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J. Biol. Chem., Vol. 264, Issue 7, 3786-3793, 03, 1989
Y Lange, MH Swaisgood, BV Ramos and TL Steck
The literature suggests that cholesterol and sphingomyelin might be
essentially confined to plasma membranes in mammalian cells; however, this
premise has thus far escaped a direct test. We explored the issue in three
ways. First, we fractionated whole homogenates of cultured human
fibroblasts by equilibrium sucrose density gradient centrifugation. We
found that the profiles of cholesterol and sphingomyelin were
indistinguishable from those of two plasma membrane markers, 5'
nucleotidase and [3H]galactose, which was conjugated to the surface of
intact cells from an exogenous donor by galactosyltransferase. Second, we
determined the relative surface areas of intact cells from their uptake of
1-(4-trimethyl-amino)phenyl-6- phenylhexa-1,3,5-triene, a cationic
fluorescent dye which partitions into but does not cross plasma membranes.
Relative to human red cell ghosts, the apparent surface area of the
fibroblasts was 17,500 microns2/cell while for canine hepatocytes, the
value was 11,500 microns2/cell. The relative ratios of cell cholesterol to
dye binding (hence, surface area) were quite similar in ghosts,
fibroblasts, and liver cells; namely 1.0, 1.12, and 0.67, respectively.
Finally, we found that the specific ratios of both cholesterol and
sphingomyelin to 5' nucleotidase were only 10% less in gradient-purified
plasma membranes than in whole homogenates. Similar results were obtained
using an entirely different method of purification: two-phase aqueous
partition. The cholesterol and sphingomyelin in fractions rich in other
membranes was closely proportional to their 5' nucleotidase content,
suggesting that the presence of these lipids reflected contamination by
plasma membrane fragments. The 5' nucleotidase/phospholipid ratio in the
purified plasma membrane fraction was roughly twice that in whole cells. We
conclude that the compartment marked by 5' nucleotidase in cultured human
fibroblasts contains approximately 90% of the two named lipids and half the
cell phospholipid phosphorus.
Plasma membranes contain half the phospholipid and 90% of the cholesterol and sphingomyelin in cultured human fibroblasts
Department of Pathology, Rush-Presbyterian-St. Luke's Medical Center, Chicago, Illinois 60612.
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