The Experts below are selected from a list of 107439 Experts worldwide ranked by ideXlab platform
Willem Hendrik Gispen - One of the best experts on this subject based on the ideXlab platform.
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Purification of B-50 by 2-Mercaptoethanol extraction from rat brain synaptosomal plasma membranes
Neurochemical Research, 1993Co-Authors: Pierre N. E. Graan, Albrecht Moritz, Willem Hendrik GispenAbstract:Several methods have been described previously for the purification of the nervous-tissue specific protein kinase C substrate B-50 (GAP-43). In this paper we present a new purification method for B-50 from rat brain which employs 2-Mercaptoethanol to release the protein from isolated synaptosomal plasma membranes. Most likely, 2-Mercaptoethanol reduces disulfide bonds involved in the linkage of B-50 to the membrane. After washing the membranes with 100 mM NaCl to detach loosely bound proteins, B-50 is the major protein (and the only protein kinase C substrate) released by 0.5% 2-Mercaptoethanol treatment. Further purification to apparent homogeneity is achieved by affinity chromatography on calmodulin sepharose. B-50 binds to calmodulin in the absence of calcium and specifically elutes from the column with 3 mM calcium. The procedures described is simple, rapid and highly suitable for large scale purification of B-50 from rat brain.
Pierre N. E. Graan - One of the best experts on this subject based on the ideXlab platform.
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Purification of B-50 by 2-Mercaptoethanol extraction from rat brain synaptosomal plasma membranes
Neurochemical Research, 1993Co-Authors: Pierre N. E. Graan, Albrecht Moritz, Willem Hendrik GispenAbstract:Several methods have been described previously for the purification of the nervous-tissue specific protein kinase C substrate B-50 (GAP-43). In this paper we present a new purification method for B-50 from rat brain which employs 2-Mercaptoethanol to release the protein from isolated synaptosomal plasma membranes. Most likely, 2-Mercaptoethanol reduces disulfide bonds involved in the linkage of B-50 to the membrane. After washing the membranes with 100 mM NaCl to detach loosely bound proteins, B-50 is the major protein (and the only protein kinase C substrate) released by 0.5% 2-Mercaptoethanol treatment. Further purification to apparent homogeneity is achieved by affinity chromatography on calmodulin sepharose. B-50 binds to calmodulin in the absence of calcium and specifically elutes from the column with 3 mM calcium. The procedures described is simple, rapid and highly suitable for large scale purification of B-50 from rat brain.
Albrecht Moritz - One of the best experts on this subject based on the ideXlab platform.
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Purification of B-50 by 2-Mercaptoethanol extraction from rat brain synaptosomal plasma membranes
Neurochemical Research, 1993Co-Authors: Pierre N. E. Graan, Albrecht Moritz, Willem Hendrik GispenAbstract:Several methods have been described previously for the purification of the nervous-tissue specific protein kinase C substrate B-50 (GAP-43). In this paper we present a new purification method for B-50 from rat brain which employs 2-Mercaptoethanol to release the protein from isolated synaptosomal plasma membranes. Most likely, 2-Mercaptoethanol reduces disulfide bonds involved in the linkage of B-50 to the membrane. After washing the membranes with 100 mM NaCl to detach loosely bound proteins, B-50 is the major protein (and the only protein kinase C substrate) released by 0.5% 2-Mercaptoethanol treatment. Further purification to apparent homogeneity is achieved by affinity chromatography on calmodulin sepharose. B-50 binds to calmodulin in the absence of calcium and specifically elutes from the column with 3 mM calcium. The procedures described is simple, rapid and highly suitable for large scale purification of B-50 from rat brain.
Claes B. Wollheim - One of the best experts on this subject based on the ideXlab platform.
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Effect of 2-Mercaptoethanol on glutathione levels, cystine uptake and insulin secretion in insulin-secreting cells.
European journal of biochemistry, 1992Co-Authors: D. Janjic, Claes B. WollheimAbstract:The role of glutathione (GSH) in the differentiated state of insulin-secreting cells was studied using 2-Mercaptoethanol as a means of varying intracellular GSH levels. 2-Mercaptoethanol (50 microM) caused a marked increase of GSH in two rat insulinoma cell lines, RINm5F and INS-1, the latter being dependent on the presence of 2-Mercaptoethanol for survival in tissue culture. The effect of 2-Mercaptoethanol on GSH was shared by other thiol compounds. Since in other cell types 2-Mercaptoethanol is thought to act on cystine transport, thereby increasing the supply of cysteine for GSH synthesis, we have studied [35S]cystine-uptake in INS-1 cells. At equimolar concentrations to cystine, 2-Mercaptoethanol caused stimulation of [35S]cystine-uptake. The effect persisted in the absence of extracellular Na+, probably suggesting the involvement of the Xc- carrier system. INS-1 cells with a high GSH level, cultured 48 h with 2-Mercaptoethanol, displayed a lower cystine uptake than control cells with a low GSH content. The effect of variations of the GSH levels on short-term insulin release was studied. No alteration of glyceraldehyde-induced or KCl-induced insulin release in RINm5F cells was detected. In contrast, both in islets and in INS-1 cells, a high GSH level was associated with a slightly lower insulin release. In INS-1 cells the effect was more marked at low glucose concentrations, resulting in an improved stimulation of insulin secretion. On the other hand, in islets, a decrease in the incremental insulin release evoked by glucose was seen. As in other cell types, oxidized glutathione (GSSG) was less than 5% of total GSH, and in INS-1 cells no change in the GSH/GSSG ratio was detected during glucose-induced or 3-isobutyl-1-methylxanthine-induced insulin release. In conclusion, 2-Mercaptoethanol-dependent INS-1 cells, as well as RINm5F cells and islets of Langerhans, display a low capacity in maintaining intracellular levels of GSH in tissue culture without extracellular thiol supplementation; 2-Mercaptoethanol possibly acts by promoting cyst(e)ine transport; changes in GSH levels caused a moderate effect on the differentiated function of insulin-secreting cells.
Walter Vetter - One of the best experts on this subject based on the ideXlab platform.
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A method for counting disulfide bridges in small proteins by reduction with Mercaptoethanol and electrospray mass spectrometry
Journal of Mass Spectrometry, 1995Co-Authors: M. Svoboda, Walter Meister, Walter VetterAbstract:A simple and rapid method for counting the number of internal disulfide bridges in a protein by incubation with 2-Mercaptoethanol and electrospray mass spectrometry analysis of the products was developed. 2-Mercaptoethanol yields intermediate mixed disulfides during reduction of a protein. This results in a molecular weight increase of the protein by 78 Da per disulfide bond, which can easily be determined by electrospray mass spectrometry (ESMS). The number of Mercaptoethanol adducts observed by ESMS reveals the number of disulfide bridges in the peptide or protein. Since the protein–Mercaptoethanol–disulfide bonds are themselves further reduced by excess Mercaptoethanol, the course of the reaction has to be followed in order to detect the maximum number of intermediates. Owing to the volatility of Mercaptoethanol, samples can be taken out of the reaction solution for MS analysis without prior purification. Successful experiments were carried out using proteins with one, two, four or six SS-bonds, covering a mass range from about 1 to over 23 kDa.
