Utilization of exogenous GDP-mannose for the synthesis of mannose-containing lipids and glycoproteins by oviduct cells.
Suspensions of oviduct cells were prepared by subjecting oviduct tissue to sequential incubations with EDTA, alpha-chymotrypsin, and crude collagenase, followed by a final incubation with EDTA. Cells isolated in this way incorporate mannose from exogenous GDP-mannose into mannosyl-lipid, oligosaccharide-lipid, and glycoprotein(s). Based on several criteria, the mannosyl-lipid is identical with mannosyl-phosphoryldolichol. Similarly, the oligosaccharide-lipid appears to be identical with the oligosaccharide-lipid synthesized in vitro (Lucas, J. J., Waechter, C. J., and Lennarz, W. J. (1975) J. Biol. Chem. 250, 1992-2002). In contrast, the glycoproteins are much lower in molecular weight than those labeled in cell-free preparations. Using intact oviduct cell suspensions it was found that: (a) exogenous GDP-mannose, not its breakdown products, serves as the direct mannosyl donor; (b) experiments using mixtures of known proportions of broken and intact cells, as well as studies with metabolic inhibitors, indicate that greater than 50% of the observed incorporation of mannose from GDP-mannose was catalyzed by enzymes associated with intact cells, rather than broken cells or membrane fragments; (c) incorporation of mannose from GDP-mannose into the mannosyl acceptors does not require energy and proceeds without significant uptake of GDP-mannose into trichloroacetic acid-soluble components of the cells; (d) under conditions where labeled guanosine incorporation into nucleic acids is readily detected, no incorporation of the guanosine moiety of [3H]GDP-mannose is observed. These results indicate that the enzymes catalyzing synthesis of lipid-linked intermediates involved in glycoprotein synthesis are not only associated with intracellular membranes, but with the plasma membrane as well.
D000818:Animals; D002645:Chickens; D005260:Female; D006017:Glycolipids / Q000096:biosynthesis*; D006023:Glycoproteins / Q000096:biosynthesis*; D006155:Guanosine Diphosphate Mannose / Q000378:metabolism*; D007700:Kinetics; D008358:Mannose / Q000378:metabolism; D008858:Microscopy, Phase-Contrast; D009702:Nucleoside Diphosphate Sugars / Q000378:metabolism*; D010057:Oviducts / Q000378:metabolism* / Q000648:ultrastructure; D010649:Phenylalanine / Q000378:metabolism
D006017:Glycolipids; D006023:Glycoproteins; D009702:Nucleoside Diphosphate Sugars; D006155:Guanosine Diphosphate Mannose; D010649:Phenylalanine; D008358:Mannose