Wakabayashi S, Goshima K
Biochim Biophys Acta. 1982 Dec 8;693(1):125-33. doi: 10.1016/0005-2736(82)90478-3.
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.
为了研究钠钙交换，在磷脂存在的情况下，用胆酸盐溶解来自鸡心脏的膜囊泡蛋白，并用链霉蛋白酶处理胆酸盐提取物。通过随后的稀释和离心以除去胆酸盐来重构这些纯化的蛋白脂质体，其催化的钙离子摄取取决于脂质体内的钠离子（Nai +）浓度。脂质体中积累的钙离子最大量为140 nmol/mg蛋白质，在25℃下，依赖于Nai +的钙离子摄取的初始速率通常为每3秒20至40 nmol/mg，但对于未经链霉蛋白酶处理的胆酸盐提取物中的粗蛋白脂质体，每3秒仅为2至4 nmol/mg。因此，链霉蛋白酶处理导致了10倍的纯化。纯化的蛋白脂质体对Nai +依赖的钙离子摄取比初始膜囊泡高30至50倍。纯化的蛋白脂质体中对Nai +依赖的钙离子摄取的基本特性，如对钙离子的Km值、对钠离子的敏感性和pH依赖性，与膜囊泡和粗蛋白脂质体中的几乎相等。因此，链霉蛋白酶处理对于获得含有功能完整的高度富集的钠钙反向转运体的重构脂质体非常有用。
