Isolation and characterization of plasma membranes from human leukemic lymphocytes.

Summary A method for the isolation of lymphocyte plasma membranes from patients with chronic lymphocytic leukemia is described. Lymphocytes were disrupted in a hypotonic bicarbonate medium using a Dounce homogenizer. The plasma membranes fraction was finally collected from a continuous sucrose gradient at density 1.115 (g/ml). Electron micrographs of this material showed membranes and small dense vesicles of unidentified origin. The enrichment of this fraction in adenosine 5′-monophosphatase, Mg2+:Na+:K+:adenosine triphosphatase, and uridine diphosphatase, considered as plasma membrane markers, was, respectively, 43-, 23-, and 42-fold. The only noticeable contamination of plasma membranes was lysosomal material as attested by a 4.7-fold increase in β-glucuronidase specific activity. It was calculated, however, that lysosomes accounted for less than 10% in the plasma membrane fraction. The concentration of cholesterol and total phospholipids per mg of plasma membrane protein was, respectively, 185 and 965 µg, thus 10- and 6-fold higher than in the whole homogenate. The molar ratio of cholesterol to phospholipids was 0.38.