Biosynthesis of heparin: II. Formation of sulfamino groups

U Lindahl, G Bäckström, L Jansson, A Hallén - Journal of Biological …, 1973 - Elsevier
U Lindahl, G Bäckström, L Jansson, A Hallén
Journal of Biological Chemistry, 1973Elsevier
Microsomal fraction from mouse mastocytoma was incubated for 60 min with UDP-[14 C]
glucuronic acid and unlabeled UDP-N-acetylglucosamine, producing nonsulfated labeled
polysaccharide. Continued incubation for an additional 60-min period, in the presence of
3′-phosphoadenylylsulfate, yielded sulfated, heparin-like polymer. The incorporation of 14
C was terminated at the beginning of the sulfation period, by including an excess of
unlabeled UDP-glucuronic acid in the incubation mixtures. The distribution in the …
Microsomal fraction from mouse mastocytoma was incubated for 60 min with UDP-[14C]glucuronic acid and unlabeled UDP-N-acetylglucosamine, producing nonsulfated labeled polysaccharide. Continued incubation for an additional 60-min period, in the presence of 3′-phosphoadenylylsulfate, yielded sulfated, heparin-like polymer. The incorporation of 14C was terminated at the beginning of the sulfation period, by including an excess of unlabeled UDP-glucuronic acid in the incubation mixtures.
The distribution in the nonsulfated and chase-sulfated 14C-polysaccharides, respectively, of N-unsubstituted, N-acetylated, and N-sulfated glucosamine residues was investigated by selective deamination with nitrous acid. Samples were treated at room temperature with 0.24 m NaNO2 in 1.8 m acetic acid for 80 min (Reaction A) or with 3.9 m NaNO2 in 0.28 m acetic acid for 10 min (Reaction B). In Reaction A the glycosidic bonds of glucosamine residues having either unsubstituted or sulfated amino groups were cleaved, whereas in Reaction B N-unsubstituted glucosamine units only were attacked. The effect of deamination on the labeled polysaccharide preparations was studied by gel chromatography of the products, on Sephadex G-25.
Characterization of the nonsulfated polysaccharide, by use of these methods, indicated that about half of the glucosamine residues had unsubstituted amino groups. This result was confirmed by ion exchange chromatography, on DEAE-cellulose, which showed the presence of a polymer with less polyanion character than standard hyaluronic acid. After N-acetylation, the elution pattern of this intermediary polysaccharide was the same as that of hyaluronic acid.
The sulfated polysaccharide contained N-sulfated and N-acetylated but no N-unsubstituted glucosamine units.
It is concluded that N-deacetylation of the heparin precursor polysaccharide occurs in the absence of 3′-phosphoadenylylsulfate and that the amino groups thus exposed can serve as acceptors of sulfate residues.
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