共查询到20条相似文献,搜索用时 31 毫秒
1.
Arnout P. Kalverda James Gowdy Gary S. Thompson Steve W. Homans Peter J. F. Henderson 《Molecular membrane biology》2014,31(4):131-140
Using the sugar transport protein, GalP, from Escherichia coli, which is a homologue of human GLUT transporters, we have overcome the challenges for achieving high-resolution [15N-1H]- and [13C-1H]-methyl-TROSY NMR spectra with a 52?kDa membrane protein that putatively has 12 transmembrane-spanning α-helices and used the spectra to detect inhibitor binding. The protein reconstituted in DDM detergent micelles retained structural and functional integrity for at least 48?h at a temperature of 25?°C as demonstrated by circular dichroism spectroscopy and fluorescence measurements of ligand binding, respectively. Selective labelling of tryptophan residues reproducibly gave 12 resolved signals for tryptophan 15N backbone positions and also resolved signals for 15N side-chain positions. For improved sensitivity isoleucine, leucine and valine (ILV) methyl-labelled protein was prepared, which produced unexpectedly well resolved [13C-1H]-methyl-TROSY spectra showing clear signals for the majority of methyl groups. The GalP/GLUT inhibitor forskolin was added to the ILV-labelled sample inducing a pronounced chemical shift change in one Ile residue and more subtle changes in other methyl groups. This work demonstrates that high-resolution TROSY NMR spectra can be achieved with large complex α-helical membrane proteins without the use of elevated temperatures. This is a prerequisite to applying further labelling strategies and NMR experiments for measurement of dynamics, structure elucidation and use of the spectra to screen ligand binding. 相似文献
2.
Nicotinic acetylcholine receptor is involved in acetylcholine regulating stomatal movement 总被引:4,自引:0,他引:4
Receptor,theprimarysiteofcellperceivingexternalsignals,controlsthecommunicationbetweencellsandenvironment[1].Acetylcholineisanimportantneurotransmitterinvolvedinthetransmissionofsignalsatjunctionsbetweennervesandbetweennerveandmuscle.Theactionofacetylch… 相似文献
3.
The high-affinity binding site for [3H] diazepam has been solubilized from rat brain using 0.5% Lubrol-PX. Using a polyethylene glycol (PEG)-γ-globulin assay, it has been possible to demonstrate solubilization of about 60% of the binding sites in a single step. The solubilized binding site possesses a KD of 11 nM for [3H] diazepam compared to approximately 4 nM for the membrane-bound form, and binding is to a single class of sites. The order of potency of benzodiazepines is identical for the solubilized receptor and the membrane-bound form. Binding of [3H] diazepam is temperature dependent and higher at 4° than 37°C. Both urea and guanidine-HC1 were capable of totally inhibiting binding, and this inhibition was partly reversible; neither sulfhydryl groups nor carbohydrate moieties seem to be important for binding. γ-Aminobutyric acid which enhanced [3H] diazepam binding to membrane fractions was without effect on the solubilized binding site. 相似文献
4.
David J. Chitwood 《Critical reviews in biochemistry and molecular biology》2013,48(4):273-284
AbstractElucidation of the molecular mechanisms that govern ligand-receptor recognition is essential to the rational design of specific pharmacological reagents. Whereas often the receptor and its binding site are the target of investigation, study of the ligand in its free and bound state can also reveal important information regarding this recognition process. Nuclear magnetic resonance (NMR) spectroscopy can be extremely useful for such studies. In this review, we discuss the attributes of NMR in the study of ligand receptor interactions. The cholinergic receptor and its binding to the neurotransmitter, acetylcholine, and cholinergic antagonists serve as a model system, illustrating the power of ligand analysis by NMR. The results discussed prove that the region of residues a 180–205 of the nicotinic acetylcholine receptor are an essential component of the cholinergic binding site and that ligand binding involves a positively charged hydrophobic motif. 相似文献
5.
Abstract— The acetylcholine receptor of the bovine adrenal medulla was studied by specific binding of [1251]α-bungarotoxin to membrane fractions and by perfusion of the isolated gland. The subcellular distribution of the acetylcholine receptor paralleled the distribution of the plasma membrane markers, acetylcholinesterase and calciumstimulated ATPase. The dissociation constant for the binding of α-bungarotoxin to a purified plasma membrane fraction was calculated from Scatchard plots to be 1.6 nM, with a concentration of 190 fmol of binding sites/mg of membrane protein. Correcting for recovery, this corresponds to 0.9 pmol acetylcholine receptor/g adrenal medulla. In decreasing order of effectiveness, d-tubocurarine, nicotine, acetylcholine, carbamylcholine, acetate plus choline, decamethonium, atropine and hexamethonium inhibited binding of α-bungarotoxin. Perfusion experiments showed the acetylcholine receptor to be entirely nicotinic. Stimulation by nicotine was inhibited by atropine and decamethonium, as well as by hexamethonium. Calculated dissociation constants for these antagonist-receptor interactions were in the range of 1 to 3 × 10?5 m. α-Bungarotoxin failed to inhibit nicotine-stimulated catecholamine release in the perfused adrenal, most likely because of its limited diffusion into the gland. 相似文献
6.
《Journal of receptor and signal transduction research》2013,33(3):369-387
AbstractBinding of (125I) iodocyanopindolol (ICYP) and (3H) CGP-12177 to rat brain homogenates was characterized and compared. ICYP was shown to bind to both ß-adrenergic and serotonin1B (5HT1B) receptors whereas (3H)CGP-12177 only labelled the first ones. The addition of 10 μM serotonin (5HT) prevented ICYP binding to 5HT receptors and under these experimental conditions both ligands labelled a similar total number of ß-adrenoceptors in the different rat brain regions. ICYP displayed a higher affinity for cerebellar (mainly ß2-subtype) than for cerebral cortex ß-adrenoceptors (mainly ß-subtype) suggesting a subtype selectivity. A multiple displacement binding approach using CGP-20712A, a ß1-subtype ligand, as competitor revealed a 2.6 fold selectivity of ICYP for the ß2-adrenoceptor subtype. On the other hand, (3H)CGP-12177 binds only to ß-adrenoceptors and is not subtype selective in the rat brain homogenate. Considering both its high specificity and its lack of subtype selectivity (3H)CGP-12177 seems to be a more suitable ligand than ICYP to non-selectively label ß-adrenoceptors in rat brain. 相似文献
7.
The parathyrin receptor in renal cortex has been investigated by studying the binding of 125I-labelled parathyrin, or of unlabelled parathyrin detected with 125I-labelled antibodies, to a partially purified plasma membrane fraction. The kinetics of hormone uptake demonstrated a biphasic response in both systems at 22 °C but this phenomenon was not detectable at 37 °C. Specific displacement of lactoperoxidase labelled 125I-labelled parathyrin occurred with 8 ng unlabelled bovine parathyrin. The apparent affinity constant was and the apparent binding capacity of the membranes 1.25 pmol/mg protein. Using the labelled antibody technique the receptor showed maximal binding at pH 7.0–7.5. As little as 80 pg bovine parathyrin produced a significant increase in binding of labelled anti-bovine parathyrin antibody and saturation of binding sites was demonstrated at 2.5 pmol/mg protein. Oxidized hormone showed undetectable binding. Treatment of membranes with phospholipases A or D, or Trypsin greatly reduced subsequent hormone binding. Prior incubation of membranes with 1–34 synthetic parathyrin decreased the binding of intact hormone whereas gastrin, insulin and glueagon had no effect. Growth hormone and calcitonin slightly increased parathyrin binding. 相似文献
8.
A reported loss in the binding capacity to ConA of thawed human peripheral blood lymphocytes has been investigated using two methods. With acetyl-3H ConA there was an apparent loss in the total binding of ConA to 2 × 105 dye-excluding cells thawed from liquid nitrogen, after cooling with a two-step procedure of 10 min at ?26 °C in 5% DMSO. Using the same cooling method, this apparent loss of binding capacity was not confirmed when a Fluorescence Activated Cell Sorter was used to measure the binding of fluorescent labelled ConA to thawed cells that are shown to be within the light scatter range of unfrozen lymphocytes. This second method, therefore, shows that a large population of lymphocytes can be recovered after thawing without any loss of receptors for ConA. The loss of binding measured by the radioactive method may be due to damaged lymphocytes and also to the loss of the small numbers of residual granulocytes. 相似文献
9.
The beta-adrenoceptors of intact human lymphocytes were investigated by binding assays with a radiolabeled β-adrenergic antagonists, (?)3H-dihydroalprenolol (DHA). Results shown are 1.48 ± 0.57 × 104 binding sites for the ligand per cell. Binding of (?)3H-DHA to the membrane fractions of human lymphocytes was studied for comparison. The affinity of (?)3H-DHA for membrane was similar to that for whole lymphocytes, but the binding to the membranes was inhibited more strongly by β-adrenergic agonist. Moreover, the cytoplasmic fraction of human lymphocytes decreased the inhibition by (?)-isoproterenol of (?)3H-DHA binding to the membrane fraction. The results imply that the cytoplasmic fraction of human lymphocytes lowered the affinity of (?)-isoproterenol to (?)3H-DHA binding sites and left no effect on the binding of the β-adrenergic antagonist to the sites. 相似文献
10.
William M. Moore Robert N. Brady 《Biochimica et Biophysica Acta (BBA)/General Subjects》1977,498(2):331-340
The pharmacological specificity of the binding of 125I-labeled α-bungarotoxin to a 1% Emulphogene BC-720 extract of a rat brain particulate fraction has been investigated. The extract contains a component which possesses the binding characteristics of a nicotinic acetylcholine receptor protein. The crude soluble acetylcholine receptor protein was purified by affinity chromatography utilizing the α-neurotoxin of Naja naja siamensis as ligand and 1.0 M carbamylcholine chloride as eluant. A single, batch-wise, affinity chromatography procedure yields an average purification of 510-fold. When this purified material is treated a second time by affinity chromatography, purification as high as 12 600-fold has been obtained. Binding of 125I-labeled α-bungarotoxin to this purified acetylcholine receptor protein is saturable with a Kd of 1·10?8 M. Nicotine and acetylcholine iodide at concentrations of 10?5 M inhibit 125I-labeled toxin-acetylcholine receptor protein complex formation by 41 and 61% respectively. At 10?4 M, carbamylcholine chloride and (+)-tubocurarine chloride give respectively 52 and 82% inhibition. Eserine sulfate and atropine sulfate have no effect on complex formation at a concentration of 10?4 M. These data support the isolation of partially purified nicotinic acetylcholine receptor protein. 相似文献
11.
Ostuni MA Tumilasci OR Péranzi G Cardoso EM Contreras LN Arregger AL Papadopoulos V Lacapere JJ 《Biology of the cell / under the auspices of the European Cell Biology Organization》2008,100(7):427-439
Background information. TSPO (translocator protein), previously known as PBR (peripheral‐type benzodiazepine receptor), is a ubiquitous 18 kDa transmembrane protein that participates in diverse cell functions. High‐affinity TSPO ligands are best known for their ability to stimulate cholesterol transport in organs synthesizing steroids and bile salts, although they modulate other physiological functions, including cell proliferation, apoptosis and calcium‐dependent transepithelial ion secretion. In present study, we investigated the localization and function of TSPO in salivary glands. Results. Immunohistochemical analysis of TSPO in rat salivary glands revealed that TSPO and its endogenous ligand, DBI (diazepam‐binding inhibitor), were present in duct and mucous acinar cells. TSPO was localized to the mitochondria of these cells, whereas DBI was cytosolic. As expected, mitochondrial membrane preparations, which were enriched in TSPO, exhibited a high affinity for the TSPO drug ligand, 3H‐labelled PK 11195, as shown by Bmax and Kd values of 10.0±0.5 pmol/mg and 4.0±1.0 nM respectively. Intravenous perfusion of PK 11195 increased the salivary flow rate that was induced by muscarinic and α‐adrenergic agonists, whereas it had no effect when administered alone. Addition of PK 11195 also increased the K+, Na+, Cl− and protein content of saliva, indicating that this ligand modulated secretion by acini and duct cells. Conclusions. High‐affinity ligand binding to mitochondrial TSPO modulates neurotransmitter‐induced salivary secretion by duct and mucous acinar cells of rat submandibular glands. 相似文献
12.
Kun Zhao Xue Wang Hing C. Wong Robert Wohlhueter Michael P. Kirberger Guantao Chen Jenny J. Yang 《Proteins》2012,80(12):2666-2679
Identifying Ca2+‐binding sites in proteins is the first step toward understanding the molecular basis of diseases related to Ca2+‐binding proteins. Currently, these sites are identified in structures either through X‐ray crystallography or NMR analysis. However, Ca2+‐binding sites are not always visible in X‐ray structures due to flexibility in the binding region or low occupancy in a Ca2+‐binding site. Similarly, both Ca2+ and its ligand oxygens are not directly observed in NMR structures. To improve our ability to predict Ca2+‐binding sites in both X‐ray and NMR structures, we report a new graph theory algorithm (MUGC) to predict Ca2+‐binding sites. Using carbon atoms covalently bonded to the chelating oxygen atoms, and without explicit reference to side‐chain oxygen ligand co‐ordinates, MUGC is able to achieve 94% sensitivity with 76% selectivity on a dataset of X‐ray structures composed of 43 Ca2+‐binding proteins. Additionally, prediction of Ca2+‐binding sites in NMR structures was obtained by MUGC using a different set of parameters, which were determined by the analysis of both Ca2+‐constrained and unconstrained Ca2+‐loaded structures derived from NMR data. MUGC identified 20 of 21 Ca2+‐binding sites in NMR structures inferred without the use of Ca2+ constraints. MUGC predictions are also highly selective for Ca2+‐binding sites as analyses of binding sites for Mg2+, Zn2+, and Pb2+ were not identified as Ca2+‐binding sites. These results indicate that the geometric arrangement of the second‐shell carbon cluster is sufficient not only for accurate identification of Ca2+‐binding sites in NMR and X‐ray structures but also for selective differentiation between Ca2+ and other relevant divalent cations. © Proteins 2012. © 2012 Wiley Periodicals, Inc. 相似文献
13.
Neville N. Osborne 《Cellular and molecular neurobiology》1981,1(2):167-174
The binding of3H-ADTN, a potent dopamine receptor agonist, to crude membrane preparations of bovine retina was studied, using a filtration method to isolate membrane-bound ligand. Specific binding was found to be saturable and occurred at a single binding site with an affinity constant of 7.3 nM. Binding was sodium-independent, slightly enhanced by Triton X-100 treatment, but drastically reduced by both trypsin and sodium laurylsulphate. The binding sites demonstrated a high degree of pharmacological specificity, with dopamine, apomorphine, and epinine being potent displacers of3H-ADTN. A higher degree of3H-ADTN binding was associated with subcellular fractions enriched with conventional synaptosomes rather than with fractions enriched with photoreceptor synaptosomes. 相似文献
14.
Ai Niitsu Ayako Egawa Keisuke Ikeda Kazuo Tachibana Toshimichi Fujiwara 《Bioorganic & medicinal chemistry》2018,26(21):5644-5653
The multi-step ligand action to a target protein is an important aspect when understanding mechanisms of ligand binding and discovering new drugs. However, structurally capturing such complex mechanisms is challenging. This is particularly true for interactions between large membrane proteins and small molecules. One such large membrane of interest is Nav1.4, a eukaryotic voltage-gated sodium channel. Domain 4 segment 6 (D4S6) of Nav1.4 is a transmembrane α-helical segment playing a key role in channel gating regulation, and is targeted by a neurotoxin, veratridine (VTD). VTD has been suggested to exhibit a two-step action to activate Nav1.4. Here, we determine the NMR structure of a selectively 13C-labeled peptide corresponding to D4S6 and its VTD binding site in lipid bilayers determined by using magic-angle spinning solid-state NMR. By 13C NMR, we obtain NMR structural constraints as 13C chemical shifts and the 1H-2H dipolar couplings between the peptide and deuterated lipids. The peptide backbone structure and its location with respect to the membrane are determined under the obtained NMR structural constraints aided by replica exchange molecular dynamics simulations with an implicit membrane/solvent system. Further, by measuring the 1H-2H dipolar couplings to monitor the peptide-lipid interaction, we identify a VTD binding site on D4S6. When superimposed to a crystal structure of a bacterial sodium channel NavRh, the determined binding site is the only surface exposed to the protein exterior and localizes beside the second-step binding site reported in the past. Based on these results, we propose that VTD initially binds to these newly-determined residues on D4S6 from the membrane hydrophobic domain, which induces the first-step channel opening followed by the second-step blocking of channel inactivation of Nav1.4. Our findings provide new detailed insights of the VTD action mechanism, which could be useful in designing new drugs targeting D4S6. 相似文献
15.
Dmitry M. Lesovoy Ekaterina N. Lyukmanova Mikhail A. Shulepko Mikhail P. Kirpichnikov Alexander S. Arseniev 《Biophysical journal》2009,97(7):2089-2097
The action of three-finger snake α-neurotoxins at their targets, nicotinic acetylcholine receptors (nAChR), is widely studied because of its biological and pharmacological relevance. Most such studies deal only with ligands and receptor models; however, for many ligand/receptor systems the membrane environment may affect ligand binding. In this work we focused on binding of short-chain α-neurotoxin II (NTII) from Naja oxiana to the native-like lipid bilayer, and the possible role played by the membrane in delivering the toxin to nAChR. Experimental (NMR and mutagenesis) and molecular modeling (molecular-dynamics simulation) studies revealed a specific interaction of the toxin molecule with the phosphatidylserine headgroup of lipids, resulting in the proper topology of NTII on lipid bilayers favoring the attack of nAChR. Analysis of short-chain α-neurotoxins showed that most of them possess a high positive charge and sequence homology in the lipid-binding motif of NTII, implying that interaction with the membrane surrounding nAChR may be common for the toxin family. 相似文献
16.
A specific interaction of human erythrocyte catalase with the inner surface of the red cell membrane was demonstrated. The dependency of catalase affinity on pH and ionic strength implies that the interaction is dominated by electrostatic forces. Scatchard analysis of the binding at pH 6.0 and 5 mm Mes buffer reveals a single class of approximately 106 binding sites/ghost with an association constant of 2.5 × 107m?1. The membrane-bound catalase retains its enzymatic activity. Competition binding studies of catalase and other proteins known to associate with the membrane inner surface were carried out. It was found that the binding of catalase is inhibited by aldolase, glyceraldehyde-3-phosphate dehydrogenase as well as by hemoglobin. The advantage of membrane-bound catalase in protection of the cell membrane against peroxidative damages is discussed. 相似文献
17.
Ronald J. Lukasiewicz Edward L. Bennett 《Biochimica et Biophysica Acta (BBA)/General Subjects》1978,544(2):294-308
High-affinity, specific binding of radiolabeled α-bungarotoxin to particulate fractions derived from rat brain shows saturability (Bmax ≈ 37fmol/mg, KDapp = 1.7 nM) and insensitivity to ionic strength, and is essentially irreversible (Kon = 5 · 106 min?1 · mol?1; Kdisplacement = 1.9 · 10?4 min?1, τ1/2 = 62 h). Subcellular distribution of specific sites is consistent with their location on synaptic junctional complex and post-synaptic membranes. These membrane-bound binding sites exhibit unique sensitivity to cholinergic ligands; pretreatment of membranes with cholinergic agonists (but not antagonists) induces transformation of α-bungarotoxin binding sites to a high affinity form toward agonist. The effect is most marked for the natural agonist, acetylcholine. These results strongly support the notion that the entity under study is an authentic nicotinic acetylcholine receptor. 相似文献
18.
Adenosine binding sites on 108CC15 neuroblastoma × glioma hybrid cells and rat brain membranes were investigated using [3H]adenosine as labelled ligand. Both the hybrid cells and brain membranes were found to have a high affinity binding site, Kd 0.8 and 3 nM respectively. The same ligand was used to demonstrate two lower affinity binding sites on brain membranes, Kds 1.4 and 29.1 μM and a single low affinity site on the hybrid cells, Kd 2.6 μM. Structure activity studies of the low affinity binding site on hybrid cells showed this to be an ‘R’ adenosine receptor of the A2 subtype. It is concluded that [3H]adenosine can be used to demonstrate both high and low affinity binding sites and that 108CC15 hybrid cells provide a valuable system for studying adenosine receptors. 相似文献
19.
《生物化学与生物物理学报:生物膜》2015,1848(2):712-720
The surrounding environment has significant consequences for the structural and functional properties of membrane proteins. While native structure and function can be reconstituted in lipid bilayer membranes, the detergents used for protein solubilization are not always compatible with biological activity and, hence, not always appropriate for direct detection of ligand binding by NMR spectroscopy. Here we describe how the sample environment affects the activity of the outer membrane protein Ail (attachment invasion locus) from Yersinia pestis. Although Ail adopts the correct β-barrel fold in micelles, the high detergent concentrations required for NMR structural studies are not compatible with the ligand binding functionality of the protein. We also describe preparations of Ail embedded in phospholipid bilayer nanodiscs, optimized for NMR studies and ligand binding activity assays. Ail in nanodiscs is capable of binding its human ligand fibronectin and also yields high quality NMR spectra that reflect the proper fold. Binding activity assays, developed to be performed directly with the NMR samples, show that ligand binding involves the extracellular loops of Ail. The data show that even when detergent micelles support the protein fold, detergents can interfere with activity in subtle ways. 相似文献
20.
—Cortex slices of rat brain were incubated with glucose mixed-labelled with 3H and 14C in the 6-position and the 3H/14C ratios of lactate, acetate, citrate and acetylcholine were determined. The values obtained were: lactate 0·95, acetate 0·85, citrate 0·65 and acetylcholine 0·67 when expressed in relation to a glucose 3H/14C ratio of 1·00. When brain slices were incubated with [2-14C, 2-3H]acetate in the presence of unlabelled glucose, labelled acetylcholine was formed with a 3H/14C ratio not significantly different from the labelled substrate. The results indicate that citrate is a precursor to the acetyl moiety of acetylcholine. 相似文献