A new de novo pathway for the formation of 1-alkyl-2-acetyl-sn-glycerols, precursors of platelet activating factor. Biochemical characterization of 1-alkyl-2-lyso-sn-glycero-3-P:acetyl-CoA acetyltransferase in rat spleen.
1-Alkyl-2-acetyl-sn-glycero-3-phosphocholine (alkylacetyl-GPC, platelet activating factor (PAF] can be biosynthesized either by acetylation of alkyllyso-GPC 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 novo 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 some 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 degrees C). Alkyllyso-GP:acetyl-CoA acetyltransferase had a pH optimum of 8.4 at 23 degrees C and was located in the microsomal fraction. The apparent Km for acetyl-CoA under these conditions was 226 microM and the optimal concentration of alkyllyso-GP ranged between 16 and 25 microM. 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.
D000105:Acetyl Coenzyme A / Q000378:metabolism; D000123:Acetyltransferases / Q000378:metabolism*; D000818:Animals; D002855:Chromatography, Thin Layer; D003978:Diazomethane / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D008861:Microsomes / Q000201:enzymology; D009251:NADPH-Ferrihemoprotein Reductase / Q000378:metabolism; D010744:Phosphoric Monoester Hydrolases / Q000378:metabolism; D010972:Platelet Activating Factor / Q000096:biosynthesis*; D051381:Rats; D013154:Spleen / Q000201:enzymology*; D013696:Temperature; D014638:Vanadates; D014639:Vanadium / Q000494:pharmacology
D010972:Platelet Activating Factor; D014639:Vanadium; D014638:Vanadates; D003978:Diazomethane; D000105:Acetyl Coenzyme A; D009251:NADPH-Ferrihemoprotein Reductase; D000123:Acetyltransferases; C027628:acetyl-CoA-1-alkyl-2-lyso-sn-glycero-3-phosphocholine acetyltransferase; D010744:Phosphoric Monoester Hydrolases; C048557:alkylacetylglycerophosphatase