ISSN:
1573-6903
Keywords:
Purification
;
2-mercaptoethanol
;
B-50/GAP-43
;
synaptosomal plasma membranes
;
PKC substrate
Source:
Springer Online Journal Archives 1860-2000
Topics:
Medicine
Notes:
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.
Type of Medium:
Electronic Resource
URL:
http://dx.doi.org/10.1007/BF00998271
