Amphomycin inhibition of mannose and GlcNAc incorporation into lipid-linked saccharides.

We previously reported that the peptide antibiotic, amphomycin, inhibited the transfer of mannose from GDP-[‘4C]mannose and GlcNAc from UDP-[3H]GlcNAc to the lipid-linked saccharides by enzyme preparations of pig aorta (Kang, M. S., Spencer, J. P., and Elbein, A. D. (1978) Biochem. Biophys. Res. Commun. 82,568-574). With the particulate enzyme, the transfer of mannose from GDP-[‘*C)mannose to mannosyl-phosphoryl-dolichol (Dol-P-Man) was much more sensitive to inhibition than was mannose transfer to lipid-linked oligosaccharides. Thus, at amphomycin concentrations which completely block the formation of Dol-P-Man, mannose was still transferred to lipid-linked oligosaccharides suggesting that some of the mannose residues come directly from GDP-mannose. Under these conditions, essentially all of the radioactivity from GDP-[14C]mannose was found in one oligosaccharide with the properties of a heptasaccharide. This is the only oligosaccharide which becomes labeled from GDP-mannose in the presence of EDTA, another condition in which the formation of Dol-P-Man is inhibited. While amphomytin inhibited mannose transfer from GDP-mannose to Dol-P-Man, it did not inhibit mannose transfer when Dol-P-Man or lipid-linked oligosaccharides were used as the substrates. Using a solubilized enzyme preparation obtained by detergent treatment of aorta microsomes, a variety of parameters were examined in an attempt to define the mechanism of inhibition. However, the inhibition of mannose or GlcNAc transfer to dolichyl-phosphate could not be overcome by high concentrations of dolichyl-phosphate or of Mn2+ or by increasing the concentrations of the two together. Various sugar nucleotides were added to the incubations to see whether these might complex with the antibiotic and overcome the inhibition, but all of these were ineffective. Thus, amphomycin inhibition does not appear to be of a competitive nature.