Purification of the bovine rod outer segment Na+/Ca2+ exchanger.
Optimal conditions for solubilization and stabilization of the Na+/Ca2+ exchanger from rod outer segments were examined. The exchanger was found to be most stable at low detergent concentrations (7.5 mM 3-[(3-cholamidopropyl)dimethylammonio]-1-propane-sulfonate), greater than or equal to 100 mM NaCl, pH 7.0-7.5, and with 0.1% added soybean asolectin. The sulfhydryl-modifying reagent, dithiothreitol, caused a loss of exchanger activity and was omitted throughout the purification procedure. These conditions were used to purify the Na+/Ca2+ exchanger from rod outer segments by a combination of selective solubilization, ion exchange, and wheat germ agglutinin chromatography. The procedure achieves a 336-fold increase in exchanger specific activity. The presence of exchanger activity most closely correlates with a polypeptide of molecular mass 215-kDa. Exchanger activity in both the crude rod outer segments and the purified exchanger is specifically dependent upon the presence of K+ in the assay medium; neither choline nor Li+ can substitute for K+.
Nicoll
D A
DA
Department of Ophthalmology, University of Chicago, Illinois 60637.
Applebury
M L
ML
D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002352:Carrier Proteins / Q000302:isolation & purification* / Q000378:metabolism; D002417:Cattle; D002499:Centrifugation, Density Gradient; D002846:Chromatography, Affinity; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D007700:Kinetics; D008970:Molecular Weight; D010786:Photoreceptor Cells / Q000378:metabolism*; D012374:Rod Cell Outer Segment / Q000378:metabolism*; D019831:Sodium-Calcium Exchanger
D002352:Carrier Proteins; D019831:Sodium-Calcium Exchanger; D002118:Calcium