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Protein tritiation

Figure 4. Binding was measured in rat brain synaptosomes using a rapid centrifugation technique. Total ( ), and nonspecific ( ) binding of tritiated PbTx-3 were measured, their difference representing specific binding (A). Rosenthal analysis yields a of 2.6 nM and a B of 6.0 pmol toxin bound/mg protein. Figure 4. Binding was measured in rat brain synaptosomes using a rapid centrifugation technique. Total ( ), and nonspecific ( ) binding of tritiated PbTx-3 were measured, their difference representing specific binding (A). Rosenthal analysis yields a of 2.6 nM and a B of 6.0 pmol toxin bound/mg protein.
Figure 6. Effect of brevetoxins on tritiated PbTx-3 binding to rat brain synaptosomes. Incubations, in the presence of 50 fig synaptosomal protein and 16 nM tritiated PbTx-3 with increasing amounts of unlabeled PbTx-1 ( ), PbTx-2 ( ), PbTx-3 ( ), PbTx-5 (A), PbTx-6 ( ), or PbTx-7 (o) were for 1 hr at 4 C. Each point represents the mean of three triplicate determinations. Figure 6. Effect of brevetoxins on tritiated PbTx-3 binding to rat brain synaptosomes. Incubations, in the presence of 50 fig synaptosomal protein and 16 nM tritiated PbTx-3 with increasing amounts of unlabeled PbTx-1 ( ), PbTx-2 ( ), PbTx-3 ( ), PbTx-5 (A), PbTx-6 ( ), or PbTx-7 (o) were for 1 hr at 4 C. Each point represents the mean of three triplicate determinations.
The aforementioned observations imply that the high affinity PCP binding sites in the brain are K channels. Therefore, as a prelude to the eventual isolation and characterization of these PCP "receptors VK channels, we employed a tritiated photo affinity analogue of PCP, m-azido-3H-PCP (3H-Az-PCP) to label rat brain, synaptic membrane proteins. The results indicate that this analogue binds specifically to two membrane polypeptides of a parent Mr 80,000 and 95,000 Daltons (Sorensen and Blaustein 1985b). [Pg.51]

Leukotriene B4 has been implicated in inflammatory processes and chemo-taxis. The G-protein-coupled receptor in human myeloid cells was postulated to possess two distinct binding sites. Photoaffinity studies using unmodified tritiated LTB4 as photoreactive species labeled two different proteins in the presence (53 kDa) and in the absence of a non-cleavable GTP analogue (56 kDa). This means that stabilization of the G-protein in the GTP-bound state resulted in an interconversion of the first high affinity binding site to an alternative low-affinity binding site [99]. [Pg.199]

The reactivity of compounds such as 28 was clearly demonstrated by the peroxidase-catalyzed covalent binding of A -methyW-hydroxyellipticine (27) to proteins (756). Using horseradish peroxidase and hydrogen peroxide, tritiated-27 was converted to the 9-oxoellipticine derivative in the presence of bovine serum albumin (BSA) and human antibovine IgG in vitro. Covalent binding to these proteins was confirmed by gel electrophoresis, combustion, and liquid scintillation analysis. Dissolution of the BSA-ellipticinium derivative with pronase and... [Pg.362]

Human KB carcinoma cells resistant to vinblastine and other drugs have been shown to exhibit increased membrane vesicular binding of tritiated vinblastine, and this binding is correlated with photoaffinity labeling of a 150,000- to 170,000-dalton protein in the vesicles. Labeling of this protein is inhibited by vinblastine, vincristine, and verapamil but not by colchicine (79). The failure of colchicine to inhibit the labeling of the membrane protein is unexpected since the cells from which the protein was isolated are resistant to colchicine as well as vinblastine. [Pg.212]

Cell number frequency distribution of nuclear DNA content of cell population, protein content, protein synthesis (e.g., 14C-labeled methionine incorporation), DNA synthesis (e.g., tritiated thymidine incorporation), DNA stains (e.g., Hoechst 33 342 4, 6-diamidino-2-phe-nylindole, DAPI picogreen) mass tracker dyes (e.g., LysoTracker green for lysosomes, MitoTracker deep red for mitochondria, ER-Tracker blue-white DPX for endoplasmic reticulum)... [Pg.335]

Thymidine Uptake Studies. Tritiated thymidine (52 mCi/ anole 0.1 /rCi/ml) was added to cells for 60 min at 37°C. Cells were then washed with PBS, incubated at 4°C for 15 min in the presence of ice cold 5% TCA, rinsed with TCA and scraped from the flasks with a rubber policeman. Cells were again washed with PBS, and solubilized in 0.1N NaOH overnight. Aliquots were assayed for protein (62) and radioactivity (scintillation fluid 100 ml Biosolve (Beckman, Fullerton, CA.), 7g of PPO and 0.6 g of POPOP per liter of toluene). [Pg.248]

Correlating positively with the hydrophobicity of the solvent, different fractions of inactivated active centers were measured in different solvents with solid-state NMR spectroscopy (13C-cross-polarization/magic angle spinning (MAS) NMR) (Burke, 1992). Just as with tritiated water (see above), immediate desorption of water molecules from the protein surface was observed after addition to the organic solvent. [Pg.347]

Figure 17.3 Binding experiments between tritiated compounds and RP-HPLC-purified antennal proteins. Each fraction corresponds to an aliquot of the purification. One pCi of each pheromonal compound was incubated with 20 pL of each fraction (sample buffer). The films were exposed to the... Figure 17.3 Binding experiments between tritiated compounds and RP-HPLC-purified antennal proteins. Each fraction corresponds to an aliquot of the purification. One pCi of each pheromonal compound was incubated with 20 pL of each fraction (sample buffer). The films were exposed to the...

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See also in sourсe #XX -- [ Pg.359 ]




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