Isolation and characterization of plasma membranes and intact nuclei from lymphoid cells

Abstract

A method has been developed for the rapid large scale isolation of plasma membranes and intact nuclei from RAJI lymphoid cells utilizing hypotonic lysis of cells after intracellular loading with glycerol followed by combined flotation-sedimentation within a discontinuous sucrose gradient. Nuclei may be isolated in about 1 h and plasma membranes in about 6 h from 1 to 20 g of cells. Intact nuclei, obtained in 90 to 95% yield based on lysed cells, was isolated by differential centrifugation and contained 16% DNA and about 30% of total cell sialic acid. A crude plasma membrane fraction was isolated by centrifugation onto a cushion of 38% sucrose (d 1.1683) and subsequently resolved into two subfractions. The less dense vesicles had an average d 1.127 and showed a 7-fold increase in specific activity for thymidine phosphodiesterase while the more dense (d 1.151) had a 20-fold concentration of enzyme activity. Activity of enzymes indicative of contamination with lysosomes, microsomes, mitochondria, and cytoplasm was negligible in these plasma membrane fractions. The less dense vesicles had a cholesterol:phospholipid ratio of 0.97 which was higher than that of the more dense vesicles (0.69). Otherwise, the analytical values for the two types of membrane vesicles were similar as both fractions contained like percentages of protein (approximately 30%), lipid (approximately 30%), and carbohydrate (approximately 15%) with trace amounts of RNA and DNA. Twenty-five per cent of the total cell sialic acid was in the plasma membrane fractions.