Purification of a synaptic membrane Na+/Ca2+ antiporter and immunoextraction with antibodies to a 36-kDa protein.
The conditions for optimal solubilization and reconstitution of bovine brain synaptic plasma membrane Na+/Ca2+ 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 LaCl3 led to the isolation of a fraction highly enriched in both Na+/Ca2+ 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 greater than 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+/Ca2+ antiporter.
Michaelis
M L
ML
Department of Pharmacology, University of Kansas, Lawrence 66047.
Nunley
E W
EW
Jayawickreme
C
C
Hurlbert
M
M
Schueler
S
S
Guilly
C
C
D000818:Animals; D000906:Antibodies / Q000276:immunology; D002118:Calcium / Q000378:metabolism; D002352:Carrier Proteins / Q000276:immunology / Q000302:isolation & purification*; D002417:Cattle; D002462:Cell Membrane / Q000378:metabolism; D007158:Immunologic Techniques; D008565:Membrane Proteins / Q000276:immunology / Q000302:isolation & purification*; D008970:Molecular Weight; D019831:Sodium-Calcium Exchanger; D012995:Solubility; D013570:Synaptic Membranes / Q000378:metabolism*
D000906:Antibodies; D002352:Carrier Proteins; D008565:Membrane Proteins; D019831:Sodium-Calcium Exchanger; D002118:Calcium