BIOSYNTHESIS OF PLATELET-ACTIVATING-FACTOR IN CULTURED MAST-CELLS - INVOLVEMENT OF THE COA-INDEPENDENT TRANSACYLASE DEMONSTRATED BY ANALYSIS OF THE MOLECULAR-SPECIES OF PLATELET-ACTIVATING-FACTOR

We have recently demonstrated that arachidonate [20:4(5,8,11,14)] was primarily linked to the hexadecyl (16:0) and octadecenyl (18:1) specie s of alkylacyl derivatives of glycerolphosphocholine (GroPCho). Consis tent with the involvement of arachidonate-specific CoA-independent tra nsacylase in the synthesis of platelet-activating factor (PAF; 1-0-alk yl-2-acetyl-GroPcho), 16:0 and 18:1 PAF species were formed upon antig en stimulation [Joly, F., Breton, M., Wolf, C., Ninio, E. & Colard, 0. (1992) Biochim. Biophys. Acta 1125, 305-312]. In the present work, ad dition of lyso-PAF to mast cells resulted in PAF production. We analyz ed the PAF species formed in the presence of a defined lyso-PAF molecu lar species in order to differentiate between either direct acetylatio n or involvement of the membrane precursor. The 18:1 lyso-PAF was more effective than the 16:0 in producing PAF which was composed of 95% 18 :1 PAF, the balance being 16:0, indicating that part of the acetylated lyso-PAF originated from the cellular pool of alkyl-arachidonyl-GroPC ho in resting cells. Consistent with alkyl-arachidonyl-GroPCho species content and acetyltransferase specificity, similar amounts of 16:0 an d 18:1 PAF species were formed when mast cells were stimulated with an tigen. Supplemented with 16:0 or 18:1 lyso-PAF, antigen-stimulated mas t cells responded by 230% and 125% increase in PAF synthesis, respecti vely. As expected, the amount of the PAF species corresponding to the added lyso-PAF was increased. More interestingly, addition of 16:0 lys o-PAF almost doubled the amount of 18:1 PAF content as compared to ant igen alone, thus indicating that the lyso-PAF formed via the CoA-indep endent transacylase was significantly used for PAF synthesis, despite a large excess of exogenous lyso-PAF. The CoA-independent transacylase , measured using [H-3]lyso-PAF as a substrate in sonicates from antige n-stimulated cells, was decreased concurrently with PAF formation. In conclusion, we show that when lyso-PAF is added to mast cells, a direc t acetylation may occur. However, PAF is preferentially synthesized th rough a mechanism involving the CoA-independent transacylase reaction.