Rapid isolation and lipid characterization of plasma membranes from normal and malignant lymphoid cells of mouse.
A rapid isolation method was developed for plasma membranes from mouse lymphoid cells such as lymph node lymphocytes, thymocytes, radiation-induced thymoma cells and L1210 cells. Lysates of these lymphoid cells were prepared by Dounce homogenization under hypotonic conditions and directly layered on sucrose step density gradients containing 2 mM CaCl2 and 5 mM MgCl2, and centrifuged at 52 000 X g for 1 h. Plasma membrane fractions appeared at the interface between 20 and 42% sucrose in the gradients. The procedure permitted purified membranes from cells to be obtained within 3 h, and the preparations appeared to be uniform by electron microscopy. Specific activities of (Na+ + K+)-ATPase, Mg2+-ATPase and 5'-nucleotidase of the isolated plasma membranes were enriched 23- to 61-fold, 12- to 15-fold and 18- to 34-fold, respectively, in comparison with those of the corresponding cell homogenates. Cholesterol content of the malignant cell membranes was lower than that of the normal membranes and the molar ratio of cholesterol to phospholipid of the malignant cell membranes was also lower than that of the normal membranes. A decreased plasmalogen content was observed in the malignant plasma membranes, together with a higher percentage of phosphatidylethanolamine and a lower percentage of phosphatidylserine. In the normal cell membranes, thymocytes contained a higher percentage of phosphatidylcholine and a lower percentage of sphingomyelin than those of the lymph node lymphocytes. At all temperature ranges (5 to 40 degrees C) the plasma membranes of the malignant cells had lower microviscosity than those of the normal cells.
D000818:Animals; D002462:Cell Membrane / Q000648:ultrastructure*; D002499:Centrifugation, Density Gradient / Q000379:methods; D008214:Lymphocytes / Q000648:ultrastructure*; D008563:Membrane Lipids / Q000032:analysis*; D051379:Mice; D008810:Mice, Inbred C57BL; D008854:Microscopy, Electron; D009374:Neoplasms, Experimental / Q000648:ultrastructure; D009381:Neoplasms, Radiation-Induced / Q000648:ultrastructure*; D010743:Phospholipids / Q000032:analysis; D013050:Spectrometry, Fluorescence; D013696:Temperature; D013945:Thymoma / Q000648:ultrastructure*; D013950:Thymus Gland / Q000648:ultrastructure*; D013953:Thymus Neoplasms / Q000648:ultrastructure*; D014783:Viscosity
D008563:Membrane Lipids; D010743:Phospholipids