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 εm²/cell while for canine hepatocytes, the value was 11,500 εm²/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.