margaret M. Shaffer Desmond N. Carney Louis Y. Korman Gail S. Lebovic Terry W. Moody 《Peptides》1987,8(6):1101-1106
The binding of 125I-VIP to human lung cancer cell lines was investigated. Radiolabeled VIP bound to adenocarcinoma, squamous cell carcinoma, large cell carcinoma and small cell lung cancer (SCLC) cell lines. As SCLC cell line NCI-N592 bound radiolabeled VIP well, its binding was further characterized. 125I-VIP bound to membranes in a specific and time dependent manner. 125I-VIP bound with high (Kd=0.8 nM) and moderate affinity (Kd=66 nM) to two classes of sites. Pharmacology studies indicated that the order of peptide potency was VIP PHI > secretin > VIP10–28. Because VIP receptors are present on human lung cancer cells, VIP may function as a regulatory peptide in lung cancer. 相似文献
9.
M. Riad S.El. Mestikawy D. Verge H. Gozlan M. Hamon 《Neurochemistry international》1991,19(4):413-423
Polyclonal antibodies were raised by the repeated injection of rabbits with synthetic peptides corresponding to selective portions (peptide 1: aminoacid residues 12–23, and peptide 2: aminoacid residues 243–268) of the aminoacid sequence of the rat 5-HT1A receptor. Both antisera allowed the immunoprecipitation of 5-HT1A receptors but not of other 5-HT receptor types and adrenergic receptors solubilized from rat hippocampal membranes. Immunoblots demonstrated that a single protein of 63 kDa, corresponding to the molecular weight of the rat 5-HT1A receptor binding subunit, was recognized by each antiserum. Immunoautoradiographic labelling of rat brain sections with the anti-peptide 2-antiserum exhibited the same regional distribution as 5-HT1A sites labelled by selective radioligands such as [3H]8-OH-DPAT and [125I]BH-8-MeO-N-PAT. However regional differences apparently existed between the respective intensity of labelling by the agonist radioligands and the antiserum, which might be explained by variations in the degree of coupling of 5-HT1A receptor binding subunits with G proteins from one brain area to another. 相似文献
10.
José C. Calvo Bernardo Yusta Francisco Mora Enrique Blázquez 《Journal of neurochemistry》1995,64(1):299-306
Abstract: Specific binding of glucagon-like peptide (GLP)-1(7–36)amide was detected in several rat brain areas, with the highest values being found in hypothalamic nuclei and the nucleus of the solitary tract. In hypothalamus and brainstem homogenate binding of 125I-GLP-1(7–36)amide was time, temperature, and protein content dependent and was inhibited by unlabeled proglucagon-derived peptides. The rank order of potency was GLP-1(7–36)amide ? GLP-1(1–36)amide > GLP-1(1–37) ? GLP-2 > glucagon. Scatchard analysis of the steady-state binding data was consistent with the presence of both high- and low-affinity binding sites in hypothalamus and brainstem. Brain 125I-GLP-1(7–36)amide-binding protein complexes were covalently cross-linked using disuccinimidyl suberate and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A single radiolabeled band of Mr 56,000 identified in both hypothalamus and brainstem homogenates was unaffected by reducing agents. An excess of unlabeled GLP-1(7–36)amide abolished the band labeling, whereas glucagon had no effect. Other unlabeled GLPs inhibited Mr 56,000 complex labeling with the following order of potency: GLP-1(1–36)amide > GLP-1(1–37) > GLP-2. The binding of 125I-GLP-1(7–36)amide and the intensity of the cross-linked band were similarly inhibited in a dose-response manner by increasing concentrations of unlabeled GLP-1(7–36)amide. Covalent Mr 56,000 125I-GLP-1(7–36)amide-binding protein complexes solubilized by Triton X-100 were adsorbed onto wheat germ agglutinin. Our results suggest that the GLP-1(7–36)amide receptor in rat brain is a glycoprotein with a single binding subunit that has a greater molecular weight but binding features and ligand specificity similar to those of its peripheral tissue counterparts. 相似文献
11.
Roger Lema-Kisoka Nathalie Hayez Ingrid Langer Patrick Robberecht Eric Sariban Christine Delporte 《Peptides》2001,22(12):2155-2162
R. LEMA-KISOKA, N. HAYEZ, I. LANGER, P. ROBBERECHT, E. SARIBAN AND C. DELPORTE. Characterization of functional VIP/PACAP receptors in the human erythroleukemic HEL cell line. PEPTIDES. The presence of VIP/PACAP receptors was investigated on the human erythroleukemic cell line HEL. Specific binding of [125I]-PACAP or [125I]-VIP on HEL cells or membranes was very low and did not allow to perform competition curves. At 37°C PACAP transiently increased cAMP levels in the presence of the non-specific phosphodiesterase inhibitor IBMX, suggesting rapid desensitization. Kinetic studies revealed that optimal conditions to measure the EC50 of PACAP(1–27) were 10 min at 20°C. Under those conditions, PACAP-related peptides increased cAMP levels with EC50 in agreement with the pharmacological profile of the VPAC1 receptor subtype: PACAP = VIP > [K15, R16, L27]VIP(1–7)/GRF(8–27) = [R16]ChSn (two VPAC1 agonists) HELODERMIN = secretin. RO 25–1553, a selective activator of VPAC2 receptor was inactive at 1 μM. Dose-response curves of VPAC1 agonist molecules (PACAP, VIP, [K15, R16, L27]VIP(1–7)/GRF(8–27), [R16]ChSn) were shifted to the right by the VPAC1 receptor antagonist [AcHis1, D-Phe2, Lys15, Leu17]VIP(3–7)/GRF(8–27), with a Ki of 3 ± 1 nM (n = 3). The presence of VPAC1 receptor mRNA was confirmed by RT-PCR. Preincubation with PACAP or PMA showed that VPAC1 receptors underwent homologous and heterologous desensitization.
This study provides the first evidence for the expression of functional VPAC1 receptors undergoing rapid desensitization in HEL cells. 相似文献
12.
Autoradiographic analysis of 1, 8, 16 and 26-day-old rat brains showed 3H-arginine8-vasopressin (3H-AVP) binding to the cingulate gyrus-dorsal hippocampus (CG) only in the 8-day-old rat brain. Saturation analysis of CG membranes prepared from pups (7–10 days) and adults (90 days) revealed a small but significant increase in binding site concentration in adults compared to pups. However, the Kd of the 3H-AVP binding site increased significantly with age. The Kd of 3H-AVP binding to pup CG membranes was 0.9±0.1 nM, while the adult CG was 5.7±1.0 nM. The pharmacological specificity of 3H-AVP binding sites in the pup and adult CG was similar, but differed markedly from the profile observed in adult septal membranes. The primary specificity difference between the pup CG and septum was the reduced potency of certain V1 receptor antagonists. In competition experiments the CG binding site showed a reduced affinity for the V1 antagonist, [d(CH2)5, Tyr(Me)]AVP. This reduced affinity for the V1 antagonist was also documented autoradiographically using 3H-[d(CH2)5, Tyr(Me)]AVP. The data suggest that the 3H-AVP binding site expressed in the pup CG is not identical to the V1 type receptor present in the periphery and brain of the adult rat. 相似文献
13.
Hazem A. Hassan Hansen M. Hsiung Xing-Yue Zhang Dennis P. Smith David L. Smiley Mark L. Heiman 《Peptides》1995,16(8):1469-1473
To study structure-activity relationships of growth hormone-releasing hormone (GHRH), a competitive binding assay was developed using cloned porcine adenopituitary GHRH receptors expressed in human kidney 293 cells. Specific binding of [His1,125I-Tyr10,Nle27]hGHRH(1–32)-NH2 increased linearly with protein concentration (10–45 μg protein/tube). Binding reached equilibrium after 90 min at 30°C and remained constant for at least 240 min. Binding was reversible to one class of high-affinity sites (Kd = 1.04 ± 0.19 nM, Bmax = 3.9 ± 0.53 pmol/mg protein). Binding was selective with a rank order of affinity (IC50) for porcine GHRH (2.8 ± 0.51 nM), rat GHRH (3.1 ± 0.69 nM), [N-Ac-Tyr1,
-Arg2]hGHRH(3–29)-NH2 (3.9 ± 0.58 nM), and [
-Thr7]GHRH(1–29)-NH2 (189.7 ± 14.3 nM), consistent with their binding to a GHRH receptor. Nonhydrolyzable guanine nucleotides inhibited binding. These data describe a selective and reliable method for a competitive GHRH binding assay that for the first time utilizes rapid filtration to terminate the binding assay. 相似文献
14.
Gino Giannaccini Laura Betti Massimo Correani Marco Dini Laura Giusti Antonio Lucacchini 《Neurochemistry international》1997,31(6):781-787
The distribution and the pharmacological properties of the binding of the benzodiazepine receptor antagonist [3H]-Ro 15–1788 (8-fluoro-3-carboethoxy-5,6-dihydro-5-methyl-6-oxo-4H imidazol [1,5-a] 1,4 benzodiazepine) were compared in some brain membranes of the saltwater teleost fish, Mullus surmuletus: only a single population of [3H]-Ro 15–1788 binding sites was detected. The binding was saturable and reversible with a high affinity, revealing a significant population of binding sites (Kd value of 2.1 ± 0.2 nM and Bmax value of 1400-900 fmol mg−1 of protein, depending on fish length). The highest concentration of benzodiazepine recognition sites labelled with [3H]-Ro 15–1788 was present in the optic lobe and the olfactory bulb and the lowest concentration was found in the medulla oblongata, cerebellum and spinal cord. In order to explore behavioural selectivity as a consequence of multiple receptor subtypes, six benzodiazepine receptor ligands, flunitrazepam (5-(2-fluoro-phenyl)-1,3,dihydro-1-methyl-7-nitro-2H-1,4-benzodiazepine-2-one), alpidem, (N,N-dipropyl-6-chloro-2-(4-chlorophenyl) imidazo [1,2-a] pyridine-3-acetamide) zolpidem {N,N,6, trimethyl-2-(4-methyl-phenyl) imidazo [1,2-a] pyridine-3-acetamide hemitartrate}, methyl β carboline-3-carboxylate (βCCM), Ro 15–1788 and Ro 5–4864 (4′-chlorodiazepam), were tested in vitro by binding of [3H]-Ro 15–1788 to membrane preparations from various brain areas of Mullus surmuletus. Displacement studies showed a similar rank order of efficacy of various unlabelled ligands. In all regions of the brain and in the spinal cord, GABA potentiate [3H]-flunitrazepam binding in a similar order, suggesting that the BDZ recognition sites are part of the GABAA receptor structure. These results suggest that central-type benzodiazepine receptors are present in one class of benzodiazepine binding sites in the saltwater teleost fish brain of Mullus surmuletus (type I-like). Here we report initial evidence of homogeneity of subtypes of central benzodiazepine receptors in the spinal cord of the saltwater teleost fish, Mullus surmuletus. 相似文献
15.
Q. Ni Heng Xu John S. Partilla Brian R. De Costa Kenner C. Rice Richard B. Rothman 《Peptides》1993,14(6):1279-1293
Recent studies from our laboratory resolved two subtypes of the κ2 binding site, termed κ2a and κ2b, using guinea pig, rat, and human brain membranes depleted of μ and δ receptors by pretreatment with the site-directed acylating agents BIT (μ-selective) and FIT (δ-selective). 6β-Iodo-3,14-dihydroxy-17-cyclopropylmethyl-4,5-epoxymorphinan (IOXY), an opioid antagonist that has high affinity for κ2 sites, was radioiodinated to maximum specific activity (2200 Ci/mmol) and purified by high pressure liquid chromotography and used to characterize multiple κ2 binding sites. The results indicated that [125I]IOXY, like [3H]bremazocine, selectively labels κ2 binding sites in rat brain membranes pretreated with BIT and FIT. Using 100 nM [
-Ala2-MePhe4,Gly-ol5]enkephalin to block [125I]IOXY binding to the κ2b site, two subtypes of the κ2a binding site were resolved, both in the absence and presence of 50 μM 5′-guanylyimidodiphosphate. Viewed collectively, these results provide further evidence for heterogeneity of the κ opioid receptor, which may provide new targets for drug design, synthesis, and therapeutics. 相似文献
16.
Vasoactive intestinal peptide (VIP) bound with high affinity (Kd 0.13 nmol/l) to receptors on the human glioma cell line U-343 MG Cl 2:6. The receptors bound the related peptides helodermin. PHM and secretin with 10, 400 and 5000 times lower affinity, respectively. Deamidated VIP (VIP-COOH) and [des-His1]VIP bound with 10 and 100 times lower affinity. The fragment VIP(7–28) displaced 25% of the receptor-bound 125I-VIP whereas VIP(16–28) and VIP(1–22-NH2) were inactive. The binding of 125I-VIP could be completely inhibited by 10 μmol/l of the antagonists [N-Ac-Tyr1,D-Phe2]GRF(1–29)-NH2, [pCl-D-Phe6,Leu17]VIP and VIP(10–28); in contrast, the antagonist L-8-K was inactive. Affinity labeling showed that VIP bound to proteins with Mr's of 75 kDa, 66 kDa and 50 kDa, respectively. Following binding, the peptide was rapidly internalized, and at steady-state only 20% of cell-associated 125I-VIP was bound to receptors on the cell surface. The internalized 125I-VIP was completely degraded to 125I-tyrosine which was released from the cells. Degradation of internalized 125I-VIP was significantly reduced by chloroquine phenantroline and pepstatin-A. Surface binding and internalization of 125I-VIP was increased 3 times by phenantroline, and pepstatin-A caused a 5 times increase in surface binding. Chloroquine reduced surface-bound 125I-VIP, but caused retention of internalized 125I-VIP. 相似文献
17.
Functional binding sites for [125I]IAPP and [125I]CGRP were solubilized from rat lung membranes with CHAPSO (10 mM). Rat IAPP had a higher affinity (Ki = 22.9 nM) for [125I]IAPP binding and rat CGRP (Ki = 0.904 nM) had a higher affinity for [125I]CGRP binding over related peptides. [125I]IAPP binding was unaffected by GTPγS, but [125I]CGRP binding was 50% inhibited, indicating solubilization of a G-protein-receptor complex for CGRP but not IAPP binding. Wheat germ agglutinin affinity columns gave a 25-fold purification of IAPP binding sites, but no CGRP binding sites were eluted from the column, indicating different patterns of glycosylation of the two sites. 相似文献
18.
19.
To investigate receptor selectivity and possible species selectivity of a number of NPY analogues and fragments, receptor binding studies were performed using cell lines and membranes of several species. NPY displays 4–25-fold higher affinity for the Y2 receptor than for the Y1 receptor. The affinity of [Leu31,Pro34]NPY is 7–60-fold higher for the Y1 receptor when compared with the Y2 subtype. Species selectivity within the Y2 receptors is demonstrated by PYY(3–36), NPY(2–36), NPY(22–36), and NPY(26–36). It is shown that NPY(22–36) is species selective for the human Y2 subtype (Ki of 0.3 nM) compared with the rabbit and rat Y2 receptor (Ki of 2 and 10 nM, respectively). PYY(3–36) displays highest affinity for the human and rabbit Y2 subtype (Ki of 0.03 and 0.17 nM). The screening of NPY analogues and fragments revealed that highest affinity for the human Y2 receptor is shown by NPY(2–36) and PYY(3–36). In addition, PYY(3–36) and NPY(2–36) are not only subtype selective, but also species selective. 相似文献
20.
M. Zeggari N. Viguerie Ch. Susini J. P. Esteve N. Vaysse J. Rivier E. Wunsch A. Ribet 《Peptides》1986,7(6):953-959
Somatostatin binding to guinea pig pancreatic acinar cell plasma membranes was characterized with an iodinated stable analog of somatostatin 28 (S28): 125I-[Leu8, DTrp22,Tyr25] S28. The binding was highly dependent on calcium ions. In 0.2 mM free Ca2+ medium, binding at 37°C was saturable, slowly reversible and exhibited a single class of high affinity binding sites (KD=0.05±0.01 nM, Bmax=157±33 fmol/mg protein). Dissociation of bound radioactivity occurred with biphasic kinetics. Rate of dissociation increased when dissociation was measured at a time before equilibrium binding was reached. In 30 nM free Ca2+ medium, binding affinity and maximal binding capacity were decreased by about 4-fold. Decreasing calcium concentrations increased the amount of rapidly dissociating form of the receptor. Somatostatin 14 antagonist, Des AA1,2[AzaAla4–5,DTrp8,Phe12–13]-somatostatin was active at the membrane level in inhibiting the binding. We conclude that using 125I-[Leu8,DTrp22,Tyr25]S28 as radioligand allows us to characterize a population of specific somatostatin receptors which are not different from those we previously described with the radioligand 125I-[Tyr11]-somatostatin. Somatostatin receptors could exist in two interconvertible forms. Calcium ions are an essential component in the regulation of the conformational change of somatostatin receptors. 相似文献