Isolation and characterization of plasma membranes from friend erythroleukaemic cells. A study with sphingomyelinase C

Abstract

Plasma membranes have been prepared from Friend erythroleukaemic cells using a Dounce homogenization technique followed by differential and sucrose gradient centrifugations. (1) A plasma membrane fraction was obtained which showed a 20- to 30-fold enrichment in 5′-nucleotidase, alkaline phosphodiesterase I, alkaline phosphatase and in 32P-labeled (poly)phosphoinositides. About 1% of the total protein, 6–7% of phospholipid, 8–9% of cholesterol and 12–15% of each of the above markers were recovered in the plasma membrane fraction with an average yield of 15–20%. The plasma membrane was characterized by a high cholesterol to phospholipid molar ratio (0.626), a 2-fold enrichment in sphingomyelin and in phosphatidylserine as compared to the whole cell and by the complete absence of diphosphatidylglycerol. (2) When compared to the phospholipid composition of the mature mouse erythrocyte membrane, the plasma membrane of the Friend cell only differs by a higher phosphatidylcholine and a lower phosphatidylethanolamine content, whereas the levels of sphingomyelin and phosphatidylinositol plus phosphatidylserine are similar. (3) Friend cells were treated with sphingomyelinase C ( S. aureus) under non-lytic conditions and subsequently submitted to subcellular fractionation. The results showed that the plasma membrane accounted for 38.5% of the total phospholipid, 64.1% of the total cholesterol and about 4.4% of the total protein content of Friend cells. (4) Sphingomyelin appeared to be asymmetrically distributed in the plasma membrane of Friend cells, with about 85% of this phospholipid being present in the outer monolayer.