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Similar Affinities (similar + affinity)
Selected AbstractsAuthentic interdomain communication in an RNA helicase reconstituted by expressed protein ligation of two helicase domainsFEBS JOURNAL, Issue 2 2007Anne R. Karow RNA helicases mediate structural rearrangements of RNA or RNA,protein complexes at the expense of ATP hydrolysis. Members of the DEAD box helicase family consist of two flexibly connected helicase domains. They share nine conserved sequence motifs that are involved in nucleotide binding and hydrolysis, RNA binding, and helicase activity. Most of these motifs line the cleft between the two helicase domains, and extensive communication between them is required for RNA unwinding. The two helicase domains of the Bacillus subtilis RNA helicase YxiN were produced separately as intein fusions, and a functional RNA helicase was generated by expressed protein ligation. The ligated helicase binds adenine nucleotides with very similar affinities to the wild-type protein. Importantly, its intrinsically low ATPase activity is stimulated by RNA, and the Michaelis,Menten parameters are similar to those of the wild-type. Finally, ligated YxiN unwinds a minimal RNA substrate to an extent comparable to that of the wild-type helicase, confirming authentic interdomain communication. [source] The uptake by cells of 2-arachidonoylglycerol, an endogenous agonist of cannabinoid receptorsFEBS JOURNAL, Issue 7 2001Tiziana Bisogno It is not yet clear if the endocannabinoid 2-arachidonoylglycerol (2-AG) is transported into cells through the same membrane transporter mediating the uptake of the other endogenous cannabinoid, anandamide (N -arachidonoylethanolamine, AEA), and whether this process (a) is regulated by cells and (b) limits 2-AG pharmacological actions. We have studied simultaneously the facilitated transport of [14C]AEA and [3H]2-AG into rat C6 glioma cells and found uptake mechanisms with different efficacies but similar affinities for the two compounds (Km 11.0 ± 2.0 and 15.3 ± 3.1 µm, Bmax 1.70 ± 0.30 and 0.24 ± 0.04 nmol·min,1·mg protein,1, respectively). Despite these similar Km values, 2-AG inhibits [14C]AEA uptake by cells at concentrations (Ki = 30.1 ± 3.9 µm) significantly higher than those required to either 2-AG or AEA to inhibit [3H]2-AG uptake (Ki = 18.9 ± 1.8 and 20.5 ± 3.2 µm, respectively). Furthermore: (a) if C6 cells are incubated simultaneously with identical concentrations of [14C]AEA and [3H]2-AG, only the uptake of the latter compound is significantly decreased as compared to that observed with [3H]2-AG alone; (b) the uptake of [14C]AEA and [3H]2-AG by cells is inhibited with the same potency by AM404 (Ki = 7.5 ± 0.7 and 10.2 ± 1.7 µm, respectively) and linvanil (Ki = 9.5 ± 0.7 and 6.4 ± 1.2 µm, respectively), two inhibitors of the AEA membrane transporter; (c) nitric oxide (NO) donors enhance the uptake of both [14C]AEA and [3H]2-AG, thus suggesting that 2-AG action can be regulated through NO release; (d) AEA and 2-AG induce a weak release of NO that can be blocked by a CB1 cannabinoid receptor antagonist, and significantly enhanced in the presence of AM404 and linvanil, thus suggesting that transport into C6 cells limits the action of both endocannabinoids. [source] Coupling Efficacy and Selectivity of the Human ,-Opioid Receptor Expressed as Receptor,G, Fusion Proteins in Escherichia coliJOURNAL OF NEUROCHEMISTRY, Issue 3 2000Laura Stanasila Abstract: Two constructs encoding the human ,-opioid receptor (hMOR) fused at its C terminus to either one of two G, subunits, G,o1 (hMOR-G,o1) and G,i2 (hMOR-G,i2), were expressed in Escherichia coli at levels suitable for pharmacological studies (0.4-0.5 pmol/mg). Receptors fused to G,o1 or to G,i2 maintained high-affinity binding of the antagonist diprenorphine. Affinities of the ,-selective agonists morphine, [D-Ala2,N -Me-Phe4,Gly5 -ol]enkephalin (DAMGO), and endomorphins as well as their potencies and intrinsic activities in stimulating guanosine 5,- O -(3-[35S]thiotriphosphate) ([35S]GTP,S) binding were assessed in the presence of added purified G,, subunits. Both fusion proteins displayed high-affinity agonist binding and agonist-stimulated [35S]GTP,S binding. In the presence of G,, dimers, the affinities of DAMGO and endomorphin-1 and -2 were higher at hMOR-G,i2 than at hMOR-G,o1, whereas morphine displayed similar affinities at the two chimeras. Potencies of the four agonists in stimulating [35S]GTP,S binding at hMOR-G,o1 were similar, whereas at hMOR-G,i2, endomorphin-1 and morphine were more potent than DAMGO and endomorphin-2. The intrinsic activities of the four agonists at the two fusion constructs were similar. The results confirm hMOR coupling to G,o1 and G,i2 and support the hypothesis of the existence of multiple receptor conformational states, depending on the nature of the G protein to which it is coupled. [source] Efficient synthesis and comparative studies of the arginine and N,,N, -dimethylarginine forms of the human nucleolin glycine/arginine rich domainJOURNAL OF PEPTIDE SCIENCE, Issue 1 2005Dr Sotir Zahariev Abstract The Gly- and Arg-rich C -terminal region of human nucleolin is a 61-residue long domain involved in a number of protein,protein and protein,nucleic acid interactions. This domain contains 10 aDma residues in the form of aDma-GG repeats interspersed with Phe residues. The exact role of Arg dimethylation is not known, partly because of the lack of efficient synthetic methods. This work describes an effective synthetic strategy, generally applicable to long RGG peptides, based on side-chain protected aDma and backbone protected dipeptide Fmoc-Gly-(Dmob)Gly-OH. This strategy allowed us to synthesize both the unmodified (N61Arg) and the dimethylated (N61aDma) peptides with high yield (,26%) and purity. As detected by NMR spectroscopy, N61Arg does not possess any stable secondary or tertiary structure in solution and N,,N, -dimethylation of the guanidino group does not alter the overall conformational propensity of this peptide. While both peptides bind single-stranded nucleic acids with similar affinities (Kd = 1.5 × 10,7M), they exhibit a different behaviour in ssDNA affinity chromatography consistent with the difference in pKa values. It has been previously shown that N61Arg inhibits HIV infection at the stage of HIV attachment to cells. This study demonstrates that Arg-dimethylated C -terminal domain lacks any inhibition activity, raising the question of whether nucleolin expressed on the cell-surface is indeed dimethylated. Copyright © 2004 European Peptide Society and John Wiley & Sons, Ltd. [source] Histidine-stimulated divalent metal uptake in human erythrocytes and in the erythroleukaemic cell line HEL.92.1.7THE JOURNAL OF PHYSIOLOGY, Issue 2 2004F. Oakley The uptake of 65Zn by human erythrocytes was investigated in the presence of high (40 mm) and low (5 mm) concentrations of histidine and 0,500 ,m cobalt, nickel, manganese and zinc. Varying concentrations of metal mono- and bis-histidine complexes will be formed and the inhibition of 65Zn uptake could be correlated with the calculated complex concentrations to investigate competition between metals. For each metal, the calculated concentrations of bis-histidine complex giving 50% inhibition of 65Zn uptake were similar at both 5 mm and 40 mm histidine. Manganese,bis-histidine appeared to have a much higher affinity for the binding site than the other metal,bis-histidine complexes, which had similar affinities to each other. Studies of the inhibition of histidine-stimulated 54Mn uptake by the addition of manganese confirmed that manganese,bis-histidine does act as a substrate for the transporter in a similar fashion to the other metals studied. In addition, human erythroleukaemic cells (HEL cells) were used as a model for erythroid precursor cells. l -histidine, but not d -histidine, stimulated 65Zn uptake in a saturable fashion. The other metals competed with zinc in a similar manner to that seen in erythrocytes, and the affinity for manganese,bis-histidine was much greater than for the bis-histidine complexes of the other three metals. Both the capacity for metal transport per cell, and the affinity of the transporter for the metal,bis-histidine complexes, were much greater in the HEL cells than in the erythrocyte. It is suggested that histidine-stimulated metal transport may play a role in the supply of metals to maturing erythroid cells. [source] Alpha-2 adrenoceptors are present in rat aorta smooth muscle cells, and their action is mediated by ATP-sensitive K+ channelsBRITISH JOURNAL OF PHARMACOLOGY, Issue 4 2000Grasiele Fauaz The role of ,2 -adrenoceptors in the response of aorta smooth muscle rings to the ,2 -adrenoceptors agonists UK 14,304 and clonidine was studied. Stimulation by 1,10 nM UK 14,304 caused dose-dependent relaxant responses in BaCl2 -contracted endothelium-denuded aorta rings, and hyperpolarization in rings with or without endothelium, which were inhibited by yohimbine and glibenclamide, but not affected by prazosin, propranolol, apamin or iberiotoxin. At higher concentrations (10 nM,10 ,M) UK 14,304 also induced a depolarizing effect which was potentiated by yohimbine and inhibited by prazosin. These results indicate that UK 14,304 acts on ,2 -adrenoceptors at lower concentrations and on both ,1 - and ,2 -adrenoceptors above 10 nM. In rings, with or without endothelium, noradrenaline had a depolarizing effect which was inhibited by prazosin. Adrenaline did not affect the membrane potential but in the presence of prazosin caused hyperpolarization, which was inhibited by yohimbine and glibenclamide. These results indicate that noradrenaline is more selective for ,1 -, whereas adrenaline has similar affinities for ,1 - and ,2 -adrenoceptors. In aortae with endothelium, L -NNA caused a small depolarization but did not affect the hyperpolarization induced by UK 14,304, indicating that NO is not involved in that response. Glibenclamide induced a small depolarization in aortae, with or without endothelium, indicating that ATP-sensitive K+ channels may play a role in maintaining the smooth muscle's membrane potential. Our results indicate that, in rat aorta, ,2 -adrenoceptors are also present in the smooth muscle, and that these receptors act through small-conductance ATP-sensitive K+ channels. British Journal of Pharmacology (2000) 131, 788,794; doi:10.1038/sj.bjp.0703630 [source] A Tale of Two Targets: Differential RNA Selectivity of Nucleobase,Aminoglycoside ConjugatesCHEMBIOCHEM, Issue 10 2006Kenneth F. Blount Dr. Aminoglycoside antibiotics are RNA-binding polyamines that can bind with similar affinities to structurally diverse RNA targets. To design new semisynthetic aminoglycosides with improved target selectivity, it is important to understand the energetic and structural basis by which diverse RNA targets recognize similar ligands. It is also imperative to discover how novel aminoglycosides could be rationally designed to have enhanced selectivity for a given target. Two RNA drug targets, the prokaryotic ribosomal A-site and the HIV-1 TAR, provide an excellent model system in which to dissect the issue of target selectivity, in that they each have distinctive interactions with aminoglycosides. We report herein the design, synthesis, and binding activity of novel nucleobase,aminoglycoside conjugates that were engineered to be more selective for the A-site binding pocket. Contrary to the structural design, the conjugates bind the A-site more weakly than does the parent compound and bind the TAR more tightly than the parent compound. This result implies that the two RNA targets differ in their ability to adapt to structurally diverse ligands and thus have inherently different selectivities. This work emphasizes the importance of considering the inherent selectivity traits of the RNA target when engineering new ligands. [source] Comparison of the pharmacological properties of GK11 and MK801, two NMDA receptor antagonists: towards an explanation for the lack of intrinsic neurotoxicity of GK11JOURNAL OF NEUROCHEMISTRY, Issue 4 2007D. Vandame Abstract Over-stimulation of NMDA receptors (NMDARs) is involved in many neurodegenerative disorders. Thus, developing safe NMDAR antagonists is of high therapeutic interest. GK11 is a high affinity uncompetitive NMDAR antagonist with low intrinsic neurotoxicity, shown to be promising for treating CNS trauma. In the present study, we investigated the molecular basis of its interaction with NMDARs and compared this with the reference molecule MK801. We show, on primary cultures of hippocampal neurons, that GK11 exhibits neuroprotection properties similar to those of MK801, but in contrast with MK801, GK11 is not toxic to neurons. Using patch-clamp techniques, we also show that on NR1a/NR2B receptors, GK11 totally blocks the NMDA-mediated currents but has a six-fold lower IC50 than MK801. On NR1a/NR2A receptors, it displays similar affinity but fails to totally prevent the currents. As NR2A is preferentially localized at synapses and NR2B at extrasynaptic sites, we investigated, using calcium imaging and patch-clamp approaches, the effects of GK11 on either synaptic or extrasynaptic NMDA-mediated responses. Here we demonstrate that in contrast with MK801, GK11 better preserve the synaptic NMDA-mediated currents. Our study supports that the selectivity of GK11 for NR2B containing receptors accounts contributes, at least partially, for its safer pharmacological profile. [source] Responsiveness, affinity constants and receptor reserves for serotonin on aortae of aged normotensive and hypertensive ratsJOURNAL OF PHARMACY AND PHARMACOLOGY: AN INTERNATI ONAL JOURNAL OF PHARMACEUTICAL SCIENCE, Issue 10 2001Sheila A. Doggrell We have previously shown that the potency and affinity constants (KA values) for serotonin (5-HT) are greater, and the 5-HT2A -receptor reserve is lesser, on the aorta of 6-month-old spontaneously hypertensive rats (SHRs) compared with age-matched Wistar Kyoto normotensive (WKY) rats. The present study was undertaken to investigate whether these parameters are altered on the aorta with ageing and as hypertension progresses to heart failure. The effects of phenoxybenzamine on the serotonergic responses of the aortae of 24-month-old WKY rats and SHRs were determined. On WKY rat aorta, ageing from 6 to 24 months was associated with an increase in sensitivity and affinity for serotonin, and a loss of 5-HT2A -receptor reserve. On SHR aorta, ageing from 6 to 24 months was also associated with an increase in sensitivity and affinity for serotonin, but a loss of 5-HT2A -receptor reserve. The sensitivity to serotonin was greater on the 24-month-old SHR aorta (pD2 6.53) than age-matched WKY rat aorta (pD2 5.89). On the aorta of the 24-month-old WKY rats, the KA value for serotonin was 4.5 times 10,6 M, and the receptor occupancies required for 20 and 50% maximum responses were 12 and 29%, respectively. There was a similar affinity, but greater receptor reserves, for serotonin on the aorta of age-matched SHRs. In summary, we have shown changes in sensitivity, affinity and 5-HT2A -receptor reserves for serotonin on the aorta with ageing and in hypertension/heart failure. [source] Cloning and paratope analysis of an antibody fragment, a rational approach for the design of a PAI-1 inhibitorJOURNAL OF THROMBOSIS AND HAEMOSTASIS, Issue 2 2004K. Verbeke Summary., This study reports the cloning, characterization and paratope analysis of the plasminogen activator inhibitor-1 (PAI-1) neutralizing single-chain variable fragment 56A7C10 (scFv-56A7C10). ScFv-56A7C10-wt exhibits a similar affinity (KA = 1.01 ± 0.3 × 109 m,1) and PAI-1 inhibitory capacity (90 ± 6% PAI-1 inhibition at a 16-fold molar excess and IC50 = 44 ± 14 ng mL,1) as MA-56A7C10 (KA = 1.43 ± 0.4 × 109 m,1, 90 ± 2% PAI-1 inhibition at a 16-fold molar excess and IC50 = 122 ± 26 ng mL,1). Subsequently, alanine scanning of the six complementarity determining regions (CDRs) was performed and the scFv-56A7C10-mutants (n = 26) were analyzed for their PAI-1 binding and PAI-1 inhibitory properties. Mutation of the residues Y32 and V33 in the CDR1 of the heavy chain (HCDR1) and the residues R98, H99, W100 or F100a (HCDR3) resulted in reduced PAI-1 inhibitory capacities (IC50 , 418 ng mL,1), confirmed by reduced affinities (14-, 17-, 7-, 9- and 16-fold reduced, respectively, vs. scFv-56A7C10-wt). In the light chain, mutation of the residues W50 (LCDR2), H91, Y92, D93, or W96 (LCDR3) resulted in reduced PAI-1 inhibitory properties (IC50 , 160 ng mL,1) and decreased affinities (i.e. 4-, 9-, 3-, 3- and 2-fold reduced affinity, respectively, vs. scFv-56A7C10-wt). Furthermore, an overlapping peptide scan confirmed the importance of the HCDR3 region. These data, combined with a three-dimensional model of scFv-56A7C10, reveal the molecular and structural properties of the paratope and contribute to the rational design of PAI-1 neutralizing compounds. [source] Synthesis and assembly of a full-length human monoclonal antibody in algal chloroplastsBIOTECHNOLOGY & BIOENGINEERING, Issue 4 2009Miller Tran Abstract Monoclonal antibodies can be effective therapeutics against a variety of human diseases, but currently marketed antibody-based drugs are very expensive compared to other therapeutic options. Here, we show that the eukaryotic green algae Chlamydomonas reinhardtii is capable of synthesizing and assembling a full-length IgG1 human monoclonal antibody (mAb) in transgenic chloroplasts. This antibody, 83K7C, is derived from a human IgG1 directed against anthrax protective antigen 83 (PA83), and has been shown to block the effects of anthrax toxin in animal models. Here we show that 83K7C heavy and light chain proteins expressed in the chloroplast accumulate as soluble proteins that assemble into complexes containing two heavy and two light chain proteins. The algal-expressed 83K7C binds PA83 in vitro with similar affinity to the mammalian-expressed 83K7C antibody. In addition, a second human IgG1 and a mouse IgG1 were also expressed and shown to properly assemble in algal chloroplast. These results show that chloroplasts have the ability to fold and assemble full-length human mAbs, and suggest the potential of algae as a platform for the cost effective production of complex human therapeutic proteins. Biotechnol. Bioeng. 2009; 104: 663,673 © 2009 Wiley Periodicals, Inc. [source] Prediction of the Three-Dimensional Structure for the Rat Urotensin,II Receptor, and Comparison of the Antagonist Binding Sites and Binding Selectivity between Human and Rat Receptors from Atomistic SimulationsCHEMMEDCHEM, Issue 9 2010Soo-Kyung Kim Dr. Abstract Urotensin-II (U-II) has been shown to be the most potent mammalian vasoconstrictor known. Thus, a U-II antagonist might be of therapeutic value in a number of cardiovascular disorders. However, interspecies variability of several nonpeptidic ligands complicates the interpretation of in vivo studies of such antagonists in preclinical animal disease models. ACT058362 is a selective antagonist for the human U-II receptor (hUT2R) with a reported Kd value of ,4,nM in a molecular binding assay, but it is reported to bind weakly to rat UT2R (rUT2R), with a Kd value of ,1,500,nM. In contrast, the arylsulphonamide SB706375 is a selective antagonist against both hUT2R (Kd=,9,nM) and rUT2R (Kd=,21,nM). To understand the species selectivity of the UT2R, we investigated the binding site of ACT058362 and SB706375 in both hUT2R and rUT2R to explain the dramatically lower (,400-fold) affinity of ACT058362 for rUT2R and the similar affinity (,10,nM) of SB706375 for both UT2Rs. These studies used MembStruk and MSCDock to predict the UT2R structure and the binding site of ACT058362 and SB706375. Based on binding energies, we found two binding modes each with D1303.32 as the crucial anchoring point (Ballesteros,Weinstein numbering given in superscript). We predict that ACT058362 (an aryl,amine,aryl or ANA ligand) binds in the transmembrane (TM) 3456 region, while SB706375 (an aryl,aryl,amine or AAN ligand) binds in the TM 1237 region. These predicted sites explain the known differences in binding of the ANA ligand to rat and human receptors, while explaining the similar binding of the AAN compound to rat and human receptors. Moreover the predictions explain currently available structure,activity relationship (SAR) data. To further validate the predicted binding sites of these ligands in hUT2R and rUT2R, we propose several mutations that would help define the structural origins of differential responses between UT2R of different species, potentially indicating novel UT2R antagonists with cross-species high affinity. [source] | ||||||||||||||||