Search results for "affinity"
showing 10 items of 313 documents
Photoaffinity Labeling and Photoaffinity Cross-Linking of Phosphofructokinase-1 from Saccharomyces cerevisiae by 8-Azidoadeninenucleotides
2001
Phosphofructokinase-1 from Saccharomyces cerevisiae is composed of four alpha- and four beta-subunits, each of them carrying catalytic and regulatory bindings sites for MgATP. In this paper, various photoaffinity labels, such as 8-azidoadenosine 5'-triphosphate, 8-azido-1,N6-ethenoadenosine 5'-triphosphate, and 8-N3-3'(2')-O-biotinyl-8-azidoadenosine 5'-triphosphate have been used to study their interaction with the enzyme in the dark and during irradiation. All nucleotidetriphosphates function as phosphate donor forming fructose 1,6-bisphosphate from fructose 6-phosphate. However, the kinetic analysis revealed distinctly differences between them. Photolabeling causes a decrease in enzyme a…
Identification and characterization of a novel Ets-2-related nuclear complex implicated in the activation of the human interleukin-12 p40 gene promot…
1997
Interleukin-12 (IL-12) is a proinflammatory cytokine produced by antigen-presenting cells in response to many microbial infections. IL-12 plays an important role in the generation of T helper type-1 cells, which favor cell-mediated immune response. IL-12 is composed of two different subunits, p40 and p35, whose expression can be regulated concomitantly or differentially. Monocytic cells, the major producers of IL-12, can be primed by interferon-gamma (IFN-gamma) to produce optimal amounts of IL-12 in response to LPS stimulation as a consequence of bacterial infection. The priming effect is exerted primarily at the transcriptional level on the p40 promoter in conjunction with the effects of …
Purification, subunit structure, and kinetics of the chloroform-released F1ATPase complex from Rhodospirillum rubrum and its comparison with F1ATPase…
1979
Abstract A stable and homogeneous adenosine-5ʹ-triphosphatase (ATPase, EC 3.6.1.3) has been solubilized from Rhodospirillum rubrum (R . rubrum) chromatophores by chloroform extraction. Purification of the Ca2+-dependent ATPase activity was 200-fold. Ca2+ can be replaced by Mg2+, Cd2+, and Mn2+ .The Km for Ca-ATP (0.17 mᴍ) is increased about 5-fold during solubilization of the enzyme, whereas the Km values for Mg-ATP (0.029 mᴍ) and Cd-ATP (0.014 mᴍ) are not affected. The chloroform-released ATPase has a molecular weight of 400,000 ± 30,000 and consists of the following subunits (molecular weights in parenthesis): α (58,000), β (53,500), γ (39,000), δ (18,500), and ε (14,000). The amino acid …
8-Azido-adenosine 5'-triphosphate as a Photoaffinity Label for Bacterial F1 ATPase
1978
1. 8-Azido-adenosine 5'-triphosphate (n83ATP) is a suitable photoaffinity label for F1 ATPase from Micrococcus luteus. The nucleotide is a substrate in the presence of bivalent cations and inhibits the enzyme irreversibly upon irradiation with ultraviolet light above 300 nm. 2. More than 80% of the label is covalently bound to the beta subunits in the presence of bivalent cations. Labeling and inactivation is decreased by protection with ADP, ATP or adenyl-5'-yl imidodiphosphate. To a much smaller degree the alpha subunits also become labeled. 3. n83AMP does not specifically bind to the beta subunits upon irradiation. Like n83ATP and n83ADP, it also labels the alpha subunits to a small exte…
Synthesis and characterization of biotinylated and photoactivatable neuroleptics. Novel bifunctional probes for dopamine receptors
1992
Abstract We have synthesized and characterized a series of novel derivatives of established antagonists of the neurotransmitter dopamine, i.e. butyrophenones, hexahydrocarbolines and phenothiazenes. All derivatives were biotinylated, some of them carried an additional (photoactivatable) azido group. In the case of butyrophenones, the structural modifications were introduced at the aliphatic keto group and/or the heterocyclic ring system, both modifications resulting in significant decreases in binding affinity to dopamine D 2 and dopamine D 1 receptor subtypes. Biotinylation of hexahydrocarbolines significantly increased their binding affinity to D 1 receptors, with the affinity for D 2 rec…
Performance analysis of two immunochromatographic assays for the diagnosis of rotavirus infection
2017
Group A rotaviruses (RVAs) are the primary cause of acute gastroenteritis (AGE) in young children worldwide. Several commercial tests including latex agglutination, enzyme-linked assays (ELISA) and immunochromatographic tests (ICT) have been developed for the diagnosis of RVA infection. In the present study, the performance of two commercially available one-step chromatographic immunoassays, CerTest Rotavirus + Adenovirus (Biotec S.L, Zaragoza, Spain) and Vikia Rota-Adeno (bioMerieux SA, Lyon, France) were retrospectively evaluated using Real-time PCR as reference test. Re-testing by Real-time PCR of 2096 stool samples of children hospitalized with AGE previously screened by ICTs (1467 by C…
Organotypic rat cerebellar slice culture as a model to analyze the molecular pharmacology of GABAA receptors
2002
The preservation of the neuronal circuitry in rat cerebellar slice cultures provides an advantage in monitoring the development and characterizing the pharmacology of GABA(A) receptor subtypes. Sprague-Dawley rats, 8-11 days of age, were decapitated, their cerebella were cut into 400-microm slices and transferred into culture dishes. Cell viability and organotypic cerebellar organization of the culture remained well preserved up to 3 weeks. Autoradiographic procedures were introduced in these advanced culture technique and employed [(3)H]Ro 15-4513 in the absence and presence of 10 microM diazepam to visualize all benzodiazepine (BZD) and diazepam-insensitive (DIS) binding sites, respective…
Characterization of γ-aminobutyrate type A receptors with atypical coupling between agonist and convulsant binding sites in discrete brain regions
2001
Abstract γ-Aminobutyric acid type A (GABA A ) receptor ionophore ligand t -[ 35 S]butylbicyclophosphorothionate ([ 35 S]TBPS) was used in an autoradiographic assay on brain cryostat sections to visualize and characterize atypical GABA-insensitive [ 35 S]TBPS binding previously described in certain recombinant GABA A receptors and the cerebellar granule cell layer. Picrotoxinin-sensitive but 1-mM GABA-insensitive [ 35 S]TBPS binding was present in the rat cerebellar granule cell layer, many thalamic nuclei, subiculum and the internal rim of the cerebral cortex, amounting in these regions up to 6% of the basal binding determined in the absence of exogenous GABA. Similar binding properties wer…
Altered receptor subtypes in the forebrain of GABAA receptor δ subunit-deficient mice: recruitment of γ2 subunits
2002
A GABA(A) receptor delta subunit-deficient mouse line was created by homologous recombination in embryonic stem cells to investigate the role of the subunit in the brain GABA(A) receptors. High-affinity [(3)H]muscimol binding to GABA sites as studied by ligand autoradiography was reduced in various brain regions of delta(-/-) animals. [(3)H]Ro 15-4513 binding to benzodiazepine sites was increased in delta(-/-) animals, partly due to an increment of diazepam-insensitive receptors, indicating an augmented forebrain assembly of gamma 2 subunits with alpha 4 subunits. In the western blots of forebrain membranes of delta(-/-) animals, the level of gamma 2 subunit was increased and that of alpha …
Synthesis and evaluation in rats of the dopamine D2/3 receptor agonist 18F-AMC20 as a potential radioligand for PET
2015
Dopamine D2/3 receptor (D2/3R) agonist PET tracers are better suited for the imaging of synaptic dopaminergic neurotransmission than D2/3R antagonists and may also offer the opportunity to study in vivo the high-affinity state of D2/3R (D2/3RHigh). With the aim to develop 18F-labeled D2/3R agonists suitable for widespread clinical application, we report here on the synthesis and in vitro and in vivo evaluation of a D2/3R agonist ligand from the aminomethyl chromane (AMC) class-(R)-2-[(4- 18Fluorobenzylamino)methyl]chroman-7-ol (18F-AMC20). Methods: In vitro affinities of AMC20 toward dopaminergic receptor subtypes were measured in membrane homogenates prepared from HEK293 cells expressing h…