Abstract
Plasma membranes, isolated from Ehrlich ascites tumor cells, were dissolved in 2% cholate, 4 M urea and then reformed into liposomes upon dialysis at 4 degrees with exogenous phospholipids. Reconstituted vesicles regain the ability to transport amino acids. Na+ was shown to accelerate the uptake of alpha-aminoisobutyrate, phenylalanine, and methionine, but not leucine or epsilon-aminohexanoic acid. With the reconstituted vesicles, methionine, but not leucine, inhibited the uptake of alpha-aminoisobutyrate. An apparent Km value for alpha-aminoisobutyrate uptake of 3.0 mM was obtained. This value is close to that observed with the intact cells and the native membrane vesicles. A Na+ gradient (high Na+ outside) increased alpha-aminoisobutyrate uptake, whereas a reversed gradient (high Na+ inside) increased alpha-aminoisobutyrate efflux. The latter flux was increased by valinomycin, suggesting electrogenic transport. A modest extent of coupling between a Na+ gradient and uphill flow of alpha-aminoisobutyrate was observed.
Highlights
Since we have developed a method for the large scale isolation of plasma membrane vesicles (261 which show Na+-stimulated and Na+ gradient-stimulated amino acid uptake and exchange diffusion of amino acids [27, 28], it appeared to us worthwhile to determine whether the membranes from Ehrlich cells could be solubilized and reconstituted into vesicles capable of amino acid transport
The data obtained showed that the a-aminoisobutyrate transport system was saturable (Fig. 7) and that the K, obtained (-3 mM) was close to that observed with the original membrane vesicles [27] and the intact cells [42, 47]
In dealing with transport systems which do not involve a chemical transformation of either the solute or carrier, isolation of a specific protein with transport function from dissolved membranes has been achieved with very few systems
Summary
With several of these lipids there was no evidence for time-dependent uptake of Na+ or amino acids, 10 to 20% of the original protein was found in the vesicles. To obtain such evidence the following experiments were performed: (a) vesicles were incubated for 30 min in K+ medium with a-amino[Wlisobutyrate to equilibrate the vesicles with these solutes;
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
