Site density of the sodium-calcium exchange carrier in reconstituted vesicles from bovine cardiac sarcolemma.
The site density of the Na2+-Ca2+ exchanger in bovine cardiac sarcolemma was estimated from measurements of the fraction of reconstituted proteoliposomes exhibiting exchange activity. Sarcolemmal vesicles were solubilized with 1% Triton X-100 in the presence of either 100 mM NaCl or 100 mM KCl; after a 20-40-min incubation period on ice, sufficient KCl, NaCl, CaCl2, and soybean phospholipids were added to each extract to give final concentrations of 40 mM NaCl, 120 mM KCl, 0.1 mM CaCl2, and 10 mg/ml phospholipid. These mixtures were then reconstituted into proteoliposomes, and the rate of 45Ca2+ isotopic exchange was measured under equilibrium conditions. Control studies showed that Na+-Ca2+ exchange activity was completely lost if Na+ was not present during solubilization. The difference in 45Ca2+ uptake between vesicles initially solubilized in the presence or absence of NaCl therefore reflected exchange activity and corresponded to 3.1 +/- 0.3% of the total 45Ca2+ uptake by the entire population of vesicles, as measured in the presence of the Ca2+ ionophore A23187. Assuming that each vesicle with exchange activity contained 1 molecule of the Na+-Ca2+ exchange carrier, a site density of 10-20 pmol/mg of protein for the exchanger was calculated. The Vmax for Na+-Ca2+ exchange activity in the proteoliposomes was approximately 20 nmol/mg of protein.s which indicates that the turnover number of the exchange carrier is 1000 s-1 or more. Thus, the Na+-Ca2+ exchanger is a low density, high turnover transport system.
Cheon
J
J
Roche Institute of Molecular Biology, Roche Research Center, Nutley, New Jersey 07110.
Reeves
J P
JP
D000818:Animals; D002352:Carrier Proteins / Q000378:metabolism*; D002417:Cattle; D009206:Myocardium / Q000378:metabolism*; D010743:Phospholipids / Q000378:metabolism; D011092:Polyethylene Glycols; D011506:Proteins / Q000378:metabolism; D011510:Proteolipids / Q000032:analysis; D012508:Sarcolemma / Q000378:metabolism; D019831:Sodium-Calcium Exchanger; D012995:Solubility
D002352:Carrier Proteins; D010743:Phospholipids; D011506:Proteins; D011510:Proteolipids; D019831:Sodium-Calcium Exchanger; C020485:proteoliposomes; D011092:Polyethylene Glycols