Nonidet P-40 extraction of lymphocyte plasma membrane. Characterization of the insoluble residue

Abstract

Purified preparations of lymphocyte plasma membrane were extracted exhaustively with Nonidet P-40 in Dulbecco''s phosphate-buffered saline medium. The insoluble fraction, as defined by sedimentation at 106 g-min, contained about 10% of the membrane protein as well as cholesterol and phospholipid. The lipid/protein ratio, cholesterol/phospholipid ratio and sphingomyelin content were increased in the residue. Density-gradient centrifugation suggested that the lipid and protein form a common entity. As judged by sodium dodecyl sulfate/polyacrylamide-gel electrophoresis, the Nonidet P-40-insoluble fractions of the plasma membranes of human B lymphoblastoid cells and pig mesenteric lymph node lymphocytes possessed similar qualitative polypeptide compositions but differed quantitatively. Both residues comprised major polypeptides of MW 28,000, 33,000, 45,000 and 68,000, together with a prominent band of MW 120,000 in the human and of MW 200,000 in the pig. The polypeptides of MW 28,000, 33,000, 68,000 and 120,000 were probably located exclusively in the Nonidet P-40-insoluble, residue, which also possessed a 4-fold increase in 5''-nucleotidase specific activity. A reproducible fraction of lymphocyte plasma membrane is insoluble in non-ionic detergents and this fraction possesses a unique polypeptide composition. By analogy with similar studies with erythrocyte ghosts, it appears likely that the polypeptides are located on the plasma membrane''s cytoplasmic face.