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

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Abstract
N-Acetyl-[3H]glucosamine supplied to intact 3 d old castor bean endosperm tissue was incorporated into TCA-insoluble product presumed to be glycoprotein. After an incubation time of 2 h the major paniculate location of this product within the cell was the endoplasmic reticulum. Cell-free preparations containing particulate enzymes transferred N-acetyl-[14C]glucosamine from UDP-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 hydrolysis released the saccharide moieties from the lipids. Paper chromatographic analysis of the released saccharides established that the C/M-soluble products contained both N-acetyl-[14C]glucosamine and N, N'-diacetyl-[14C]chitobiose. In contrast, N-acetyl-[14C]glucosamine released from the C/M/W-soluble product was contained in an oligosaccharide, probably in association with unlabelled mannose residues. The stimulatory effect of dolichol monophosphate and the inhibitory effect of tunicamycin on saccharide-lipid synthesis indicated that N-acetyl-glucosamine is transferred to a glycopolymer by the established reactions of the dolichol monophosphate pathway. The enzymes catalysing the constituent reactions of this pathway were exclusively located in the ER.