Nonsulfated, partially N deacetylated, 14C labeled polysaccharide was formed by incubation of a mouse mastocytoma microsomal fraction with UDP [14C] glucuronic acid and UDP N acetylglucosamine. After 60 min at 37° the incorporation of radioactivity was interrupted, and sulfation was initiated, by the addition of unlabeled UDP glucuronic acid and 3 phosphoadenylysulfate, respectively. After varying periods of incubation with the nucleotide sulfate, polysaccharide was isolated and analyzed by chromatography on DEAE cellulose. During incubation with 3 phosphoadenylylsulfate, sulfated 14C polysaccharides were formed, while the nonsulfated 14C polysaccharide was eliminated, each type of component retaining the same elution position throughout the entire sulfation period (60 min). No accumulation of low sulfated material was observed. Fully sulfated heparin was detected after only 0.5 min of sulfation. These results suggest that the sulfation of heparin is a rapid process, involving a limited number of polysaccharide molecules at a time. Besides heparin like products, having both N and O sulfate groups, and [14C] iduronic acid as predominant uronic acid constituent, an additional sulfated component was detected, having N sulfate but no O sulfate groups. This novel polysaccharide contained [14C] glucuronic acid but little or no [14C] iduronic acid. Previous studies have shown that L iduronic acid residues in heparin are formed by C 5 inversion of D glucuronic acid units, previously incorporated into the polymer; the inversion reaction requires concomitant sulfation of the polymer. The present results demonstrate that the formation of iduronic acid residues is related to the sulfation of hydroxyl groups. Pulse chase experiments with 3' phosphoadenylyl [35S] sulfate indicated that the N sulfated polymer, lacking O sulfate groups, could be converted into heparin like products by further sulfation of hydroxyl groups only. The biosynthesis of heparin may thus involve a pathway, in which the formation of N sulfated intermediates precedes the introduction of O sulfate groups.
|Original language||English (US)|
|Number of pages||7|
|Journal||Journal of Biological Chemistry|
|State||Published - 1975|
ASJC Scopus subject areas
- Molecular Biology
- Cell Biology