Sodium-dependent neutral amino acid transport in native and reconstituted membrane vesicles from Ehrlich cells.

Cholate extracts of a highly purified plasma membrane preparation from Ehrlich ascites cells were reassembled by a Sephadex G-50 column chromatography procedure into small, single bilayer vesicles without supplementation with exogenous phospholipids.These reconstituted vesicles exhibited Na+-dependent, stereospecific, and rheogenic neutral amino acid transport similar to that in the native plasma membrane vesicles.The transport rate was somewhat slower in the reconstituted vesicles, however, than in the plasma membrane vesicles.Addition of phospholipid mixtures to the cholate extracts decreased the overall density of the reconstituted vesicles and changed not only their phospholipid composition, but also their protein content and composition.The reconstituted vesicles prepared with an equal mixture of phosphatidylcholine and phosphatidylethanolamine exhibited neutral amino acid transport characteristics similar to the reconstituted vesicles prepared without addition of exogenous phospholipids.By contrast, the reconstituted vesicles prepared with a 3:l mixture of phosphatidylcholine and phosphatidylserine exhibited only Na+-independent, stereospecific L-alanine uptake.This alteration of transport characteristics is attributed to changes in the protein composition rather than the phospholipid composition of the reconstituted vesicles.A similar Na+-independent stereospecific uptake of Lalanine was observed with a reconstituted subfraction of the cholate extract that was obtained by column chromatography over Bio-Gel A-15m.The observed Na+-independent uptake possibly represents the facilitated diffusion component of the Na+-dependent neutral amino acid transport system (System A) of Ehrlich cells.Plasma membrane vesicles derived from various types of animal cells have been used to investigate the mechanism of neutral amino acid transport (1-11).These studies indicate that, as in intact mammalian cells (12-15), accumulation of neutral amino acids in membrane vesicles requires a sodium gradient (outside greater than inside) and is stimulated by an electrical potential (negative inside).Reconstituted systems prepared from a cholate-solubilized membrane fraction of Ehrlich ascites cells or mouse fibroblasts also exhibit sodiumdependent neutral amino acid uptake.The transport properties of these reconstituted systems are very similar to those