Isolation of plasma membranes from cultured glioma cells and application to evaluation of membrane sphingomyelin turnover

Journal Article · Mon Oct 31 23:00:00 EST 1988 · Anal. Biochem.; (United States)

DOI:https://doi.org/10.1016/0003-2697(88)90055-3· OSTI ID:5990737

Cook, H W; Palmer, F B; Byers, D M; Spence, M W

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-/sup 3/H)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.

• Research Organization: Dalhousie Univ., Halifax, Nova Scotia (Canada)
• OSTI ID: 5990737
• Journal Information: Anal. Biochem.; (United States), Journal Name: Anal. Biochem.; (United States) Vol. 174:2; ISSN ANBCA
• Country of Publication: United States
• Language: English

Related Subjects

550201* -- Biochemistry-- Tracer Techniques
59 BASIC BIOLOGICAL SCIENCES
ALCOHOLS
AMINES
AMMONIUM COMPOUNDS
ANIMAL CELLS
ANIMALS
BIOLOGICAL MARKERS
BIOSYNTHESIS
CELL CONSTITUENTS
CELL CULTURES
CELL MEMBRANES
CENTRIFUGATION
CHOLINE
CONNECTIVE TISSUE CELLS
DISEASES
DRUGS
ENDOPLASMIC RETICULUM
ENZYMES
ESTERS
FIBROBLASTS
FRACTIONATION
GLIOMAS
HYDROXY COMPOUNDS
ISOTOPE APPLICATIONS
LABELLED COMPOUNDS
LIPIDS
LIPOTROPIC FACTORS
MAMMALS
MAN
MEMBRANES
MICROSOMES
NEOPLASMS
ORGANIC COMPOUNDS
ORGANIC PHOSPHORUS COMPOUNDS
ORGANOIDS
PHOSPHOLIPIDS
PRIMATES
QUATERNARY COMPOUNDS
SEPARATION PROCESSES
SOMATIC CELLS
SPHINGOMYELINS
SYNTHESIS
TRACER TECHNIQUES
TRITIUM COMPOUNDS
TUMOR CELLS
ULTRACENTRIFUGATION
VERTEBRATES