Effect of alkali cations on freeze-thaw-dependent reconstitution of amino acid transport from Ehrlich ascites cell plasma membrane.
Na+-dependent amino acid transport can be reconstituted by gel filtration of disaggregated plasma membrane and asolectin vesicles coupled to a freeze-thaw cycle. The resultant transport activity is markedly affected by the nature of the reconstitution medium. Reconstitution in K+ permits the formation of active liposomes, whereas reconstitution in Na+, Li+, or choline does not. Electron micrographs of K+ liposomes show a wide variation in liposome sizes. Ficoll density gradient fractionation of K+ liposomes shows that the largest vesicles are lipid rich, have the lowest density, and have the highest level of Na+-dependent amino acid transport. Liposomes formed in Na+ have a 34% smaller trapped volume than K+ liposomes and lack a population of large vesicles. A second freeze-thaw in K+ restores activity to Na+ liposomes which now contain large low density active vesicles. Fluorescence measurements of freeze-thaw-induced mixing of vesicle lipids indicates that the absence of large vesicles in Na+ liposomes is due to inhibition by Na+ of lipid vesicle fusion events during freezing and thawing. The large vesicle fraction is enriched in a 125-kDa peptide. It has not yet been established whether this peptide is part of the transport system for neutral amino acids.
D000596:Amino Acids / Q000378:metabolism*; D000621:Aminoisobutyric Acids / Q000378:metabolism; D000818:Animals; D001693:Biological Transport, Active / Q000187:drug effects; D002286:Carcinoma, Ehrlich Tumor / Q000378:metabolism*; D002499:Centrifugation, Density Gradient; D002794:Choline / Q000494:pharmacology; D003470:Culture Media; D004591:Electrophoresis, Polyacrylamide Gel; D005615:Freezing; D006863:Hydrogen-Ion Concentration; D008094:Lithium / Q000494:pharmacology; D051379:Mice; D008854:Microscopy, Electron; D011188:Potassium / Q000494:pharmacology; D012964:Sodium / Q000378:metabolism*
D000596:Amino Acids; D000621:Aminoisobutyric Acids; D003470:Culture Media; C100049:2-aminoisobutyric acid; D008094:Lithium; D012964:Sodium; D002794:Choline; D011188:Potassium