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Equilibrium Dissociation Constant (equilibrium + dissociation_constant)
Selected AbstractsSolid,liquid equilibrium of substrates and products of the enzymatic synthesis of ampicillinAICHE JOURNAL, Issue 6 2010Mônica Santana Abstract The solid,liquid equilibrium of precursors and products of the enzymatic synthesis of ampicillin (AMP) [6-aminopencillanic acid (6-APA) and D(,)phenylglycine (PG)] was investigated at different temperatures (283,298 K) and pHs (5.5,7.5). Solubility data were obtained using an analytical methodology. Equilibrium dissociation constants were experimentally measured at several temperatures for AMP, 6-APA, PG, and D(,)phenylglycine methyl ester. A model based on the simplified perturbed hard sphere theory proposed by Khoshkbarchi and Vera (Ind Eng Chem Res. 1996;35:4319-4327) was fitted against solubility data. The model could describe the water solubility behavior for AMP and PG as function of pH and temperature, but a bias was observed when fitting the model to the solubility of 6-APA. © 2009 American Institute of Chemical Engineers AIChE J, 2010 [source] Binding properties of peptidic affinity ligands for plasmid DNA capture and detectionAICHE JOURNAL, Issue 2 2009Ying Han Abstract Peptides constructed from ,-helical subunits of the Lac repressor protein (LacI) were designed then tailored to achieve particular binding kinetics and dissociation constants for plasmid DNA purification and detection. Surface plasmon resonance was employed for quantification and characterization of the binding of double stranded Escherichia coli plasmid DNA (pUC19) via the lac operon (lacO) to "biomimics" of the DNA binding domain of LacI. Equilibrium dissociation constants (KD), association (ka), and dissociation rates (kd) for the interaction between a suite of peptide sequences and pUC19 were determined. KD values measured for the binding of pUC19 to the 47mer, 27mer, 16mer, and 14mer peptides were 8.8 ± 1.3 × 10,10 M, 7.2 ± 0.6 × 10,10 M, 4.5 ± 0.5 × 10,8 M, and 6.2 ± 0.9 × 10,6 M, respectively. These findings show that affinity peptides, composed of subunits from a naturally occurring operon,repressor interaction, can be designed to achieve binding characteristics suitable for affinity chromatography and biosensor devices. © 2008 American Institute of Chemical Engineers AIChE J, 2009 [source] Competitive binding comparison of endocrine-disrupting compounds to recombinant androgen receptor from fathead minnow, rainbow trout, and humanENVIRONMENTAL TOXICOLOGY & CHEMISTRY, Issue 9 2007Vickie S. Wilson Abstract Typically, in vitro hazard assessments for the identification of endocrine-disrupting compounds (EDCs), including those outlined in the Endocrine Disruptor Screening and Testing Advisory Committee (EDSTAC) Tier 1 Screening protocols, utilize mammalian receptors. Evidence, however, exists that fish sex steroid hormone receptors differ from mammalian receptors both structurally and in their binding affinities for some steroids and environmental chemicals. Most of the binding studies to date have been conducted using cytosolic preparations from various tissues. In the present study, we compare competitive binding of a set of compounds to full-length recombinant rainbow trout androgen receptor , (rtAR), fathead minnow androgen receptor (fhAR), and human androgen receptor (hAR), each expressed in COS cells. Saturation binding and subsequent Scatchard analysis using [3H]R1881, a high-affinity synthetic androgen, revealed an equilibrium dissociation constant (Kd) of 0.11 nM for the rtAR, 1.8 nM for the fhAR, and 0.84 nM for the hAR. Compounds, including endogenous and synthetic steroids, known mammalian antiandrogens, and environmental compounds, were tested for competitive binding to each of the three receptors. Overall, agreement existed across receptors as to binding versus nonbinding for all compounds tested in this study. Minor differences, however, were found in the relative order of binding of the compounds to the individual receptors. Studies such as these will facilitate the identification of EDCs that may differentially affect specific species and aid in the development and support of future risk assessment protocols. [source] Oligohis-tags: mechanisms of binding to Ni2+ -NTA surfacesJOURNAL OF MOLECULAR RECOGNITION, Issue 4 2009Steven Knecht Abstract Since immobilized metal ion affinity chromatography (IMAC) was first reported, several modifications have been developed. Among them, Ni2+ immobilized by chelation with nitrilotriacetic acid (NTA) bound to a solid support has become the most common method for the purification of proteins carrying either a C - or N -terminal histidine (His) tag. Despite its broad application in protein purification, only little is known about the binding properties of the His-tag, and therefore almost no thermodynamic and kinetic data are available. In this study, we investigated the binding mechanism of His-tags to Ni2+ -NTA. Different series of oligohistidines and mixed oligohistidines/oligoalanines were synthesized using automated solid-phase peptide synthesis (SPPS). Binding to Ni2+ -NTA was analyzed both qualitatively and quantitatively with surface plasmon resonance (SPR) using commercially available NTA sensor chips from Biacore. The hexahistidine tag shows an apparent equilibrium dissociation constant (KD) of 14,±,1,nM and thus the highest affinity of the peptides synthesized in this study. Furthermore, we could demonstrate that two His separated by either one or four residues are the preferred binding motifs within hexahis tag. Finally, elongation of these referred motifs decreased affinity, probably due to increased entropy costs upon binding. Copyright © 2009 John Wiley & Sons, Ltd. [source] Real-time monitoring of the membrane-binding and insertion properties of the cholesterol-dependent cytolysin anthrolysin O from Bacillus anthracis,JOURNAL OF MOLECULAR RECOGNITION, Issue 4 2006Simon Cocklin Abstract Bacillus anthracis has recently been shown to secrete a potently hemolytic/cytolytic protein that has been designated anthrolysin O (ALO). In this work, we initiated a study of this potential anthrax virulence factor in an effort to understand the membrane,binding properties of this protein. Recombinant anthrolysin O (rALO35,512) and two N-terminally truncated versions of ALO (rALO390,512 and rALO403,512) from B. anthracis were overproduced in Escherichia coli and purified to homogeneity. The role of cholesterol in the cytolytic activity of ALO was probed in cellular cholesterol depletion assays using mouse and human macrophage-like lines, and also Drosophila Schneider 2 cells. Challenging the macrophage cells with rALO35,512, but not rALO390,512 or rALO403,512, resulted in cell death by lysis, with this cytolysis being abolished by depletion of the membrane cholesterol. Drosophila cells, which contain ergosterol as their major membrane sterol, were resistant to rALO-mediated cytolysis. In order to determine the molecular mechanism of this resistance, the interaction of rALO with model membranes comprised of POPC alone, or with a variety of structurally similar sterols including ergosterol, was probed using Biacore. Both rALO35,512 and rALO403,512 demonstrated robust binding to model membranes composed of POPC and cholesterol, with amount of protein bound proportional to the cholesterol content. Ergosterol supported greatly reduced binding of both rALO35,512 and rALO403,512, whereas other sterols tested did not support binding. The rALO403,512,membrane interaction demonstrated an equilibrium dissociation constant (KD) in the low nanomolar range, whereas rALO35,512 exhibited complex kinetics likely due to the multiple events involved in pore formation. These results establish the pivotal role of cholesterol in the action of rALO. The biosensor method developed to measure ALO recognition of cholesterol in a membrane environment could be extended to provide a platform for the screening of inhibitors of other membrane-binding proteins and peptides. Copyright© 2006 John Wiley & Sons, Ltd. [source] The Presence and Role of the Dopamine DA-2 Receptor in the Human DeciduaJOURNAL OF OBSTETRICS AND GYNAECOLOGY RESEARCH (ELECTRONIC), Issue 6 2000Fujimi Arai Abstract Objectives: Our objectives were to identify the presence of the dopamine DA-2 receptor in human decidua and to study its function in human parturition. Methods: Human term decidual tissues were obtained during vaginal delivery and then homogenized. The P3 fraction was prepared for a radiolabeled receptor assay with [3H] spiperone as the ligand. Human decidual tissues obtained at cesarean section before the onset of labor were incubated in Krebs-Ringer buffer at 37°C for 30 minutes in the presence of dopamine with or without (,)-sulpiride. The level of prostaglandin (PG) F in the medium was measured with a RIA kit. Differences were assessed with the Wilcoxon non-parametric test. Results: Scatchard analysis showed a single class of binding sites having an equilibrium dissociation constant (Kd) of 2.25 + 0.59 nm (mean + SD) and a maximum binding capacity (Bmax) value of 166.5 + 77.7 fmol/mg protein (n = 3). Dopamine significantly increased the production of PGF. This stimulatory effect of dopamine was suppressed by (,)-sulpiride (p < 0.05; n = 7). Conclusion: The DA-2 receptor was demonstrated in the human decidua. Dopamine can stimulate PGF production via this receptor. [source] Hemorrhagic stroke associated with antidepressant use in patients with depression: does degree of serotonin reuptake inhibition matter?PHARMACOEPIDEMIOLOGY AND DRUG SAFETY, Issue 3 2009Yan Chen MD Abstract Objective This study aimed to determine whether the degree of serotonin (5-HT) reuptake inhibition affects risk of hemorrhagic stroke associated with antidepressant use in patients with depression. Method A population-based, nested case-control study was performed using a managed care medical claims database. Ninety two depressed patients with a diagnosis of hemorrhagic stroke were identified and matched with 552 controls by age, sex, and year of index date of depression (IDD). Diagnoses of depression, hemorrhagic stroke, and other medical comorbidities were identified using ICD-9 codes. Antidepressants were classified as high, medium, or low reuptake inhibition based on their affinities for the 5-HT reuptake transporter, determined using their respective equilibrium dissociation constants (KD; high: KD,<,1,nM; medium: 1,,,KD,<,10,nM; low: KD,,,10,nM). Conditional logistic regression analysis was performed to estimate the crude and adjusted odds ratios (ORs) with 95% confidence intervals (CIs) of the risk of hemorrhagic stroke. Results Compared to non-users of antidepressants, risk of hemorrhagic stroke did not significantly differ between patients who had ever used antidepressants with high (OR,=,0.82; 95% CI,=,0.44,1.55), medium (OR,=,0.93; 95% CI,=,0.37,2.31), or low (OR,=,0.38; 95% CI,=,0.11,1.41) 5-HTT inhibition. Conclusion Risk of hemorrhagic stroke associated with antidepressant use may not be related to an antidepressant's degree of 5-HT reuptake inhibition. Given the limitations of this study, additional studies are needed to confirm these findings. Copyright © 2009 John Wiley & Sons, Ltd. [source] Tissue transglutaminase modulates ,-synuclein oligomerizationPROTEIN SCIENCE, Issue 8 2008Ine M.J. Segers-Nolten Abstract We have studied the interaction of the enzyme tissue transglutaminase (tTG), catalyzing cross-link formation between protein-bound glutamine residues and primary amines, with Parkinson's disease-associated ,-synuclein protein variants at physiologically relevant concentrations. We have, for the first time, determined binding affinities of tTG for wild-type and mutant ,-synucleins using surface plasmon resonance approaches, revealing high-affinity nanomolar equilibrium dissociation constants. Nanomolar tTG concentrations were sufficient for complete inhibition of fibrillization by effective ,-synuclein cross-linking, resulting predominantly in intramolecularly cross-linked monomers accompanied by an oligomeric fraction. Since oligomeric species have a pathophysiological relevance we further investigated the properties of the tTG/,-synuclein oligomers. Atomic force microscopy revealed morphologically similar structures for oligomers from all ,-synuclein variants; the extent of oligomer formation was found to correlate with tTG concentration. Unlike normal ,-synuclein oligomers the resultant structures were extremely stable and resistant to GdnHCl and SDS. In contrast to normal ,-sheet-containing oligomers, the tTG/,-synuclein oligomers appear to be unstructured and are unable to disrupt phospholipid vesicles. These data suggest that tTG binds equally effective to wild-type and disease mutant ,-synuclein variants. We propose that tTG cross-linking imposes structural constraints on ,-synuclein, preventing the assembly of structured oligomers required for disruption of membranes and for progression into fibrils. In general, cross-linking of amyloid forming proteins by tTG may prevent the progression into pathogenic species. [source] Kinetic analysis of the binding of monomeric and dimeric ephrins to Eph receptors: Correlation to function in a growth cone collapse assayPROTEIN SCIENCE, Issue 3 2007Kumar B. Pabbisetty Abstract Eph receptors and ephrins play important roles in regulating cell migration and positioning during both normal and oncogenic tissue development. Using a surface plasma resonance (SPR) biosensor, we examined the binding kinetics of representative monomeric and dimeric ephrins to their corresponding Eph receptors and correlated the apparent binding affinity with their functional activity in a neuronal growth cone collapse assay. Our results indicate that the Eph receptor binding of dimeric ephrins, formed through fusion with disulfide-linked Fc fragments, is best described using a bivalent analyte model as a two-step process involving an initial monovalent 2:1 binding followed by a second bivalent 2:2 binding. The bivalent binding dramatically decreases the apparent dissociation rate constants with little effect on the initial association rate constants, resulting in a 30- to 6000-fold decrease in apparent equilibrium dissociation constants for the binding of dimeric ephrins to Eph receptors relative to their monomeric counterparts. Interestingly, the change was more prominent in the A-class ephrin/Eph interactions than in the B-class of ephrins to Eph receptors. The increase in apparent binding affinities correlated well with increased activation of Eph receptors and the resulting growth cone collapse. Our kinetic analysis and correlation of binding affinity with function helped us better understand the interactions between ephrins and Eph receptors and should be useful in the design of inhibitors that interfere with the interactions. [source] | |||||||||||||