Abstract
An oligosaccharide-P-P-lipid has been isolated from porcine liver by extraction with organic solvents and purified by chromatography on silica gel and DEAE-cellulose. The purified oligosaccharide-lipid was shown to contain mannose and N-acetylglucosamine in an approximate ratio of 1:1 and our results suggest that the major oligosaccharide component in the preparation was a tetrasaccharide with the composition (Man)2 (GlcNAc)2. When the oligosaccharide-lipid was incubated with GDP-[14C]mannose and a solubilized enzyme preparation from rabbit liver in the presence of MgCl2, three radioactive products could be isolated. The oligosaccharides in the products could be identified as a penta-, a hexa-, and a heptasaccharide. These products were formed by the stepwise addition of mannose to the growing oligosaccharide chain and GDP-mannose was indicated as the glycosyl donor in each reaction.
Highlights
An oligosaccharide-P-P-lipidhas been isolated from a liquid scintillation counter using premixed scintillation fluid (Scinporcine liver by extraction with organic solvents and tiverse, Fisher)
Samples were analyzed for di-N-acetylchitobiose on a GDP-["C]mannoseand a solubilized enzyme prepara- column (6 feet X 2 mm) of 3% OV-17on SO/l00 meshChromosorb W
The organic phase was applied to a column of Silica Gel 60
Summary
Purification of Oligosaccharide-lipid Acceptor-oligosaccharide-lipids were extracted from porcine liver as described under “Materials and Methods.” After saponification, the organic phase was applied to a column of Silica Gel 60 The organic phase was applied to a column of Silica Gel 60 (Fig. l ) , eluted with ahear gradient of water in CHC4/CH30H (l:l), and column fractions were assayed for acceptor activity using standard assays. Acceptor activity eluted as a rather broad peak fromthis final silica gel column and Fractions 10 to 37 were pooled and concentrated.
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