Reconstitution of the sodium-calcium exchanger from cardiac sarcolemmal vesicles.
The Na+-Ca2+ exchanger was extracted from cardiac sarcolemmal vesicles and reconstituted into phospholipid vesicles by a cholate-dialysis method. Reconstitution was attempted with different phospholipids. Phosphatidylcholine alone was ineffective, whereas phosphatidylcholine and phosphatidylethanolamine (1:1, w/w) showed high activity, but a significant Ca2+ uptake in the absence of Na+ gradient. Optimal reconstitution was obtained with a mixture of phosphatidylcholine and phosphatidylserine (9:1, mol/mol). The reconstituted proteoliposomes showed an ouabain-sensitive (Na+ + K+)-ATPase activity and a Na+-Ca2+ exchange with a specific activity comparable to that of the original vesicles. The specificity toward Na+ was also recovered. A partial purification of the exchanger was obtained by the method of transport-specificity fractionation ( Goldin , S.M. and Rhoden , V. (1978) J. Biol. Chem. 253, 2575-2583). When proteoliposomes were reconstituted with sodium oxalate inside and incubated with calcium in the presence of an outwardly directed Na+ gradient, the vesicles containing the Na+-Ca2+ exchanger specifically accumulated calcium which precipitated inside as calcium oxalate. The resulting increase in density allowed separation of the proteoliposomes containing the Na+-Ca2+ exchanger from the rest of the vesicles on a sucrose density gradient.
D000584:Amiloride / Q000494:pharmacology; D000818:Animals; D000001:Calcimycin / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism*; D002122:Calcium Chloride / Q000378:metabolism; D002352:Carrier Proteins / Q000302:isolation & purification / Q000378:metabolism*; D004285:Dogs; D007700:Kinetics; D009206:Myocardium / Q000378:metabolism*; D010743:Phospholipids; D012508:Sarcolemma / Q000187:drug effects / Q000378:metabolism*; D019831:Sodium-Calcium Exchanger; D000254:Sodium-Potassium-Exchanging ATPase / Q000378:metabolism
D002352:Carrier Proteins; D010743:Phospholipids; D019831:Sodium-Calcium Exchanger; D000001:Calcimycin; D000584:Amiloride; D000254:Sodium-Potassium-Exchanging ATPase; D002122:Calcium Chloride; D002118:Calcium