Purification of B-50 by 2-mercaptoethanol extraction from rat brain synaptosomal plasma membranes.
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.
Division of Molecular Neurobiology, Rudolf Magnus Institute, University of Utrecht, The Netherlands.
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De Graan
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Moritz
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de Wit
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Gispen
D000818:Animals; D015153:Blotting, Western; D001923:Brain Chemistry; D002147:Calmodulin; D002846:Chromatography, Affinity; D004229:Dithiothreitol; D019922:GAP-43 Protein; D008297:Male; D008562:Membrane Glycoproteins / Q000302:isolation & purification*; D008623:Mercaptoethanol; D009419:Nerve Tissue Proteins / Q000302:isolation & purification*; D010766:Phosphorylation; D011493:Protein Kinase C / Q000378:metabolism; D051381:Rats; D017208:Rats, Wistar; D013570:Synaptic Membranes / Q000737:chemistry*
D002147:Calmodulin; D019922:GAP-43 Protein; D008562:Membrane Glycoproteins; D009419:Nerve Tissue Proteins; D008623:Mercaptoethanol; D011493:Protein Kinase C; D004229:Dithiothreitol