Biosynthesis of Heparin

作者：Ulf Lindahl, Gudrun Bäckström, Leif E. Jansson, A Hallén · 发表于：Journal of Biological Chemistry · 年份：1973 · DOI：10.1016/s0021-9258(19)43383-8 · 被引用次数：271 · 研究领域：Proteoglycans and glycosaminoglycans research、Glycosylation and Glycoproteins Research、Polysaccharides and Plant Cell Walls

Abstract 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...