Abstract
To obtain information on the mechanism of glycosylation of ovalbumin, three types of experiments were performed with either hen oviduct membrane preparations or tissue slices and the antibiotic tunicamycin. First, experiments involving the addition of tunicamycin to oviduct membranes demonstrated that this antibiotic inhibited the synthesis of a N-acetylglucosaminyl-lipid with the properties of N-acetyl-glucosaminylpyrophosphorylpolyisoprenol. No inhibitory effects on the other steps in the synthesis of oligosaccharide-lipid were observed. Thus, tunicamycin inhibits the lipid-linked pathway for membrane protein glycosylation by blocking the first step in the synthesis of oligosaccharide-lipid, namely, the synthesis of N-acetylglucosaminylpyrophosphorylpolyisoprenol. Second, in experiments using membranes prepared from oviduct tissue slices preincubated with tunicamycin, it was found that mannosylphosphoryldolichol was the only saccharide-lipid synthesized. This result indicates that tunicamycin administered in vivo depletes endogenous pools of N-acetylglucosamine-lipid precursors to oligosaccharide-lipid in a manner consistent with its activity in vitro. Finally, it was found that tissue slices incubated in the presence of tunicamycin synthesized the polypeptide chain of ovalbumin at almost normal rates. However, the protein newly synthesized in vivo did not contain labeled N-acetylglucosamine or mannose and had the properties of unglycosylated ovalbumin. These results indicate that saccharide-lipids participate in the assembly of the core oligosaccharide of the secretory glycoprotein, ovalbumin.
Highlights
To obtain information on the mechanism of glycosylation of ovalbumin, three types of experiments were performed with either hen oviduct membrane preparations or tissue slices and the antibiotic tunicamycin
The presence of tunicamycin does not significantly inhibit the labeling of these three fractions (Fig. Z?). This finding indicates that tunicamycin prevents neither the synthesis of mannosylphosphoryldolichol, nor the transfer of mannose from this monosaccharide-lipid into the endogenous precursors of oligosaccharide-lipid known to be present in the membrane preparation
Endogenous, labeled N-acetylglucosamine-lipids generated in the absence of tunicamycin can be elongated to form oligosaccharide-lipid by addition of cold GDP-mannose in the presence of the antibiotic. These results indicate that the labeling of oligosaccharide-lipid from UDP-N-acetylglucosamine requires synthesis of N-acetylglucosaminyl-lipids, and that tunicamycin inhibits the synthesis of these lipids, but not their elongation to oligosaccharide-lipid
Summary
To obtain information on the mechanism of glycosylation of ovalbumin, three types of experiments were performed with either hen oviduct membrane preparations or tissue slices and the antibiotic tunicamycin. In experiments using membranes prepared from oviduct tissue slices preincubated with tunicamycin, it was found that mannosylphosphoryldolichol was the only saccharide-lipid synthesized. This result indicates that tunicamycin administered in viva depletes endogenous pools of N-. The protein newly synthesized in uiuo did not contain labeled N-acetylglucosamine or mannose and had the properties of unglycosylated ovalbumin. These results indicate that saccharidelipids participate in the assembly of the core oligosaccharide of the secretory glycoprotein, ovalbumin. The oligosaccharide chain of ovalbumin is polydisperse, with some chains terminating with N-acetylglucosamine residues, a major component has been shown [1] to have the structure: Manul,
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