| Autor: |
Campbell, P, Hannesson, H H, Sandbäck, D, Rodén, L, Lindahl, U, Li, J P |
| Zdroj: |
Journal of Biological Chemistry; October 1994, Vol. 269 Issue: 43 p26953-26958, 6p |
| Abstrakt: |
The D-glucuronyl C-5 epimerase involved in the biosynthesis of heparin/heparan sulfate was purified from the high speed supernatant fraction of a homogenate of bovine liver by chromatography on immobilized O-desulfated heparin, red Sepharose, phenyl Sepharose, and concanavalin A-Sepharose. After close to 1 million-fold purification, in 10-15& yield, the product gave a single band on SDS-polyacrylamide gel electrophoresis with silver staining and had a mobility corresponding to an M(r) of approximately 52,000. Since the epimerase assay used in the course of purification was based on release of tritium, as [3H]H2O, from a [5-3H]uronyl-labeled substrate, it was important to establish that the purified enzyme did indeed catalyze the actual conversion of D-glucuronyl to L-iduronyl residues. Upon incubation of the purified enzyme with 3H-labeled heparosan N-sulfate, prepared by metabolic labeling (with D-[1-3H]glucose) of a capsular polysaccharide from Escherichia coli K5 and subsequent chemical partial N-deacetylation and N-sulfation, approximately 30& of the D-glucuronyl residues located between two N-sulfated glucosamine units were converted to L-iduronyl units. |
| Databáze: |
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