Home About us Contact
|
Home About us Contact | ||
|
|
||
Ligand Affinity (ligand + affinity)
Selected AbstractsDeterminants of Ligand Affinity and Heme Reactivity in H-NOX Domains,ANGEWANDTE CHEMIE, Issue 4 2010Emily Scheuer Sauerstoff: Das Einführen von sterischer Belastung in der entfernten Tasche der Thermoanaerobacter-tengcongensis -H-NOX-Domäne (H-NOX: Häm-Stickstoffmonoxid/Sauerstoff) verändert die Proteinstruktur erheblich, was drastische Unterschiede in der O2 -Bindekinetik und der Häm-Reaktivität zur Folge hat (siehe Bild). [source] Regulation of A2A adenosine receptor expression and functioning following permanent focal ischemia in rat brainJOURNAL OF NEUROCHEMISTRY, Issue 2 2008Maria L. Trincavelli Abstract Ischemia, through modulation of adenosine receptors (ARs), may influence adenosine-mediated-cellular responses. In the present study, we investigated the modulation of rat A2A receptor expression and functioning, in rat cerebral cortex and striatum, following in vivo focal ischemia (24 h). In cortex, middle cerebral artery occlusion did not induce any alterations in A2A receptor binding and functioning. On the contrary, in striatum, a significant decrease in A2A ligand affinity, associated with an increase in receptor density, were detected. In striatum, ischemia also induced a significant reduction both in G protein pool and in A2A receptor-G protein coupling. On the contrary, A2A receptor functional responsiveness, measured as stimulation of adenylyl cyclise, was not affected by ischemia, suggesting receptor up-regulation may represent a compensatory mechanism to maintain receptor functioning during cerebral damage. Immunohistochemical study showed that following 24 h middle cerebral artery occlusion, A2A ARs were definitely expressed both on neurons and activated microglia in ischemic striatum and cortex, but were not detected on astrocytes. In the non-ischemic hemisphere and in sham-operated rats A2A ARs were barely detected. Modifications of ARs may play a significant role in determining adenosine effects during ischemia and therefore should be taken into account when evaluating time-dependent protective effects of specific A2A active compounds. [source] An approach to characterizing single-subunit mutations in multimeric prepores and pores of anthrax protective antigenPROTEIN SCIENCE, Issue 2 2009Blythe E. Janowiak Abstract Heptameric pores formed by the protective antigen (PA) moiety of anthrax toxin translocate the intracellular effector moieties of the toxin across the endosomal membrane to the cytosol of mammalian cells. We devised a protocol to characterize the effects of individual mutations in a single subunit of heptameric PA prepores (pore precursors) or pores. We prepared monomeric PA containing a test mutation plus an innocuous Cys-replacement mutation at a second residue (Lys563, located on the external surface of the prepore). The introduced Cys was biotinylated, and the protein was allowed to cooligomerize with a 20-fold excess of wild-type PA. Finally, biotinylated prepores were freed from wild-type prepores by avidin affinity chromatography. For the proof of principle, we examined single-subunit mutations of Asp425 and Phe427, two residues where Ala replacements have been shown to cause strong inhibitory effects. The single-subunit D425A mutation inhibited pore formation by >104 and abrogated activity of PA almost completely in our standard cytotoxicity assay. The single-subunit F427A mutation caused ,100-fold inhibition in the cytotoxicity assay, and this effect was shown to result from a combination of strong inhibition of translocation and smaller effects on pore formation and ligand affinity. Our results show definitively that replacing a single residue in one subunit of the heptameric PA prepore can inhibit the transport activity of the oligomer almost completely,and by different mechanisms, depending on the specific residue mutated. [source] Biochemical and structural characterization of residue 96 mutants of Plasmodium falciparum triosephosphate isomerase: active-site loop conformation, hydration and identification of a dimer-interface ligand-binding siteACTA CRYSTALLOGRAPHICA SECTION D, Issue 8 2009P. Gayathri Plasmodium falciparum TIM (PfTIM) is unique in possessing a Phe residue at position 96 in place of the conserved Ser that is found in TIMs from the majority of other organisms. In order to probe the role of residue 96, three PfTIM mutants, F96S, F96H and F96W, have been biochemically and structurally characterized. The three mutants exhibited reduced catalytic efficiency and a decrease in substrate-binding affinity, with the most pronounced effects being observed for F96S and F96H. The kcat values and Km values are (2.54 ± 0.19) × 105,min,1 and 0.39 ± 0.049,mM, respectively, for the wild type; (3.72 ± 0.28) × 103,min,1 and 2.18 ± 0.028,mM, respectively, for the F96S mutant; (1.11 ± 0.03) × 104,min,1 and 2.62 ± 0.042,mM, respectively, for the F96H mutant; and (1.48 ± 0.05) × 105,min,1 and 1.20 ± 0.056,mM, respectively, for the F96W mutant. Unliganded and 3-phosphoglycerate (3PG) complexed structures are reported for the wild-type enzyme and the mutants. The ligand binds to the active sites of the wild-type enzyme (wtPfTIM) and the F96W mutant, with a loop-open state in the former and both open and closed states in the latter. In contrast, no density for the ligand could be detected at the active sites of the F96S and F96H mutants under identical conditions. The decrease in ligand affinity could be a consequence of differences in the water network connecting residue 96 to Ser73 in the vicinity of the active site. Soaking of crystals of wtPfTIM and the F96S and F96H mutants resulted in the binding of 3PG at a dimer-interface site. In addition, loop closure at the liganded active site was observed for wtPfTIM. The dimer-interface site in PfTIM shows strong electrostatic anchoring of the phosphate group involving the Arg98 and Lys112 residues of PfTIM. [source] Separation of cannabinoid receptor affinity and efficacy in delta-8-tetrahydrocannabinol side-chain analoguesBRITISH JOURNAL OF PHARMACOLOGY, Issue 2 2001Graeme Griffin The activities of a number of side-chain analogues of delta-8-tetrahydrocannabinol (,8 -THC) in rat cerebellar membrane preparations were tested. The affinities of each compound for the CB1 receptor were compared by their respective abilities to displace [3H]-SR141716A and their efficacies compared by stimulation of [35S]-GTP,S binding. It was found that the affinities varied from 0.19±0.03 nM for 3-norpentyl-3-[6,-cyano,1,,1,-dimethyl]hexyl-,8 -THC to 395±66.3 nM for 5,-[N-(4-chlorophenyl)]-1,,1,-dimethyl-carboxamido-,8 -THC. The efficacies of these compounds varied greatly, ranging from the very low efficacy exhibited to acetylenic compounds such as 1,-heptyn-,8 -THC and 4,-octyn-,8 -THC to higher efficacy compounds such as 5,-(4-cyanophenoxy)-1,,1,-dimethyl-,8 -THC and 5,-[N-(4-aminosulphonylphenyl)]-1,,1, dimethyl-carboxamido ,8 -THC. All agonist activities were antagonized by the CB1 -selective antagonist SR141716A. It was found that a ligand's CB1 affinity and efficacy are differentially altered by modifications in the side-chain. Decreasing the flexibility of the side-chain reduced efficacy but largely did not alter affinity. Additionally, the positioning of electrostatic moieties, such as cyano groups, within the side-chain also has contrasting effects on these two properties. In summary, this report details the characterization of a number of novel ,8 -THC analogues in rat cerebellar membranes. It provides the first detailed pharmacological analysis of how the inclusion of electrostatic moieties in the side-chain and also how alteration of the side-chain's flexibility may differentially affect a CB1 cannabinoid receptor ligand's affinity and efficacy. British Journal of Pharmacology (2001) 132, 525,535; doi:10.1038/sj.bjp.0703827 [source] | ||||||||||