UTILIZATION OF PHOSPHATIDYLCHOLINE AND PRODUCTION OF DIRADYLGLYCEROL AS A CONSEQUENCE OF SPHINGOMYELIN SYNTHESIS

1. After the degradation of cell-surface sphingomyelin (SM) by exogeno us sphingomyelinase (SMase), the resynthesis of SM by baby-hamster kid ney (BHK) and human leukaemia-60 (HL-60) cells was examined in relatio n to utilization of substrate phosphatidylcholine (PtdCho) and generat ion of the expected product, diradylglycerol (DRG). Using [H-3]choline -labelled BHK cells incubated in non-radioactive medium, SMase caused a release of phosphocholine, which was derived approximately equally f rom SM and PtdCho, consistent with the anticipated resynthesis of SM a t the expense of PtdCho, However, with choline-labelled cells incubate d in radioactive medium or [C-14]acetate-labelled cells treated with S Mase, no loss of radioactivity from PtdCho or accumulation of labelled DRG was observed, suggesting that any DRG produced as a consequence o f SM synthesis must have been rapidly converted back into PtdCho. In c ontrast, SMase treatment of HL-60 cells caused more than a doubling of DRG levels at the expense of PtdCho, and this appears to be the first demonstration of a rise in DRG related to the synthesis of SM. The DR G produced consisted of about 80 % 1,2-diacylglycerol and 18 % 1-O-alk yl-2-acylglycerol species, a similar composition to that of the DRG ba ckbone of total cell PtdCho, 2, The requirement for cell-surface PtdCh o in the biosynthesis of SM by BHK cells was also investigated. Treatm ent of [H-3]choline-labelled BHK cells with Bacillus cereus PtdCho-spe cific phospholipase C (PLC) rapidly degraded about 6 % of the total Pt dCho, which was assumed to represent the cell-surface pool. This did n ot appear to be the pool of PtdCho required for SM synthesis, since (a ) the released phosphocholine was additional to that derived from PtdC ho in cells treated with SMase and (b) treatment with PLC did not affe ct SM synthesis, either de novo or in response to degradation of cell- surface SM by SMase. These findings suggest either that there is no SM synthase in the plasma membrane or, if it is present, substrate.