Biosynthesis of molecular species of CDP-diglyceride from endogenously-labeled phosphatidate in rat liver microsomes.
The biosynthesis of [14C] CDP-diglyceride was studied using rat liver microsomes which were endogenously labeled with [14C] phosphatidic acid by preincubation of unlabeled microsomes with sn-[14C] glycerol-3-phosphate and appropriate cofactors. The formation of CDP-diglyceride from radioactive phosphatidate showed an absolute requirement for CTP and MgCl2. The newly formed [14C] CDP-diglyceride was characterized by thin layer chromatography (TLC), isotopic labeling from radioactive CTP, and its ability to serve as substrate for the microsomal enzyme, CDP-diglyceride: inositol phosphatidyltransferase. The distributions of radioactive glycerol-3-phosphate among the various chemical classes of microsomal [14C] phosphatidate and [14C] CDP-diglyceride were determined following argentation TLC of their 1,2-diglyceride acetate derivatives. Most of the radioactivity among the phosphatidic acids was present in the monoenoic (36%) and dienoic (33%) molecular species, whereas 10, 8, 4, and 8% were associated with the saturates, trienes, tetraenes, and polyenes, respectively. Similar distributions of radioactivity were found among the corresponding classes of newly formed CDP-diglyceride. Only a slight enrichment of radioactivity in the tetraenoic CDP-diglyceride was found relative to the corresponding phosphatidates. Therefore, under the conditions of study, the microsomal CTP: phosphatidate cytidylyltransferase produces mainly monoenoic and dienoic species of CDP-diglyceride and shows little specificity towards different molecular species of phosphatidic acids. The present results suggest also that the arachidonoyl phosphatidate derived from the microsomal acylation of sn-glycerol-3-phosphate is not likely the major source of arachidonic acid in liver phosphatidylinositol.
D000818:Animals; D003567:Cytidine Diphosphate Diglycerides / Q000096:biosynthesis* / Q000302:isolation & purification; D005231:Fatty Acids, Unsaturated / Q000378:metabolism; D066298:In Vitro Techniques; D008297:Male; D008862:Microsomes, Liver / Q000201:enzymology / Q000378:metabolism*; D009702:Nucleoside Diphosphate Sugars / Q000096:biosynthesis*; D009713:Nucleotidyltransferases / Q000378:metabolism; D010712:Phosphatidic Acids / Q000378:metabolism*; D010716:Phosphatidylinositols / Q000096:biosynthesis; D010743:Phospholipids / Q000378:metabolism*; D051381:Rats
D003567:Cytidine Diphosphate Diglycerides; D005231:Fatty Acids, Unsaturated; D009702:Nucleoside Diphosphate Sugars; D010712:Phosphatidic Acids; D010716:Phosphatidylinositols; D010743:Phospholipids; D009713:Nucleotidyltransferases