JBC, Vol. 252, Issue 8, 2498-2506, Apr, 1977
Enzymatic transfer of mannose from mannosyl-phosphoryl-polyprenol to lipid-linked oligosaccharides by pig aorta
J. Chambers, W. T. Forsee and A. D. Elbein
A particulate enzyme preparation prepared from the intimal layer of pig
aorta catalyzed the transfer of mannose from mannosyl-phosphoryl-polyprenol
(MPP) into a series of oligosaccharides that were linked to lipid. The
reaction required detergent with Triton X-100 and NP-40 being best at a
concentration of 0.5%. Several other detergents were inactive or only
slightly active. The pH optima for this activity was about 7 to 7.5 in Tris
buffer and the apparent Km for MPP was about 2 x 10(-7) M. The reaction was
not stimulated by the addition of divalent cation and, in fact, was
inhibited by the high concentrations of cation. The addition of EDTA did
not inhibit the transfer of mannose from MPP and was somewhat stimulatory.
The transferase(s) activity was "solubilized" from the particles by
treatment with Triton X-100. This solubilized enzyme still formed a series
of lipid-linked oligosaccharides from either MPP or GDP-mannose. The
oligosaccharides were released from the lipid by mild acid hydrolysis and
were separated by paper chromatography. Some five or six radioactive
oligosaccharides were formed from either MPP or from GDP-mannose and these
oligosaccharides had similar mobilities upon paper chromatography. However,
MPP was a better donor for the larger oligosaccharides (i.e. those
containing 8, 9, or 10 sugar residues), whereas GDP-mannose was better for
formation of the oligosaccharide containing 7 sugar residues. In the
presence of EDTA and detergent no MPP was formed from GDP-mannose, but
radioactivity was still incorporated into the lipid-linked
oligosaccharides. Under these conditions essentially all of the
radioactivity was in the oligosaccharide containing 7 sugar residues. Since
much of this activity could be released as mannose by acetolysis,
GDP-mannose may be the direct mannosyl donor for formation of 1 leads to 6
branches. Oligosaccharides 7, 8, 9, and 10 were isolated and partially
characterized in terms of their molecular weights, sugar composition,
susceptibility to alpha-mannosidase, and 14C products formed by acetolysis
and periodate oxidation. The molecular weights ranged from 1310 for
oligosaccharide 7 to 1750 for oligosaccharide 10. Hydrolysis of each
oligosaccharide and reduction with NaB3H4 gave the expected ratio of
[3H]hexitol to [3H]hexosaminitol based on the molecular weight of the
oligosaccharide. However, the hexitol fraction contained [3H]mannitol and
[3H]glucitol. Since the amount of radioactivity in glucitol was 2 to 4
times that in mannitol and since only glucosaminitol was found in the amino
sugar peak, it seems likely that each 14C-oligosaccharide was contaminated
with an unlabeled oligosaccharide of equal molecular weight containing
glucose and GlcNAc. Acetolysis of the 14C-oligosaccharides gave rise to 14C
peaks of mannose, mannobiose, and mannotriose. In the larger
oligosaccharides, most of the radioactivity was in mannobiose whereas in
oligosaccharide 7 most of the radioactivity was in mannose...
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