Molecular identification of nicotinic acid receptor

Nicotinic acid and its derivative, Acipimox, have been widely used in the treatment of hyperlipidemia. Pharmacological studies have demonstrated that they exert the beneficial effect through the activation of a Gi-protein-coupled receptor on adipocyte, which has remained elusive to date. Here we show that a novel GPCR, designated HM74b because of its high similarity to HM74, is a receptor for nicotinic acid. HM74b mRNA is found in human, murine, and rat adipose tissues. Nicotinic acid and Acipimox inhibit forskolin-stimulated intracellular cAMP accumulation in human HM74b-expressing cells and activate GTP gamma S binding in a dose-dependent manner. [3H]Nicotinic acid specifically binds to HM74b-expressing membrane and its binding is replaced by Acipimox. This finding will open a new phase of research on the physiological role of nicotinic acid and will be a clue to develop novel antihyperlipidemic drugs.     

Ethanol increases agonist affinity for nicotinic receptors from Torpedo

The presence of ethanol increases the apparent affinity with which acetylcholine and carbamylcholine elicit 86Rb+ flux from Torpedo nicotinic acetylcholine receptor-rich vesicles at 4 degrees C. Affinity increased exponentially with ethanol concentration, reaching nearly 200-fold by 3.0 M ethanol without sign of saturation. At submaximal agonist concentrations 50-100 mM ethanol enhanced flux by 15-35%, but the maximum agonist-induced flux was unaffected in quenched-flow assays. The effect was independent of the agonist and of the time over which flux was measured (5 ms to 10 s), indicating that ethanol acts before agonist-induced desensitization occurs. Ethanol also caused an increase in the apparent affinity with which acetylcholine caused fast desensitization. This affinity increase was equal to that for flux-response curves, but the maximum fast desensitization rate was increased 50% at 0.5 M ethanol. This was the most pronounced of ethanol's actions and has not been reported before. Prolonged preincubation with 1.0 M ethanol alone reduced agonist-induced flux activity by only 25%. The rate of agonist-induced slow desensitization was also increased, but neither of these effects was as marked as those on fast desensitization and cation flux.     

Subunit structure of the high and low affinity human interleukin-15 receptors

Radio-iodinated cytokines and monoclonal antibodies directed at the IL-2R beta- and gamma-chains were used to analyze the structure of the cell-surface IL-15 and IL-2 receptors expressed by the human lymphoma cell clone YT-2C2. YT-2C2 cells are IL-2R alpha negative and express IL-2R gamma (15,000 molecules/cell) in excess of IL-2R beta (11,000 molecules/cell). Accordingly, they display a number of beta/gamma complexes of intermediate affinity for IL-2 and IL-15 which is equivalent to the number of beta-chains. Both cytokines compete for binding to this beta/gamma complex. There are about 800 high affinity IL-15 receptors, suggesting the presence of a similar number of IL-15R alpha-chains. Within the common intermediate affinity beta/gamma-complex, the anti-beta-chain A41 mAb defines an epitope which is similarly engaged in IL-2 and IL-15 binding, whereas the anti-beta-chain 284 mAb defines an epitope which does not display similar interaction with either cytokines. Thus, although IL-2 and IL-15 compete for binding to this beta/gamma-complex, they do not use similar binding areas. Cross-linking and immunoprecipitation experiments have shown that the high affinity IL-15 receptors comprises IL-2R beta/gamma, in association with IL-15R alpha and that the three chains can be efficiently cross-linked to IL-15 and co-immunoprecipitated. Contrary to the intermediate affinity situation, high affinity IL-15 binding and subunit cross-linking were not affected by excess amounts of IL-2, A41 or 284 mAb, suggesting that when engaged in the IL-15 high affinity complex, the beta- and gamma-chains adopt different conformations, at least with respect to IL-15 binding. Finally, we provide evidence for the participation of a novel 35 kDa component within the high affinity structure. This component is immunoprecipitated with anti-IL-2R gamma mAb but not with anti-IL-2R beta mAb and might correspond to a truncated form of IL-2R gamma-chain.     

一株高效催化烟酸羟化反应菌株的筛选及鉴定

为开发生物法生产6-羟基烟酸,利用快速筛选方法从多个地区的土壤中筛到了一株能高效羟化烟酸为6-羟基烟酸的菌株BK-1,可以用菌体催化得到高纯度的6-羟基烟酸.通过对菌株BK-1形态观察和16S rDNA同源性分析,构建了系统发育树,BK-1菌株与Pseudomonas plecoglos-sicida形成一个类群,且有99.7%的同源性,初步判定为假单胞菌属(Pseudomonas).     

Bioisosteric replacement strategy for the synthesis of 1-azacyclic compounds with high affinity for the central nicotinic cholinergic receptors

Bioisosteric replacement of the isoxazole heterocycle in (3-methyl-5-isoxazolyl)methylene-azacyclic compounds with pyridine, oxadiazole, or an acyl group resulted in ligands with high to moderate affinity for the central nicotinic cholinergic receptors (IC50 = 2.0 to IC50 > 1000 nM) labeled by [3H]methylcarbamylcholine. Additionally, further support of an important distance parameter for high-affinity nicotinic compounds has been provided.     

Direct identification of the high and low affinity calcium binding sites of troponin-C

Covalent labelling of the calcium ligands of intact troponin-C (0.1 M KCl, pH 6.0) with [³H] -ethanolamine, at various ratios of calcium to troponin-C followed by analysis of the two separated cleavage products, shows that the first and second calcium binding sites of the sequence are the low affinity sites and that the third and fourth sites are the high affinity or structure defining sites of troponin-C.     

Identification of 9-fluoro substituted (-)-cytisine derivatives as ligands with high affinity for nicotinic receptors

(-)-9-Fluorocytisine, (-)-9-methylcytisine and (-)-9-trifluoromethylcytisine were synthesized from the natural product (-)-cytisine. 9-Methyl and 9-trifluoromethyl cytisines display a remarkable affinity at the α(4)β(2) nicotinic receptor subtype (0.2 nM) with a high selectivity versus the α(7) nAChR subtype. Comparison of the affinity values suggests that the size of the substituent at the 9 position of (-)-cytisine seems more important than electronic factors for efficient binding and selectivity at α(4)β(2) nAChRs.     

3H]-Methyllycaconitine: a high affinity radioligand that labels invertebrate nicotinic acetylcholine receptors

Nicotinic acetylcholine receptors (nAChR) of insect and other invertebrates are heterogeneous and new tools are needed to dissect their multiplicity. [(3)H]-Methyllycaconitine ([(3)H]-MLA) is a novel radioligand which is a potent antagonist at vertebrate alpha7-type nAChR. Putative invertebrate nAChR of the aphid Myzus persicae, the moths Heliothis virescens and Manduca sexta, the fly Lucilia sericata, and the squid Loligo vulgaris were investigated in radioligand binding studies with [(3)H]-MLA. Saturable binding was consistent with a single class of high affinity binding sites for each of these invertebrates, characterised by a dissociation constant, K(d), of approximately 1 nM and maximal binding capacities, B(max), between 749 and 1689 fmol/mg protein for the insects and 14,111 fmol/mg protein for squid. [(3)H]-MLA binding to M. persicae membranes was characterised in more detail. Kinetic analysis demonstrated rapid association in a biphasic manner and slow, monophasic dissociation. Displacement studies demonstrate the nicotinic character of [(3)H]-MLA binding sites. Data for all nicotinic ligands, except MLA itself, are consistent with displacement from a high and a low affinity site, indicating that displacement is occurring from two or more classes of nicotinic binding site that are not distinguished by MLA itself. Autoradiographic analysis of the distribution of [(3)H]-MLA binding sites in Manduca sexta shows discrete labelling of neuropil areas of the optic and antennal lobes.     

4-Bromoacetamidoprocaine: An affinity ligand for brain muscarinic and nicotinic cholinergic receptors

This study describes the synthesis, receptor binding characteristics, and some behavioral effects of p-bromoacetamidoprocaine (BAP), a new affinity ligand for brain muscarinic and nicotinic cholinergic receptors. The reversible binding of [³H]QNB to rat brain membranes was inhibited in a concentration dependent and saturable manner by both procaine and BAP, with Ki values of 4×10^–6 and 3×10^–7 M, respectively, and complete inhibition at 1×10^–5 M. Both procaine and BAP, although at much concentrations, inhibited the binding of [³H]methylcarbamylcholine in a concentration dependent manner, with Ki values of 5×10^–5 and 1×10^–5 M, respectively, and complete inhibition for both at 1×10^–3 M. Plots of the % irreversible inhibition of [³H]QNB, [³H]nicotine, and [³H]MCC vs [BAP] yielded Ki values of 7×10^–8, 1×10^–4, and 6×10^–5 M, respectively. In behavioral studies BAP was able to antagonize the QNB-induced hyperactivity in mice; however, BAP did not appear to alter nicotine-induced seizure activity or other behavioral effects in mice. A plot of the time course of inhibition by BAP for [³H]QNB binding revealed that the inhibition was almost complete within 10 min exposure at 37°. The findings indicate that BAP is a useful affinity ligand for examining the biochemical and functional characteristics of brain cholinergic receptors, particularly the muscarinic which has an affinity near the nM concentration range.     

A synthetic weak neurotoxin binds with low affinity to Torpedo and chicken alpha7 nicotinic acetylcholine receptors.

Weak neurotoxins from snake venom are small proteins with five disulfide bonds, which have been shown to be poor binders of nicotinic acetylcholine receptors. We report on the cloning and sequencing of four cDNAs encoding weak neurotoxins from Naja sputatrix venom glands. The protein encoded by one of them, Wntx-5, has been synthesized by solid-phase synthesis and characterized. The physicochemical properties of the synthetic toxin (sWntx-5) agree with those anticipated for the natural toxin. We show that this toxin interacts with relatively low affinity (K(d) = 180 nm) with the muscular-type acetylcholine receptor of the electric organ of T. marmorata, and with an even weaker affinity (90 microm) with the neuronal alpha7 receptor of chicken. Electrophysiological recordings using isolated mouse hemidiaphragm and frog cutaneous pectoris nerve-muscle preparations revealed no blocking activity of sWntx-5 at microm concentrations. Our data confirm previous observations that natural weak neurotoxins from cobras have poor affinity for nicotinic acetylcholine receptors.     

Molecular determinants of high affinity binding to group III metabotropic glutamate receptors.

The amino-terminal domain containing the ligand binding site of the G protein-coupled metabotropic glutamate receptors (mGluRs) consists of two lobes that close upon agonist binding. In this study, we explored the ligand binding pocket of the Group III mGluR4 receptor subtype using site-directed mutagenesis and radioligand binding. The selection of 16 mutations was guided by a molecular model of mGluR4, which was based on the crystal structure of the mGluR1 receptor. Lysines 74 and 405 are present on lobe I of mGluR4. The mutation of lysine 405 to alanine virtually eliminated the binding of the agonist [(3)H]l-amino-4-phosphonobutyrate ([(3)H]l-AP4). Thus lysine 405, which is conserved in all eight mGluRs, likely represents a fundamental recognition residue for ligand binding to the mGluRs. Single point mutations of lysines 74 or 317, which are not conserved in the mGluRs, to alanine had no effect on agonist affinity, whereas mutation of both residues together caused a loss of ligand binding. Mutation of lysine 74 in mGluR4, or the analogous lysine in mGluR8, to tyrosine (mimicking mGluR1 at this position) produced a large decrease in binding. The reduction in binding is likely due to steric hindrance of the phenolic side chain of tyrosine. The mutation of glutamate 287 to alanine, which is present on lobe II and is not conserved in the mGluR family, caused a loss of [(3)H]l-AP4 binding. We conclude that the determinants of high affinity ligand binding are dispersed across lobes I and II. Our results define a microenvironment within the binding pocket that encompasses several positively charged amino acids that recognize the negatively charged phosphonate group of l-AP4 or the endogenous compound l-serine-O-phosphate.     

Importance of N- and C-terminal regions of gastrin-Gly for preferential binding to high and low affinity gastrin-Gly receptors

G17-Gly has been shown to stimulate the growth of DLD-1 human colon cancer cells in a biphasic manner via high and low affinity receptors. In the current study, the existence of heterogeneous receptor populations for G17-Gly on the HT-29 human colon cancer cell line was investigated. The effect of either N- or C-terminal peptide truncation on receptor binding and cell growth stimulation was also explored. [Leu15]G17-Gly bound to both high (nM) and low (microM) affinity sites on HT-29 cells. The peptide stimulated cell growth in a dose-dependent and biphasic manner with maximal stimulation at 10(-9) M peptide concentration, suggesting that, as in the case of DLD-1 cells, it is the high affinity receptor which is responsible for the growth-promoting effects. In contrast, G17(1-12) stimulated the growth of HT-29 cells in a sigmoidal fashion with an EC50 of 4.6x10(-9) M. Sequential N-terminal truncation of [Leu15]G17-Gly results in decreased binding to the high affinity G17-Gly receptor on DLD-1 cells. [Leu15]G17(11-17)Gly bound to the low affinity G17-Gly receptor with an affinity similar to that of the full sequence peptide but was unable to displace the radioligand from high affinity sites. G17(1-6)-NH2 was unable to displace [3H]G17-Gly from either site. These results suggest that the important residues for binding to the low affinity receptor are in the C-terminal region of the peptide while those required for interaction with the high affinity receptor lie further towards the N-terminus.     

一株烟酸羟基化转化菌株的筛选和鉴定

从南京地区的土壤中筛选到一株高效转化烟酸为 6_羟基烟酸的菌株NA_1。形态及生理生化特征测定结果表明 ,NA_1菌株与假单胞菌属 (Pseudomonas)中的恶臭假单胞菌 (P .putida)种的特征基本一致。测定了该菌株的16SrDNA序列并根据 16SrDNA构建了系统发育树 ;在系统发育树中 ,NA_1菌株与恶臭假单胞菌形成一个类群 ,序列同源性为 99%。因此将NA_1菌株鉴定为恶臭假单胞菌     

Syntheses of novel high affinity ligands for opioid receptors

A series of novel high affinity opioid receptor ligands have been made whereby the phenolic-OH group of nalbuphine, naltrexone methiodide, 6-desoxonaltrexone, hydromorphone and naltrindole was replaced by a carboxamido group and the furan ring was opened to the corresponding 4-OH derivatives. These furan ring ‘open’ derivatives display very high affinity for μ and κ receptors and much less affinity for δ. The observation that these target compounds have much higher receptor affinity than the corresponding ring ‘closed’ carboxamides significantly strengthens our underlying pharmacophore hypothesis concerning the bioactive conformation of the carboxamide group.     

Possible involvement of low and high affinity GABA receptors in the chloride influx into synaptosomes

Experiments carried out in the absence or presence of GABA using a synaptosomal fraction from which endogenous GABA was as far as possibly eliminated, seem to indicate that both GABA receptors are involved in the chloride channel opening. This hypothesis is supported by results obtained in the presence of GABA agonist (muscimol) or drugs which are related to the complex GABA receptor-ionophore (diazepam and phenobarbital).     

Critical residues influence the affinity and selectivity of alpha-conotoxin MI for nicotinic acetylcholine receptors.

The mammalian skeletal muscle acetylcholine receptor contains two nonequivalent acetylcholine binding sites, one each at the alpha/delta and alpha/gamma subunit interfaces. Alpha-Conotoxin MI, a 14-amino acid competitive antagonist, binds at both interfaces but has approximately 10(4) higher affinity for the alpha/delta site. We performed an "alanine walk" to identify the residues in alpha-MI that contribute to this selective interaction with the alpha/delta site. Electrophysiological measurements with Xenopus oocytes expressing normal receptors or receptors lacking either the gamma or delta subunit were made to assay toxin-receptor interaction. Alanine substitutions in most amino acid positions had only modest effects on toxin potency at either binding site. However, substitutions in two positions, proline-6 and tyrosine-12, dramatically reduced toxin potency at the high-affinity alpha/delta site while having comparatively little effect on low-affinity alpha/gamma binding. When tyrosine-12 was replaced by alanine, the toxin's selectivity for the high-affinity site (relative to that for the low-affinity site) was reduced from 45,000- to 30-fold. A series of additional amino acid substitutions in this position showed that increasing side chain size/hydrophobicity increases toxin potency at the alpha/delta site without affecting alpha/gamma binding. In contrast, when tyrosine-12 is diiodinated, toxin binding is nearly irreversible at the alpha/delta site but also increases by approximately 500-fold at the alpha/gamma site. The effects of position 12 substitutions are accounted for almost entirely by changes in the rate of toxin dissociation from the high-affinity alpha/delta binding site.     

Molecular mechanisms and binding site locations for noncompetitive antagonists of nicotinic acetylcholine receptors

Nicotinic acetylcholine receptors are pentameric proteins that belong to the Cys-loop receptor superfamily. Their essential mechanism of functioning is to couple neurotransmitter binding, which occurs at the extracellular domain, to the opening of the membrane-spanning cation channel. The function of these receptors can be modulated by structurally different compounds called noncompetitive antagonists. Noncompetitive antagonists may act at least by two different mechanisms: a steric and/or an allosteric mechanism. The simplest idea representing a steric mechanism is that the antagonist molecule physically blocks the ion channel. On the other hand, there exist distinct allosteric mechanisms. For example, noncompetitive antagonists may bind to the receptor and stabilize a nonconducting conformational state (e.g., resting or desensitized state), and/or increase the receptor desensitization rate. Barbiturates, dissociative anesthetics, antidepressants, and neurosteroids have been shown to inhibit nicotinic receptors by allosteric mechanisms and/or by open- and closed-channel blockade. Receptor modulation has proved to be highly complex for most noncompetitive antagonists. Noncompetitive antagonists may act by more than one mechanism and at distinct sites in the same receptor subtype. The binding site location for one particular molecule depends on the conformational state of the receptor. The mechanisms of action and binding affinities of noncompetitive antagonists differ among nicotinic receptor subtypes. Knowledge of the structure of the nicotinic acetylcholine receptor, the location of its noncompetitive antagonist binding sites, and the mechanisms of inhibition will aid the design of new and more efficacious drugs for treatment of neurological diseases.