2 SEPARATE POOLS OF SPHINGOMYELIN IN BHK CELLS

In BHK cells labelled to equilibrium with [H-3]choline and treated with sphingomyelinase the surface pool of sphingomyelin is degraded very rapidly (half-time 10 min) but the internal pool of sphingomyelin which accounts for about 30% of the total is only degraded slowly (half-time about 80 h) showing that the internal pool does not normally reach the surface. In [H-3]choline incorporation experiments the internal pool begins to accumulate radioactivity at about the same time as phosphatidylcholine (30 min) but label does not enter the surface pool of sphingomyelin for a further 90 min. The internal and external pools reach the same specific activity only after about 20 h. Pulse-chase analysis with [H-3]choline shows that radioactivity in each pool of sphingomyelin continues to increase when the specific radioactivity of phosphatidylcholine is decreasing, consistent with both pools being synthesised from a phosphatidylcholine precursor. The results suggest that sphingomyelin in BHK cells is present not only in the plasma membrane but also in a more rapidly labelling pool which does not mix with the surface pool.