Identification and partial purification of the cardiac sodium-calcium exchange protein.
Cardiac sarcolemmal vesicles were solubilized in 2% sodium cholate/0.5 M NaCl/soybean phospholipids (25 mg/ml), and reconstituted following the procedure of Miyamoto and Racker [Miyamoto, H. & Racker, E. (1980) J. Biol. Chem. 255, 2656-2658]. Initial rates of Na-Ca exchange in the reconstituted proteoliposomes were 17-fold higher than in the native vesicles. Total recovery of exchange activity often exceeded 100%, indicating that the exchange system had been activated by the reconstitution procedure. Examination of native and reconstituted vesicles by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate revealed two protein bands that were substantially enriched in the reconstituted system: one at 48 kDa and a diffuse band centered at 82 kDa. A cholate extract of the vesicles was applied to a Sephacryl S-300 column and the various eluted fractions were reconstituted, assayed for Na-Ca exchange and examined by polyacrylamide gel electrophoresis. The activity profile, after correcting for loss of activity on the column, showed a good correlation with the presence of the diffuse 82-kDa band. A cholate extract of the vesicles was treated with Pronase (1 mg/ml) for 10 min at 37 degrees C and reconstituted using a procedure similar to that described by Wakabayashi and Goshima [Wakabayashi, S. & Goshima, K. (1982) Biochim. Biophys. Acta 693, 125-133]. The resulting proteoliposomes catalyzed Na-Ca exchange with a specific activity 30- to 100-fold greater than that of native vesicles. Upon examination by polyacrylamide gel electrophoresis, these proteoliposomes exhibited a single major band at 82 kDa, with several minor bands at lower molecular weights that migrated identically to the components of Pronase. The results suggest that the 82-kDa band represents the cardiac Na-Ca exchange protein.
D000818:Animals; D002118:Calcium / Q000378:metabolism; D002352:Carrier Proteins / Q000302:isolation & purification / Q000378:metabolism*; D002417:Cattle; D019826:Cholic Acid; D002793:Cholic Acids; D002845:Chromatography; D004591:Electrophoresis, Polyacrylamide Gel; D008081:Liposomes / Q000378:metabolism; D008970:Molecular Weight; D009206:Myocardium / Q000032:analysis*; D011402:Pronase / Q000494:pharmacology; D012508:Sarcolemma / Q000032:analysis*; D019831:Sodium-Calcium Exchanger
D002352:Carrier Proteins; D002793:Cholic Acids; D008081:Liposomes; D019831:Sodium-Calcium Exchanger; D011402:Pronase; D019826:Cholic Acid; D002118:Calcium