It was previously shown that the formation of L-iduronic acid residues by C-5 epimerization of D-glucuronic acid units at the polymer level during the synthesis of heparin involves exchange of the hydrogen atom at C-5 with protons of the medium (Lindahl, U., Jacobsson, I., Hook, M., Backstrom, G., and Feingold, D. S. (1976) Biochem. Biophys, Res. Commun. 70, 492-499). When a heparin precursor polysaccharide composed of alternating D-[5-(3)H]glucuronosyl and N-sulfated D-glucosaminosyl residues was incubated with a microsomal mouse mastocytoma preparation, the (3)H located on C-5 was exchanged with protons of the medium. The aqueous portion of the reaction mixture was isolated by low temperature distillation, and the (3)H present was determined by liquid scintillation counting. The rate of (3)H release was directly proportional to the concentration of microsomal enzyme as well as to substrate concentration and was used as an assay for the uronosyl 5-epimerase. The reaction had a pH optimum close to 7.4. No requirement for specific metal ions was observed; however, the reaction rate was strongly dependent on the ionic strength of the medium. Incubation of various 5-(3)H-labeled heparin precursor polysaccharides showed that only N-sulfated preparations were substrates; the best substrate consisted largely of alternating D-glucuronosyl and N-sulfated D-glucosaminosyl moieties. Exhaustive incubation of this substrate with microxomal enzyme caused release of 60 to 70% of the (3)H originally present; concomitantly, the L-iduronic acid content increased from 18 to 29% of the total uronic acid. When the reaction mixture was supplemented with 3'-phosphoadenylylsulfate, more than half of the substrate was converted into an O-sulfated species which contained 41% L-iduronic acid. These results suggest that although exchange of the C-5 hydrogen atom always accompanies 5-epimerization, the converse does not always occur. In contrast to the exogenous substrate, the membrane-bound endogenous intermediates did not lose tritium in excess of the extent of conversion of D-glucuronic acid to L-iduronic acid. Apparently, the high degree of organization in the native biosynthetic system permits a stricter regulation such that each attack by the epimerase is carried through to inversion of configuration.
|Original language||English (US)|
|Number of pages||8|
|Journal||Journal of Biological Chemistry|
|State||Published - 1979|
ASJC Scopus subject areas
- Molecular Biology
- Cell Biology