Purification of B-50 by 2-mercaptoethanol extraction from rat brain synaptosomal plasma membranes.

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.

journal_name

Neurochem Res

journal_title

Neurochemical research

authors

De Graan PN,Moritz A,de Wit M,Gispen WH

doi

10.1007/BF00998271

subject

Has Abstract

pub_date

1993-08-01 00:00:00

pages

875-81

issue

8

eissn

0364-3190

issn

1573-6903

journal_volume

18

pub_type

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