GPI BIOSYNTHESIS IN MAMMALIAN-CELLS - COA-DEPENDENT ACYLATION OF GLCN-PI

We have used microsomes prepared from murine lymphoma cell lines to in vestigate the individual reactions by which glycosylphosphatidylinosit ol (GPI) is synthesized in mammalian cells. Previously, GTP was found to specifically stimulate the second reaction in the pathway, the deac etylation of GlcNAc-PI to GlcN-PI. An additional GPI precursor was det ected in incubations with GTP and was found to be GlcN-PI(acyl), the g lycolipid proposed to be the third intermediate in mammalian GPI biosy nthesis. Investigation into the factors that affect the formation of G lcN-PI(acyl) revealed that, in the presence of GTP, the addition of ei ther CoA or palmitoyl-Coa to the incubation greatly enhanced the amoun t of this product made. CoA stimulation of this reaction persisted eve n when ATP was depleted and no formation of acyl-CoA was possible, ind icating that the free CoA rather than an acyl-CoA is the actual effect or of GlcN-PI acylation. Therefore, we propose that the third reaction in mammalian GPI biosynthesis is catalyzed by a CoA-dependent transac ylase rather than an acyl-CoA acyltransferase.