RESUMO
We have used microsomes prepared from murine lymphoma cell lines to investigate the individual reactions by which glycosylphosphatidylinositol (GPI) is synthesized in mammalian cells. Previously, GTP was found to specifically stimulate the second reaction in the pathway, the deacetylation of GlcNAc-PI to GlcN-PI. An additional GPI precursor was detected in incubations with GTP and was found to be GlcN-PI(acyl), the glycolipid proposed to be the third intermediate in mammalian GPI biosynthesis. Investigation into the factors that affect the formation of GlcN-PI(acyl) revealed that, in the presence of GTP, the addition of either CoA or palmitoyl-CoA to the incubation greatly enhanced the amount of this product made. CoA stimulation of this reaction persisted even when ATP was depleted and no formation of acyl-CoA was possible, indicating that the free CoA rather than an acyl-CoA is the actual effector of GlcN-PI acylation. Therefore, we propose that the third reaction in mammalian GPI biosynthesis is catalyzed by a CoA-dependent transacylase rather than an acyl-CoA acyltransferase.
Assuntos
Acetilglucosamina/análogos & derivados , Glicosilfosfatidilinositóis/biossíntese , Fosfatidilinositóis/metabolismo , Precursores de Proteínas/metabolismo , Acetilglucosamina/metabolismo , Acil Coenzima A/metabolismo , Acilação , Animais , Coenzima A/fisiologia , Linfoma/metabolismo , Camundongos , Palmitoil Coenzima A/metabolismo , Células Tumorais CultivadasRESUMO
In view of the fact that mepacrine (Mp) is usually used as an inhibitor of the endogenous phospholipase A2, and since this enzyme produces the release of arachidonic acid (AA) from membrane phospholipids, we studied the effect of different concentrations of Mp on the mobilization of [1-14C]AA in rat renomedullary phospholipids. During the acylation period, 0.1 mM Mp did not produce any significant change in the incorporation of [1-14C]AA into phosphatidylcholine (PC) and phosphatidylethanolamine (PE), and only a slight increase in phosphatidylinositol (PI). Higher concentrations of Mp (0.5 to 1.0 mM) produced a decrease of radioactivity in PE and PC with an increase in PI. Using prelabeled slices, a dose-dependent decrease in the 14C-radioactivity in PE and PC was observed, with a parallel increase in PI. This effect of Mp persisted even in the presence of a physiological activator of phospholipase A2, bradykinin (BK). No change in the net amount of phospholipids was observed at any of the Mp concentrations used. The results of this study show that Mp, at concentrations generally used to inhibit phospholipase A2, produced a transfer of arachidonic acid from PE and PC to PI, rather than a blockade in the release of AA from membrane phospholipids.