A New de Novo Pathway for the Formation of 1-Alkyl-2-acetyl-sn- glycerols, Precursors of Platelet Activating Factor

l-Alkyl-2-acetyl-sn-glycero-3-phosphocholine (alkylacetyl-GPC, platelet activating factor (PAF)) can be biosynthesized either by acetylation of alkyllysoGPC through a remodeling pathway or by the transfer of phosphocholine to alkylacetyl-sn-glycerol (alkylacetyl-G) via a putative de novo pathway involving a dithiothreitol-insensitive cholinephosphotransferase. However, the relevance of the de nouo pathway in the biosynthesis of PAF depends on the existence of enzymes that can directly synthesize alkylacetyl-G from 1-alkyl-2-lyso-sn-glycero-3-P (alkyllyso-GP) or eome other source. In this study, we demonstrated that microsomal preparations of rat spleen can synthesize alkylacetyl-GP by an alkyllyso-GP:acetyl-CoA acetyltransferase and that this intermediate is subsequently dephosphorylated by an alkylacetyl-GP phosphohydrolase to generate alkylacetyl-G. The properties of alkyllyso-GP:acetyl-CoA acetyltransferase were characterized under conditions where the contaminating activity of alkylacetyl-GP phosphohydrolase was minimal; this was accomplished by inhibiting the phosphohydrolase with the addition of sodium vanadate and sodium fluoride to the assay mixtures and incubating at relatively low temperatures (23 “C). AlkyllysoGP:acetyl-CoA acetyltransferase had a pH optimum of 8.4 at 23 “C and was located in the microsomal fraction. The apparent K, for acetyl-coA under these conditions was 226 PM and the optimal concentration of alkyllyso-GP ranged between 16 and 25 PM. Based on pH optima, substrate inhibition studies, and sensitivities to preincubation temperatures of the microsomes, it appears that alkyllyso-GP:acetyl-CoA acetyltransferase differs from the acetyltransferase responsible for the transfer of acetate from acetyl-coA to alkyllyso-GPC to form PAF. A variety of tissues had high activities of alkyllyso-GP:acetyl-CoA acetyltransferase, which indicates that this pathway is operational in many cell types. Our results document the existence of a complete de novo biosynthetic pathway for the assembly of PAF, and this route could be responsible for maintaining physiological levels of platelet activating factor for normal cell function.