Simple and effective purification of a Na+-dependent amino acid transport system from Ehrlich ascites cell plasma membrane.
A reconstitution assay was used to measure transport activity during purification of a Na+-dependent amino acid transporter from Ehrlich cell plasma membrane. Cholate/urea-solubilized membranes were fractionated on a Sepharose 6B column and transport activity was recovered in the column void volume. Centrifugation of the void volume fraction at 105,000 X g and reextraction of the pellet with 1% octyl glucoside led to recovery of an extract whose specific transport activity was nearly 30-fold higher than that of the original solubilized extract with a recovery of 38% of the original activity. The properties of amino acid uptake in the purified reconstituted transporter were identical to those in native plasma membrane vesicles. The major component present in the purified fraction had a molecular mass of 120-130 kDa. Strong evidence that this 120- to 130-kDa peptide contains a component of the amino acid transporter was obtained by immunoprecipitation of transport activity from solubilized membranes with an antibody against the 120- to 130-kDa peptide. This study tentatively identifies a component of the Na+-dependent amino acid transporter as a peptide with an apparent molecular mass of 120-130 kDa.
McCormick
J I
JI
McGill University, Department of Biochemistry, Montreal, PQ Canada.
Johnstone
R M
RM
D002731:4-Chloromercuribenzenesulfonate / Q000494:pharmacology; D026905:Amino Acid Transport Systems; D000621:Aminoisobutyric Acids / Q000378:metabolism; D000818:Animals; D002286:Carcinoma, Ehrlich Tumor / Q000032:analysis*; D002352:Carrier Proteins / Q000302:isolation & purification*; D002462:Cell Membrane / Q000032:analysis; D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D005960:Glucosides; D007930:Leucine / Q000378:metabolism; D008970:Molecular Weight; D012964:Sodium / Q000378:metabolism*; D012995:Solubility
D026905:Amino Acid Transport Systems; D000621:Aminoisobutyric Acids; D002352:Carrier Proteins; D005960:Glucosides; C017911:2-(methylamino)isobutyric acid; C018619:octyl-beta-D-glucoside; D002731:4-Chloromercuribenzenesulfonate; D012964:Sodium; D007930:Leucine