Isolation and characterization of the plasma membranes of cultured lymphoblasts from patients with cystic fibrosis and normal individuals.

ABSTRACT

Plasma membranes were isolated from two cystic fibrosis lymphoblastoid cell lines and two age and sex-matched normal cell lines after controlled cell disruption. The preparations were enriched approx. 20-fold in plasma membrane markers and were essentially free of markers for other organelles except the Golgi-localized enzyme, galactosyltransferase. Sodium dodecyl sulphate polyacrylamide gel electrophoresis showed virtually identical polypeptide profiles after staining with Coomassie blue. Membranes from normal cells contained 0.49--0.67 mumol/mg of total phospholipid/mg membrane protein and those from cystic fibrosis cells, 0.53-0.67 mumol. The cholesterol content was 0.35-0.41 ummol/mg protein for normal, and 0.43--0.49 mumol for cystic fibrosis cell membranes. The proportions of individual phospholipid classes and their fatty acid compositions as well as that of the total membrane lipid also did not differ significantly in the two cell types. The bulk lipid fluidity as reflected by the fluorescence polarization of beta-parinaric acid and diphenylhexatriene was also not changed. The carbohydrate composition of cell surface glycopeptides released with papain was not consistently different with the normal and cystic fibrosis cells. The quantity of fucose in intact plasma membranes was also quantitated enzymatically and found to be very similar in both cell types.