Sequential glycosylations of endogenous glycosphingolipids in hamster fibroblasts incubated with nucleotide sugars.
Hamster fibroblasts, NIL 2cl cells, which were harvested from culture bottles by the use of chelating agents of divalent cations, incorporated [14C]galactose and N-acetyl[14C]galactosamine (GalnAc) from corresponding nucleotide 14-C-labeled sugars into endogenous glycosphingolipid acceptors. Incorporation of [14C]-GalNAc from UDP-[14C]GalNAc into endogenous acceptors to form tetra- and pentahexosylcermide with terminal GalNAc was enhanced 3.8-gold by preincubation of the cells with UDPglucose and UDPgalactose. In the whole cell reactions, sequential glycosylations of endogenous glycosphingolipids were observed by addition of whole cell reactions, sequential glycosylations of endogenous glycosphingolipids were observed by addition of appropriate nucleotide sugars to the reaction mixture: in a two step incubation experiment, mono-, di= and trihexosylceramides newly glycosylated from UDP-[14C]glucose and UDP-[14C] galactose in the first step of the whole cell reaction were effectively converted to hematoside by addition of CMP-N-[3H]acetylneuraminic acid in the second step. In the sequential glycosylation of endogenous glycolipids observed in the whole cell reaction, quantitative differences were found between cells harvested from confluent cultures and cells harvested from sparse cultures. Furthermore, a result was obtained which indicates the presence of two distinct pools of each glycosphingolipid in NIL cells. Glycolipids in the one pools represent newly glycosylated glycolipids and act as substrates for further glycosylations. On the other hand, precursor-product relationships do not exist among the main pools of each glycolipid.
D000818:Animals; D002250:Carbon Radioisotopes; D002460:Cell Line; D002999:Clone Cells; D006224:Cricetinae; D006017:Glycolipids / Q000096:biosynthesis; D006028:Glycosphingolipids / Q000378:metabolism*; D007700:Kinetics; D014531:Uridine Diphosphate Galactose / Q000378:metabolism*; D014532:Uridine Diphosphate Glucose / Q000378:metabolism*; D014536:Uridine Diphosphate N-Acetylgalactosamine / Q000378:metabolism*; D014539:Uridine Diphosphate Sugars / Q000378:metabolism*
D002250:Carbon Radioisotopes; D006017:Glycolipids; D006028:Glycosphingolipids; D014539:Uridine Diphosphate Sugars; D014531:Uridine Diphosphate Galactose; D014536:Uridine Diphosphate N-Acetylgalactosamine; D014532:Uridine Diphosphate Glucose