Partial purification of amino acid transport systems in Ehrlich ascites tumor cell plasma membranes.
Ehrlich ascites tumor cell plasma membranes were subjected to sequential selective protein extraction to identify protein components associated with amino acid transport. These membranes were extracted with Triton X-100 followed by 2,3-dimethylmaleic anhydride. Approximately 80% of the membrane proteins were extracted by these procedures while the original lipids were largely retained (approximately 70%). The quantity of carbohydrate per milligram protein in the residue increased on extraction, consistent with an enrichment of glycoprotein in the residue. The residual vesicles display the characteristic properties of Na+-coupled amino acid transport. These properties include Na+-stimulated uptake and Na+-gradient-stimulated uptake leading to an accumulation of the solute against its chemical gradient as well as inhibition of uptake by a competitive amino acid, L-methionine. The extracted vesicles exhibit a peak level of alpha-aminoisobutyrate uptake six times greater than that expected from equilibration of alpha-aminoisobutyrate. This accumulation is greater than that obtained with native vesicles, albeit slower. The accelerated exchange diffusion of L-leucine is not measurable in the residual vesicles after dimethylmaleic acid anhydride treatment, although it can be measured after Triton extraction. These results are consistent with the conclusion that the amino acid transport systems "A" (Na+-coupled) and "L" (Na+-independent) in Ehrlich cells, though having overlapping specificities for amino acids, and distinct physical entities.
D000596:Amino Acids / Q000378:metabolism*; D000818:Animals; D002286:Carcinoma, Ehrlich Tumor / Q000032:analysis*; D002352:Carrier Proteins / Q000302:isolation & purification* / Q000378:metabolism; D002458:Cell Fractionation / Q000379:methods; D002462:Cell Membrane / Q000032:analysis; D051379:Mice; D012964:Sodium / Q000378:metabolism; D014357:Trypsin / Q000494:pharmacology
D000596:Amino Acids; D002352:Carrier Proteins; D012964:Sodium; D014357:Trypsin