Isolation of plasma membranes from cultured glioma cells and application to evaluation of membrane sphingomyelin turnover.
A rapid and reliable method for the isolation of plasma membranes and microsomes of high purity and yield from cultured glioma cells is described. The procedure involves disruption by N2 cavitation, preliminary separation by centrifugation in Tricine buffer, and final separation on a gradient formed from 40% Percoll at pH 9.3. Enzyme and chemical markers indicated greater than 60% yield with six- to eightfold enrichment for plasma membranes and greater than 25% yield with three- to fourfold enrichment for a microsomal fraction consisting mainly of endoplasmic reticulum. The final fractions were obtained with high reproducibility in less than 1 h from the time of cell harvesting. Application of this procedure to human fibroblasts in culture is assessed. The isolation procedure was applied to investigations of synthesis and turnover of sphingomyelin and phosphatidylcholine in plasma membranes of glioma cells following incubation for 4-24 h with [methyl-3H]choline. These studies indicated that radioactivity from phosphatidylcholine synthesized in microsomes from exogenous choline may serve as a precursor of the head-group of sphingomyelin accumulating in the plasma membrane.
Cook
H W
HW
Atlantic Research Centre for Mental Retardation, Department of Pediatrics, Dalhousie University, Halifax, Nova Scotia, Canada.
Palmer
F B
FB
Byers
D M
DM
Spence
M W
MW
D015415:Biomarkers; D002462:Cell Membrane / Q000378:metabolism / Q000648:ultrastructure; D002478:Cells, Cultured; D002499:Centrifugation, Density Gradient / Q000379:methods*; D002794:Choline / Q000032:analysis; D004798:Enzymes / Q000378:metabolism; D005910:Glioma / Q000032:analysis / Q000648:ultrastructure*; D006801:Humans; D010713:Phosphatidylcholines / Q000378:metabolism; D013109:Sphingomyelins / Q000378:metabolism*; D014316:Tritium
D015415:Biomarkers; D004798:Enzymes; D010713:Phosphatidylcholines; D013109:Sphingomyelins; D014316:Tritium; D002794:Choline