A Role of Cytidine 5′-Diphosphocholine in the Transfer of N-Acetylglncosamine from UDP-N-acetylglucosamine into Endogenous Acceptor Lipids and Proteins in Rat and Hen Liver Microsomes

Abstract

Evidence is presented for the transfer of N-acetylglucosamine into lipids by cell-free preparations of rat and hen livers. CDP-choline stimulates the formation of this carbohydrate–lipid product. Triton is required for this reaction and for promoting the CDP-choline effect. Requirements for divalent cations, Triton, pH, and temperature optima have been established. The total microsomes, rough microsomes, and Golgi-depleted fractions of rat liver, but not the Golgi-rich membrane fractions, are active in the formation of carbohydrate–lipid product and are responsive to CDP-choline.The labeled lipid–carbohydrate product is stable to mild alkali but labile to mild acid hydrolysis. Following acid hydrolysis radioactive N-acetylglucosamine was recovered by thin-layer electrophoresis. The radioactive lipid has been separated by thin-layer chromatography. Autoradiography of thin-layer plates showed two or three (only in presence of CDP-choline) radioactive bands. Over 90% of the labeled carbohydrate–lipid product has been isolated by DEAE-cellulose acetate column chromatography by elution with ammoniacal chloroform–methanol mixture.A study of simultaneous incorporation of N-acetylglucosamine-1-14C into lipids and proteins in the microsomes showed that the peak incorporation into lipids and into proteins was 5 and 30 min, respectively. This suggests that N-acetylglucosamine incorporation into protein may be mediated through a lipid–carbohydrate intermediate.