Sodium Transport by Phospholipid Vesicles Containing Purified Sodium and Potassium Ion-activated Adenosine Triphosphatase

When the purified sodium and potassium ion-activated ATPase (NaK-ATPase) from Squalus acanthias is added to a sonicated mixture of egg lecithin and cholate and dialyzed, vesicles are formed which trap either glucose or inulin. These vesicles are capable of ATP-dependent 22Na+ uptake. Optimum 22Na+ uptake occurs after 2 days dialysis at 4°. 22Na+ uptake was considerably higher at 25° than at 37°, presumably because of a higher lability of the vesicles at the higher temperature. Optimum incubation time for 22Na+ uptake was 1 to 2 hours. The pH profiles for NaK-ATPase activity and 22Na+ uptake were the same. 22Na+ uptake showed a dependence on the presence of Mg2+ but this dependence was not absolute, presumably because of some bound Mg2+ in the preparation. There was a good parallelism in the nucleotide specificity for the NaK-ATPase reaction and 22Na+ uptake, with ATP being the most effective substrate, UTP the poorest substrate, and CTP showing intermediate activity. Internal, but not external, ouabain inhibited 22Na+ uptake, as would be predicted from other observations in the literature which show that ouabain inhibits only on the side of the membrane opposite to the ATP substrate site. The ATP-dependent, ouabain-inhibitable uptake of 22Na+ was shown to be true active transport of Na+, by forming vesicles in the presence of 22Na+ so that the specific activity and concentration of Na+ were the same on both sides of the membrane. In the presence of external ATP there was an actual increase in the concentration of internal Na+ from 20 mm to 38.4 mm. The ratio of Na+ transported to ATP hydrolyzed was 0.3 to 0.4, which is only 10 to 15% of that reported in intact erythrocytes. In the absence of K+, an Na+-Na+ exchange which was dependent on ATP and inhibited by ouabain could be demonstrated; this is similar to Na+-Na+ exchange demonstrated in erythrocytes and muscle. Na+-Na+ exchange was a small percentage of active Na+ transport in the presence of K+. These observations provide strong evidence that vesicles formed from the purified NaK-ATPase of S. acanthias are capable of active sodium transport and that this transport shows the same properties as sodium transport observed in intact cells such as the erythrocyte.