Enzymatic glucosylation of dolichol monophosphate and transfer of glucose from isolated dolichyl-D-glucosyl phosphate to ceramides by BHK-21 cell microsomes.
Enzymatic glucosylation of dolichol monophosphate (dolichol-P) from UDP-D-[3H]glucose was studied using the microsomal fraction of BHK-21 cells. The reaction product was separated by preparative thin-layer chromatography, further purified by DEAE-cellulose acetate column chromatography, and characterized as dolichyl-beta-D-glucosyl phosphate (Dol-P-Glc). The microsomal fraction of BHK cells catalyzed the incorporation of glucose from UDP-[3H]glucose into ceramides (endogenous and exogenous) and Dol-P; both reactions required Mn2+. Maximal glucosylation of Dol-P was achieved at pH 5.6-5.8 in the presence of a non-ionic detergent, Zonyl A. Glucosylation of exogenous Dol-P, from UDP-Glc, was non-competitively inhibited by exogenous ceramides. Incubation of Dol-P-[3H]Glc or Dol-P-[14C]Glc with liposomes (containing ceramides) and the microsomal fraction of BHK-21 cells resulted in the formation of a radioactive glucolipid which comigrated with the same RF value as glucosylceramide (Glc-Cer) on silica gel thin-layer chromatography. Transfer of [14C]glucose from Dol-P-[14C]Glc to exogenous ceramides was confirmed by double-labeling techniques. The pH dependence for transfer of radio-labeled glucose from Dol-P-[3H]Glc to ceramides was multi-phasic (optima at pH 4.0 and 7.0); glycosylation occurred within 5 min and Zonyl A was absolutely essential for the transfer reaction. These results indicate that Dol-P-Glc may also participate in the synthesis of ceramide hexosides.
D000818:Animals; D002412:Cations / Q000494:pharmacology; D002460:Cell Line; D002518:Ceramides / Q000378:metabolism*; D006224:Cricetinae; D003902:Detergents / Q000494:pharmacology; D004288:Dolichol Phosphates / Q000378:metabolism*; D005947:Glucose / Q000378:metabolism*; D005964:Glucosyltransferases / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007668:Kidney; D008861:Microsomes / Q000201:enzymology*; D011103:Polyisoprenyl Phosphate Monosaccharides / Q000378:metabolism*; D011105:Polyisoprenyl Phosphate Sugars / Q000378:metabolism*; D011106:Polyisoprenyl Phosphates / Q000378:metabolism*
D002412:Cations; D002518:Ceramides; D003902:Detergents; D004288:Dolichol Phosphates; D011103:Polyisoprenyl Phosphate Monosaccharides; D011105:Polyisoprenyl Phosphate Sugars; D011106:Polyisoprenyl Phosphates; C028306:dolichol monophosphate; C002968:dolichol-D-glucosylmonophosphate; D005964:Glucosyltransferases; D005947:Glucose