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Multiple Binding Sites (multiple + binding_site)
Selected AbstractsTREM-1 expression in macrophages is regulated at transcriptional level by NF-,B and PU.1EUROPEAN JOURNAL OF IMMUNOLOGY, Issue 8 2007Heng Zeng Abstract Triggering receptor expressed on myeloid cells (TREM)-1 is a recently identified immunoglobulin receptor that is expressed on neutrophils and monocytes where it amplifies the acute inflammatory response to bacteria. We examined the transcriptional regulation of TREM-1 in macrophages. Treatment of RAW cells with Escherichia coli LPS or Pseudomonas aeruginosa led to the induction of TREM-1 within 1,h with an expression lasting up to at least 24,h in vitro as detected by RT-PCR. Since the promoter of TREM-1 has multiple binding sites for NF-,B and PU.1 (one of the members of the ets family of transcription factors), we investigated the role of these transcription factors in the induction of TREM-1. Treatment of cells with NF-,B inhibitors abolished the expression of message of TREM-1 induced by LPS and P.,aeruginosa. In contrast, the expression of TREM-1 was increased after stimulation with LPS or P.,aeruginosa in cells that had gene of PU.1 silenced. Additionally, over-expression of PU.1 led to inhibition of TREM-1 induction in response to LPS and P.,aeruginosa. These data suggest that both these transcription factors are involved in the expression of TREM-1. NF-,B functions as a positive regulator whereas PU.1 is a negative regulator of the TREM-1 gene. [source] Kinetic analysis of effector modulation of butyrylcholinesterase-catalysed hydrolysis of acetanilides and homologous estersFEBS JOURNAL, Issue 10 2008Patrick Masson The effects of tyramine, serotonin and benzalkonium on the esterase and aryl acylamidase activities of wild-type human butyrylcholinesterase and its peripheral anionic site mutant, D70G, were investigated. The kinetic study was carried out under steady-state conditions with neutral and positively charged aryl acylamides [o -nitrophenylacetanilide, o -nitrotrifluorophenylacetanilide and m -(acetamido) N,N,N -trimethylanilinium] and homologous esters (o -nitrophenyl acetate and acetylthiocholine). Tyramine was an activator of hydrolysis for neutral substrates and an inhibitor of hydrolysis for positively charged substrates. The affinity of D70G for tyramine was lower than that of the wild-type enzyme. Tyramine activation of hydrolysis for neutral substrates by D70G was linear. Tyramine was found to be a pure competitive inhibitor of hydrolysis for positively charged substrates with both wild-type butyrylcholinesterase and D70G. Serotonin inhibited both esterase and aryl acylamidase activities for both positively charged and neutral substrates. Inhibition of wild-type butyrylcholinesterase was hyperbolic (i.e. partial) with neutral substrates and linear with positively charged substrates. Inhibition of D70G was linear with all substrates. A comparison of the effects of tyramine and serotonin on D70G versus the wild-type enzyme indicated that: (a) the peripheral anionic site is involved in the nonlinear activation and inhibition of the wild-type enzyme; and (b) in the presence of charged substrates, the ligand does not bind to the peripheral anionic site, so that ligand effects are linear, reflecting their sole interaction with the active site binding locus. Benzalkonium acted as an activator at low concentrations with neutral substrates. High concentrations of benzalkonium caused parabolic inhibition of the activity with neutral substrates for both wild-type butyrylcholinesterase and D70G, suggesting multiple binding sites. Benzalkonium caused linear, noncompetitive inhibition of the positively charged aryl acetanilide m -(acetamido) N,N,N -trimethylanilinium for D70G, and an unusual mixed-type inhibition/activation (, > , > 1) for wild-type butyrylcholinesterase with this substrate. No fundamental difference was observed between the effects of ligands on the butyrylcholinesterase-catalysed hydrolysis of esters and amides. Thus, butyrylcholinesterase uses the same machinery, i.e. the catalytic triad S198/H448/E325, for the hydrolysis of both types of substrate. The differences in response to ligand binding depend on whether the substrates are neutral or positively charged, i.e. the differences depend on the function of the peripheral site in wild-type butyrylcholinesterase, or the absence of its function in the D70G mutant. The complex inhibition/activation effects of effectors, depending on the integrity of the peripheral anionic site, reflect the allosteric ,cross-talk' between the peripheral anionic site and the catalytic centre. [source] SMAP-29 has two LPS-binding sites and a central hingeFEBS JOURNAL, Issue 4 2002Brian F. Tack The CD spectra of SMAP-29, an antimicrobial peptide from sheep, showed disordered structure in aqueous buffers, and significant helicity in membrane-like environments, including SDS micelles, lipopolysaccharide (LPS) dispersions, and trifluoroethanol buffer systems. A structure determined by NMR in 40% perdeuterated trifluoroethanol indicated that residues 8,17 were helical, residues 18,19 formed a hinge, and residues 20,28 formed an ordered, hydrophobic segment. SMAP-29 was flexible in 40% trifluoroethanol, forming two sets of conformers that differed in the relative orientation of the N-terminal domain. We used a chromogenic Limulus assay to determine the EC50 of the peptide (the concentration that bound 50% of the added LPS). Studies with full-length and truncated SMAP-29 molecules revealed that each end of the holopeptide contained an LPS-binding domain. The higher affinity LPS-binding domain was situated in the flexible N-terminal portion. LPS binding to full-length SMAP-29 showed positive cooperativity, so the EC50 of the peptide (2.6 µm) was considerably lower than that of the individual LPS-binding domains. LPS-binding studies with a mixture of truncated peptides revealed that this cooperativity was primarily intramolecular (i.e. involving the N- and C-terminal LPS-binding sites of the same peptide molecule). CAP-18[106,142], an antimicrobial cathelicidin peptide of rabbits, resembled SMAP-29 in that it contained N- and C-terminal LPS-binding domains, had an EC50 of 2.5 µm, and bound LPS with positive cooperativity. We conclude that the presence of multiple binding sites that function cooperatively allow peptides such as SMAP-29 and CAP-18 to bind LPS with high affinity. [source] On the calculation of the concentration dependence of drug binding to plasma proteins with multiple binding sites of different affinities: Determination of the possible variation of the unbound drug fraction and calculation of the number of binding sites of the proteinJOURNAL OF PHARMACEUTICAL SCIENCES, Issue 2 2007Leonid M. Berezhkovskiy Abstract The measurement of the unbound drug fraction in plasma is routinely performed at drug concentrations much less than that of plasma proteins. Commonly, the protein has several binding sites of different affinities. The obtained value of the unbound drug fraction does not yield the affinity of each binding site separately. For drug binding to a single type of protein, it is shown that the assumption that all binding sites of the protein have the same affinity yields the slowest possible concentration increase of the unbound drug fraction, while the assumption that a drug binds to a single binding site yields the highest possible value of the unbound fraction for a given drug concentration. The conditions to be imposed on the affinities of binding sites, to provide the fastest and the slowest possible concentration increase of the unbound drug fraction are also obtained for the case of drug binding to several types of plasma proteins. The suggested approach is applied to the determination of the number of binding sites of the protein from the measured values of the unbound drug fraction at different drug concentrations. ©2006 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 96:249,257, 2007 [source] Geschichte und Evolution der Lactose(in)toleranz.BIOLOGIE IN UNSERER ZEIT (BIUZ), Issue 6 2009Das Erbe der frühen Viehzüchter Abstract Die Fähigkeit, auch im Erwachsenenalter noch Lactose verarbeiten zu können, basiert auf Punktmutationen in einem dem Lactase-(LPH-)Gen vorgelagerten Sequenzbereich, der Bindestellen für Regulatorproteine enthält. Die Ursache für die weltweit sehr uneinheitliche Verteilung der Lactase-Persistenz liegt in der europäischen Menschheitsgeschichte: Im Verlauf des 8. vorchristlichen Jahrtausends entwickelte sich im Nahen Osten innerhalb einer größtenteils lactoseintoleranten Population eine Tradition der Milchviehzucht und des Milchverzehrs. Durch den starken Selektionsdruck auf die Lactosetoleranz verbreiteten sich die mutierten Allele sehr schnell. Während des 7. Jahrtausends v. Chr. begannen etliche dieser Populationen sukzessiv Europa zu besiedeln. Auch im nordöstlichen Afrika und auf der arabischen Halbinsel entstand eine Milchwirtschaft, die jedoch auf anderen Mutationen basiert. The ability to digest lactose in adulthood is baised on point mutations within an upstream region of the lactase-(LPH-)gene. This region contains multiple binding sites for different transcription factors. The heterogenous distribution of the lactase persistence all over the world originates from the European history of humanity: in the course of the eighth millennium BC among a mainly lactose-intolerant population in the Near East evolved a cultural practice of dairy farming and milk consumption. As a result of the strong and positive selection the mutated alleles spread out rapidly. In the course of the seventh millennium BC many of these populations gradually settled Central Europe. The beginning of dairy farming in the north east of the African continent and on the Arabian Peninsula are based upon different point mutations. [source] Anion,, Slides for Transmembrane TransportCHEMISTRY - A EUROPEAN JOURNAL, Issue 1 2009Jiri Mareda Dr. Abstract The recognition and transport of anions is usually accomplished by hydrogen bonding, ion pairing, metal coordination, and anion,dipole interactions. Here, we elaborate on the concept to use anion,, interactions for this purpose. Different to the popular cation,, interactions, applications of the complementary ,-acidic surfaces do not exist. This is understandable because the inversion of the aromatic quadrupole moment to produce ,-acidity is a rare phenomenon. Here, we suggest that ,-acidic aromatics can be linked together to produce an unbendable scaffold with multiple binding sites for anions to move along across a lipid bilayer membrane. The alignment of multiple anion,, sites is needed to introduce a cooperative multi-ion hopping mechanism. Experimental support for the validity of the concept comes from preliminary results with oligonaphthalenediimide (O-NDI) rods. Predicted by strongly positive facial quadrupole moments, the cooperativity and chloride selectivity found for anion transport by O-NDI rods were consistent with the existence of anion,, slides. The proposed mechanism for anion transport is supported by DFT results for model systems, as well as MD simulations of rigid O-NDI rods. Applicability of anion,, slides to achieve electroneutral photosynthesis is elaborated with the readily colorizable oligoperylenediimide (O-PDI) rods. To clarify validity, scope and limitations of these concepts, a collaborative research effort will be needed to address by computer modeling and experimental observations the basic questions in simple model systems and to design advanced multifunctional anion,, architectures. [source] Identification of Putative Binding Sites of P-glycoprotein Based on its Homology ModelCHEMMEDCHEM, Issue 2 2008Christoph Globisch Abstract A homology model of P-glycoprotein based on the crystal structure of the multidrug transporter Sav1866 is developed, incorporated into a membrane environment, and optimized. The resulting model is analyzed in relation to the functional state and potential binding sites. The comparison of modeled distances to distances reported in experimental studies between particular residues suggests that the model corresponds most closely to the first ATP hydrolysis step of the protein transport cycle. Comparison to the protein 3D structure confirms this suggestion. Using SiteID and Site Finder programs three membrane related binding regions are identified: a region at the interface between the membrane and cytosol and two regions located in the transmembrane domains. The regions contain binding pockets of different size, orientation, and amino acids. A binding pocket located inside the membrane cavity is also identified. The pockets are analyzed in relation to amino acids shown experimentally to influence the protein function. The results suggest that the protein has multiple binding sites and may bind and/or release substrates in multiple pathways. [source] | ||||||||||