Separation of plasma membrane domains of calf thymocytes by affinity chromatography on ouabain-Sepharose.
Highly purified plasma membranes of calf thymocytes were fractionated by means of affinity chromatography on ouabain-Sepharose. By the method used two subfractions were obtained, one eluting freely from the affinity gel (MF1oua) and a second specifically retained by matrix-bound ouabain (MF2oua), with a total recovery of 95 per cent. Fractionation required the binding of matrix-bound ouabain to its plasma membrane receptor, i.e. (Na+ + K+)-ATPase. Increasing the temperature and binding time did not significantly alter the fractionation of plasma membranes into the two subfractions. Both plasma membrane subfractions separated by ouabain-Sepharose were of plasma membrane origin, as revealed by the identical specific activities of several membrane bound enzymes, gamma-glutamyl transpeptidase, alkaline phosphatase and Mg2+-ATPase in unseparated plasma membranes and in both subfractions, and by the identical amounts of the cytoskeletal protein actin in unseparated plasma membranes and subfractions. The plasma membrane subfractions MF1oua and MF2oua showed different structural and functional properties. In SDS-polyacrylamide gel electrophoresis polypeptides of 170, 150, 110, 94, 39, and 30 kDa were several-fold enriched in the adherent fraction, MF2oua. The phospholipid fatty acid composition of the plasma membrane subfractions proved to be different, as well. MF2oua contained significantly higher amounts of saturated fatty acids as compared to MF1oua. The specific activities of (Na+ + K+)-ATPase, Ca2+-ATPase and lysolecithin acyltransferase were highly enriched in the adherent fraction MF2oua, as compared to MF1oua. The data suggest that by the means of affinity chromatography on ouabain-Sepharose plasma membrane domains of the lymphocyte plasma membrane can be isolated, most probably implicated in the initiation of lymphocyte activation.
D000818:Animals; D002417:Cattle; D002458:Cell Fractionation / Q000379:methods; D002462:Cell Membrane / Q000648:ultrastructure*; D002499:Centrifugation, Density Gradient / Q000379:methods; D002846:Chromatography, Affinity / Q000379:methods; D006023:Glycoproteins / Q000032:analysis; D008214:Lymphocytes / Q000201:enzymology / Q000648:ultrastructure*; D008565:Membrane Proteins / Q000032:analysis; D008970:Molecular Weight; D010042:Ouabain / Q000031:analogs & derivatives; D012685:Sepharose / Q000031:analogs & derivatives; D000254:Sodium-Potassium-Exchanging ATPase / Q000032:analysis
D006023:Glycoproteins; D008565:Membrane Proteins; C052670:ouabain-sepharose; D010042:Ouabain; D012685:Sepharose; D000254:Sodium-Potassium-Exchanging ATPase