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Purification of a Synaptic Membrane Na + /Ca 2+ Antiporter and Immunoextraction with Antibodies to a 36‐kDa Protein
Author(s) -
Michaelis M. L.,
Nunley E. W.,
Jayawickreme C.,
Hurlbert M.,
Schueler S.,
Guilly C.
Publication year - 1992
Publication title -
journal of neurochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.75
H-Index - 229
eISSN - 1471-4159
pISSN - 0022-3042
DOI - 10.1111/j.1471-4159.1992.tb09290.x
Subject(s) - antiporter , polyclonal antibodies , chemistry , size exclusion chromatography , fast protein liquid chromatography , biochemistry , chromatography , tris , membrane , gel electrophoresis , membrane protein , enzyme , antibody , biology , immunology
The conditions for optimal solubilization and reconstitution of bovine brain synaptic plasma membrane Na + /Ca 2+ exchange activity were examined and a series of chromatographic procedures were used for the isolation of a protein involved in this transport activity. The zwitterionic detergent 3‐[(3‐cholamidopropyl)dimethylammonio]‐1‐propanesulfonate in the presence of 20% (vol/vol) glycerol led to optimal solubilization, and soybean phospholipids in low‐pH medium were found to produce optimal reconstitution of activity after dialysis to remove the detergent. Sequential chromatography steps involving the use of gel filtration on Sephacryl S‐400 HR, ion exchange on diethylaminoethyl‐Sephacel, and metal chelate chromatography on tris‐(carboxymethyl)ethylenediamine loaded with LaCl 3 led to the isolation of a fraction highly enriched in both Na + /Ca 2+ exchange activity and two protein bands identified by denaturing electrophoresis. The estimated molecular masses of the two proteins were 50 and 36 kDa. Development of polyclonal antibodies to the 36‐kDa protein permitted immunoextraction of >95% of the antiporter activity from solubilized synaptic plasma membranes. These antibodies cross‐reacted with the electroeluted 50‐kDa protein on enzyme‐linked immunosorbent assays, suggesting a close relationship between the two proteins. These results indicate that the 36‐kDa protein is at least a component of the brain membrane Na + /Ca 2+ antiporter.