A new method for making phospholipid vesicles and the partial reconstitution of the (Na+, K+-activated ATPase

It has been found that vesicles of phospholipid (96% (w/w) phosphatidylcholine; 4% (w/W) phosphatidylserine) can be formed by dialysis of a solution of the phospholipid in the detergent, sodium deoxycholate. Depending upon the composition of the dialysis medium, small closed vesicles apparently bounded by one or two membranes or large multi-walled structures are produced. The former are predomiant if only univalent ions are present in the dialysis buffer. As the Mg²⁺ concentration is raised above about 0.1 mM multiwalled structures are found.The (Na⁺,K⁺)-ATPase (EC 3.6.1.3) from cattle brain microsomes has been solubilized with deoxycholate. Dialysis of this material after the addition of the above phospholipid mixture in detergent also produces membrane-bound vesicles. Sucrose density gradient centrifugation has been used to demonstrate that the phospholipid, (Na⁺,K⁺)-ATPase and protein reaggregate together only if the phospholipid and solubilized protein are mixed before dialysis. This method of forming artificial membranes may be a useful way of studying transport proteins in isolation as the vesicles appear to be small and closed.