Purification of B-50 by 2-mercaptoethanol extraction from rat brain synaptosomal plasma membranes |
| |
Authors: | Pierre N. E. De Graan Albrecht Moritz Marina de Wit Willem Hendrik Gispen |
| |
Affiliation: | (1) Division of Molecular Neurobiology, Rudolf Magnus Institute and Institute of Molecular Biology and Medical Biotechnology, University of Utrecht, Padualaan 8, 3584 CH Utrecht, The Netherlands |
| |
Abstract: | 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. |
| |
Keywords: | Purification 2-mercaptoethanol B-50/GAP-43 synaptosomal plasma membranes PKC substrate |
本文献已被 SpringerLink 等数据库收录! |