Cell-free labeling in thyroid rough microsomes of lipid-linked and protein-linked oligosaccharides: II. Glucosylated units

Abstract

Pig thyroid rough microsomes catalyzed the transfer of glucose from UDP-[ 14C]Glc to glycolipids extractable with chloroform/methanol, glycolipids extractable with a water-saturated chloroform/methanol and to a residual material. Kinetics of labeling were compatible with a precursor-product relationship between the second type of glycolipid and residuals. The [ 14C] Glc-glycolipids soluble in CHCl 3/CH 3OH/H 2O, 10 : 10 : 3, behaved on DEAE-cellulose mainly as pyrophospho derivatives, with some less acidic radioactivity, probably dolichol-P-[ 14 C] Glc . Their saccharide moieties released by mild acid appeared polydisperse on paper chromatography, a part of them being estimated larger than a nonasaccharide marker GlcNAc-[Man] 8. The 14C-labeled glucosylated glycoproteins have represented all the considerable polymeric label remaining after lipid extraction. Their pronase glycopeptides were submitted to a differential reductive alkaline hydrolysis and it was concluded that their [ 14C] glucose belongs mainly to N-glycosically linked units. On gel filtration, the released saccharides exhibited an average size of nine monosaccharide units (from six to twelve with a relatively high proportion of material containing more than nine sugars). In a [ 14C] Glc-microsomal extract, 29% of the non-lipid radioactivity was found immunoreactive with an antiserum to pig thyroglobulin.