Biosynthesis of heparin/heparan sulfate. Purification of the D-glucuronyl C-5 epimerase from bovine liver.
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.
Campbell
P
P
University of Alabama at Birmingham 35294.
Hannesson
H H
HH
Sandbäck
D
D
Rodén
L
L
Lindahl
U
U
Li
J P
JP
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D016667:Bacterial Capsules / Q000737:chemistry; D002238:Carbohydrate Epimerases / Q000302:isolation & purification / Q000378:metabolism*; D002417:Cattle; D002846:Chromatography, Affinity; D006493:Heparin / Q000096:biosynthesis*; D006497:Heparan Sulfate / Q000096:biosynthesis*; D006863:Hydrogen-Ion Concentration; D007067:Iduronic Acid / Q000378:metabolism; D008099:Liver / Q000201:enzymology*; D011135:Polysaccharides, Bacterial / Q000378:metabolism
D000596:Amino Acids; D002238:Carbohydrate Epimerases; D006493:Heparin; D006497:Heparan Sulfate; D007067:Iduronic Acid; D011135:Polysaccharides, Bacterial; C019591:heparosan N-sulfate D-glucuronosyl 5-epimerase