Partial purification of Na+-Ca2+ antiporter from plasma membrane of chick heart.
To study Na+-Ca2+ exchange, proteins of membrane vesicles from chick hearts were solubilized with cholate in the presence of phospholipids and the cholate extract was treated with pronase. These purified proteoliposomes, reconstituted by subsequent dilution and centrifugation to eliminate the cholate, catalyzed Ca2+ uptake depending on the intraliposomal Na+ (Nai+) concentration. The maximal amount of Ca2+ accumulating in the liposomes was 140 nmol/mg protein and the initial rates of Nai+-dependent Ca2+ uptake were routinely 20 to 40 nmol/mg per 3 s at 25 degrees C, but only 2 to 4 nmol/mg per 3 s for the crude proteoliposomes from the cholate extract not treated with pronase. Thus the pronase treatment resulted in 10-fold purification. Nai+-dependent Ca2+ uptake by purified proteoliposomes was 30- to 50-fold higher than that by the initial membrane vesicles. The fundamental properties of Nai+-dependent Ca2+ uptake in purified proteoliposomes such as Km for Ca2+, the sensitivity for Na+ and pH dependency, were nearly equal to those in membrane vesicles and crude proteoliposomes. Thus, pronase treatment was very useful for obtaining reconstituted liposomes containing highly enriched Na+-Ca2+ antiporters which were functionally intact.
D000818:Animals; D002118:Calcium / Q000378:metabolism; D002352:Carrier Proteins / Q000302:isolation & purification* / Q000378:metabolism; D002462:Cell Membrane / Q000378:metabolism; D002645:Chickens; D007700:Kinetics; D008081:Liposomes; D008565:Membrane Proteins / Q000302:isolation & purification* / Q000378:metabolism; D009206:Myocardium / Q000378:metabolism*; D010743:Phospholipids / Q000494:pharmacology; D011510:Proteolipids / Q000378:metabolism; D019831:Sodium-Calcium Exchanger
D002352:Carrier Proteins; D008081:Liposomes; D008565:Membrane Proteins; D010743:Phospholipids; D011510:Proteolipids; D019831:Sodium-Calcium Exchanger; C020485:proteoliposomes; D002118:Calcium