Reconstitution of neutral amino acid transport from partially purified membrane components from Ehrlich ascites tumor cells.
Solubilized protein fractions have been obtained from plasma membranes of Ehrlich ascites cells either by extraction with 0.5% Triton X-100 or by extraction with 2% cholate. Partial purification of the solubilized protein fraction has been obtained by utilizing a combination of ammonium sulfate precipitation and column chromatography. Leucine-binding activity has been detected in the Triton X-100 solubilized membrane fraction. The leucine-binding activity was measured by equilibrium dialysis and was saturable with high levels of leucine or phenylalanine and is not strongly effected by alanine. These properties are similar to those previously identified as System L. In addition, the cholate extracted protein fraction was partially purified and reconstituted into liposomes. Sodium dependent uptake of alanine and leucine could be demonstrated in the reconstituted vesicles. Concentrative uptake was dependent upon a sodium gradient. A membrane potential produced by valinomycin mediated potassium diffusion in the presence of sodium also stimulated amino acid transport in reconstituted liposomes.
D000409:Alanine / Q000378:metabolism; D000596:Amino Acids / Q000378:metabolism*; D000818:Animals; D001693:Biological Transport, Active; D002286:Carcinoma, Ehrlich Tumor / Q000473:pathology*; D002457:Cell Extracts; D066298:In Vitro Techniques; D007930:Leucine / Q000378:metabolism; D008081:Liposomes / Q000378:metabolism; D008564:Membrane Potentials; D008565:Membrane Proteins / Q000378:metabolism*; D008566:Membranes / Q000378:metabolism*; D008722:Methods; D013347:Subcellular Fractions / Q000378:metabolism
D000596:Amino Acids; D002457:Cell Extracts; D008081:Liposomes; D008565:Membrane Proteins; D007930:Leucine; D000409:Alanine