Search results for "Affinity label"
showing 9 items of 39 documents
The N-terminal Amino Group of [Tyr8]Bradykinin Is Bound Adjacent to Analogous Amino Acids of the Human and Rat B2 Receptor
1996
To obtain data of the bradykinin B2 receptor's agonist binding site, we used a combined approach of affinity labeling and "immunoidentification" of receptor fragments generated by cyanogen bromide cleavage. Domain-specific antibodies to the various extracellular receptor domains were applied to detect receptor fragments with covalently attached [125I-Tyr8]bradykinin. As a cross-linker we used the homobifunctional reagent disuccinimidyl tartarate (DST), which reacts preferentially with primary amines. With this technique a [125I-Tyr8]bradykinin-labeled receptor fragment derived from the third extracellular domain was identified. The epsilon-amino group of lysine (Lys172) of the human B2 rece…
Recombinant avidin and avidin-fusion proteins.
2000
Both chicken egg-white avidin and its bacterial relative streptavidin are well known for their extraordinary high affinity with biotin (Kd approximately 10(-15) M). They are widely used as tools in a number of affinity-based separations, in diagnostic assays and in a variety of other applications. These methods have collectively become known as (strept)avidin-biotin technology. Biotin can easily and effectively be attached to different molecules, termed binders and probes, without destroying their biological activity. The exceptional stability of the avidin-biotin complex and the wide range of commercially available reagents explain the popularity of this system. In order by genetic enginee…
Synthesis, X-ray Structure Determination, and Comprehensive Photochemical Characterization of (Trifluoromethyl)diazirine-Containing TRPML1 Ligands
2021
Potential (trifluoromethyl)diazirine-based TRPML1 ion channel ligands were designed and synthesized, and their structures were determined by single-crystal X-ray diffraction analysis. Photoactivation studies via 19F NMR spectroscopy and HPLC-MS analysis revealed distinct kinetical characteristics in selected solvents and favorable photochemical properties in an aqueous buffer. These photoactivatable TRPML activators represent useful and valuable tools for TRPML photoaffinity labeling combined with mass spectrometry.
Covalent modificaition of juvenile hormone binding proteins by photoaffinity labeling: An unexpected gel shift effect
1994
The 32 kD juvenile hormone binding protein (JHBP) and two 80 kD proteins in larval Manduca sexta hemolymph were labeled with [3H]FDK, a photoaffinity analog of methyl farnesoate (MF). The labeling could be completely displaced by a 30-fold excess of either MF or JH II, demonstrating that [3H]FDK binds specifically to the JH binding sites of the 32 kD JHBP and the 80 kD proteins. In addition, a high molecular-mass protein was labeled with [3H]FDK; labeling could be displaced by excess MF but not by JH II, demonstrating the selectivity in binding MF. The 32 kD JHBP also appeared to weakly bind the potent juvenoid, methoprene, at the JH binding site. Covalent modification by [3H]FDK induced a …
3'-Arylazido-beta-alanyl-2-azido ATP, a cross-linking photoaffinity label for F1ATPases.
1989
Abstract The synthesis of the 3′-arylazido-2-azido ATP derivative 3′-O-{3-[N-(4-azido-2-nitrophenyl)-amino]propionyl}2-azido-adenosine 5′-triphosphate (2,3′-DiN3ATP) is described. The bifunc tional photoreactive ATP analog is characterized spectroscopically. Photoaffinity labeling of F, ATPase from Micrococcus luteus by this analog results in the inactivation of the enzyme and in the formation of higher molecular weight cross-links,
Photoaffinity labeling of the coupling factor 1 from the thermophilic bacterum PS3 by 8-azido ATP
1984
AbstractTo localize the nucleotide binding sites of the F1ATPase (TF1) from the thermophilic bacterium PS3 we have used 14C-labeled 8-azido ATP (8-N3ATP) as photoaffmity label. 8-N3ATP is hydrolyzed by the F,ATPase in the absence of ultraviolet light. Irradiation by ultraviolet light of the enzyme in the presence of 8-N3ATP results in reduction of ATPase activity and in preferential nucleotide specific labeling of the α subunits (0.8–0.9 mol 8-N3ATP/TF1,α:β = 4:1). Inactivation and labeling do not depend on the presence of Mg2+. Both effects decrease upon addition of various nucleotide di- or triphosphates.
Photo-DHEA--a functional photoreactive dehydroepiandrosterone (DHEA) analog.
2011
Abstract The steroid hormone dehydroepiandrosterone (DHEA) has beneficial effects on vascular function, survival of neurons, and fatty acid metabolism. However, a specific receptor for DHEA has not been identified to date. Here, we describe the synthesis of a photoreactive DHEA derivative (Photo-DHEA). In Photo-DHEA, typical characteristics of DHEA are conserved: (i) a “planar” tetracyclic ring system with a Δ 5 double bond, (ii) a 3β-hydroxyl group, and (iii) a keto group at C17. In cell-based assays, Photo-DHEA showed the same properties as DHEA. We conclude that Photo-DHEA is suitable for radioiodination to yield a tool for the identification of the elusive DHEA receptor.
Insulin-like growth factors in chick embryo retina during development.
1996
Evidence exists supporting an important role for insulin-like growth factors (IGFs) during fetal growth. In the present report we performed studies to define whether developing chick retina contains IGFs and whether IGFs play a role in the growth of this tissue. We have shown that both IGF-I and IGF-II are present in chick embryo retina throughout development (7th-18th day). The highest values, when expressed as ng/g of tissue, were found in the youngest retinas studied (7th-9th day) and at 16th-18th day of development. During whole development the content of IGF-II was about two to three times higher than that ascertained for IGF-I. The tissue also contains cell-surface binding for IGFs. H…
Synthesis of biotin-labelled dexamethasone derivatives. Novel hormone-affinity probes.
1983
A new, general methodology for 'sandwich' affinity chromatography of steroid hormone receptors is proposed, the part purification of the human spleen tumor glucocorticoid receptor is quoted as an illustration. 9-Fluoro-16 alpha-methyl-11 beta, 17-dihydroxy-1,4-androstadiene-3-one-17 beta-carboxylic acid was coupled to biotin using pentamethylenediamine (BioDex 1) as a spacer. The bifunctional derivative binds to glucocorticoid receptors and avidin-Sepharose and efficiently protects the glucocorticoid receptor against inactivation when previously added during homogenisation. We have standardized the capacity and optimum conditions for elution of receptor-BioDex-1 complexes which are bound to…