N-Acetylglucosamine Transfer Reactions and Glycoprotein Biosynthesis in Castor Bean Endosperm

N-Acetyl-[³H]glucosamine supplied to intact 3 d old castor beanendosperm tissue was incorporated into TCA-insoluble productpresumed to be glycoprotein. After an incubation time of 2 hthe major paniculate location of this product within the cellwas the endoplasmic reticulum. Cell-free preparations containingparticulate enzymes transferred N-acetyl-[¹⁴C]glucosamine fromUDP-N-acetyl-[¹⁴C]glucosamine into a fraction soluble in chloroform/methanol(2: 1, by vol), a fraction soluble in chloroform/methanol/water(10: 10: 3, by vol.), and an insoluble residue. Mild acid hydrolysisreleased the saccharide moieties from the lipids. Paper chromatographicanalysis of the released saccharides established that the C/M-solubleproducts contained both N-acetyl-[¹⁴C]glucosamine and N, N'-diacetyl-[¹⁴C]chitobiose.In contrast, N-acetyl-[¹⁴C]glucosamine released from the C/M/W-solubleproduct was contained in an oligosaccharide, probably in associationwith unlabelled mannose residues. The stimulatory effect ofdolichol monophosphate and the inhibitory effect of tunicamycinon saccharide-lipid synthesis indicated that N-acetyl-glucosamineis transferred to a glycopolymer by the established reactionsof the dolichol monophosphate pathway. The enzymes catalysingthe constituent reactions of this pathway were exclusively locatedin the ER.