Identification and partial purification of the cardiac sodium-calcium exchange protein.

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Abstract
Bovine 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. 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 (PAGE) in the presence of sodium dodecyl sulfate revealed 2 protein bands that were substantially enriched in the reconstituted system: one at 48 kDa [kilodalton] 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 PAGE. 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.degree. C and reconstituted using a procedure similar to that described by Wakabayashi and Goshima. The resulting proteoliposomes catalyzed Na-Ca exchange with a specific activity 30- to 100-fold greater than that of native vesicles. Upon examination by PAGE, these proteoliposome exhibited a single major band at 82 kDa, with several minor bands at lower MW that migrated identically to the components of Pronase. The 82-kDa band probably represents the cardiac Na-Ca exchange protein.