Abstract
A solubilized mannosyl transferase(s) was obtained by treatment of the pig aorta particulate enzyme with the nonionic detergent Nonidet P-40 followed by centrifugation at 100,000 X g for 60 min. This enzyme preparation catalyzed the transfer of mannose from GDP-[14C]mannose (but not from [14C]mannosylphosphoryldolichol) to form a heptasaccharide-lipid. The synthesis of this heptasaccharide-lipid required the addition of an acceptor lipid that was isolated from pig liver. The oligosaccharide portion of the acceptor lipid appeared to be a mixture of trisaccharide and pentasaccharide. The formation of heptasaccharide-lipid did not require divalent cation, was not inhibited by EDTA, and was not inhibited by the antibiotic amphomycin. The heptasaccharide portion of the heptasaccharide-lipid had the same migration properties on paper chromatograms in two different solvent systems as a previously characterized Man5GlcNAc2 oligosaccharide [Li, E. & Kornfeld, S. (1979) J. Biol. Chem. 254, 2754-2758]. It also had the same migration properties as the oligosaccharide that accumulates in the presence of amphomycin. All three of these oligosaccharides emerged from a Bio-Gel P-4 column in the same position. Radioactive mannose was released from the heptasaccharide by digestion with alpha-mannosidase. These data demonstrate that at least some of the alpha-linked mannose residues in the heptasaccharide-lipid are donated directly from GDP-mannose.
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