Rapid isolation of neuroblastoma plasma membranes on Percoll gradients. Characterization and lipid composition

Abstract

A purified plasma membrane fraction was isolated from cultured neuroblastoma (N1E-115) cells on a discontinuous gradient of 5, 25 and 35% Percoll within 1 h of cell disruption by nitrogen cavitation. Yield of plasma membrane, banding in the 25% Percoll ( d = ; 1.051), was high as judged by the recoveries of the marker enzymes, 5′-nucleotidase ( 58.0 ± 5.4%, n = ;5), alkaline phosphatase ( 46.0 ± 3.0%, n = ; 4) and Mg 2+-stimulated neutral sphingomyelinase ( 48.0 ± 4.2%, n = ; 3); enrichment of specific activities of these enzymes relative to total cell homogenate (lysate) were 10.9 ± 1.0-, 9.1 ± 1.0- and 9.6 ± 0.4- fold , respectively. Levels of marker enzymes for other organelles were less than 3% of total activity, except for microsomes (less than 9%). The plasma membrane fraction was further characterized by 2-, 5- and 6-fold higher content (nmol/mg protein) of total phospholipids, free cholesterol and sphingomyelin, respectively, compared to lysate. Ratios of free cholesterol to phospholipids and of sphingomyelin to phosphatidylcholine in the plasma membrane fraction were about 2-fold greater than that of lysate. The cholesterol ester content of plasma membrane ( 36 ± 8 nmol / mg protein ) was 2–3-fold higher than that of lysate. Sphingomyelin of the plasma membrane fraction had a higher concentration of long-chain fatty acids (more than 18 carbon atoms) relative to lysate or microsomes. Significant differences also were observed in the fatty acyl composition of diphosphatidylglycerol, cholesterol esters and triacylglycerol of plasma membrane. Thus, we have devised a rapid and reliable method for isolation of highly purified plasma membranes of cultured neuroblastoma cells that is suitable for comparison of metabolic relationships between the plasma membrane and other cellular organelles.