Purification of rat liver N-heparan-sulfate sulfotransferase.
N-Heparan-sulfate sulfotransferase catalyzes the transfer of sulfate from 3'-phosphoadenilyl sulfate to the nitrogen of glucosamine in heparan sulfate. This reaction is an obligatory step for subsequent epimerization of D-glucuronic to L-iduronic acid and of O-sulfation of the sugar chains. We have purified this sulfotransferase from rat liver membranes to apparent homogeneity using a combination of conventional and affinity chromatography on DEAE-Sephacel, heparin-agarose, 3',5'-ADP-agarose, wheat germ-Sepharose, and finally a glycerol gradient. The pure enzyme is a glycoprotein with an apparent molecular weight of 97,000. It was enriched in specific activity 65,000-fold over the homogenate. The recovery of activity was 4% of that of the homogenate. Preliminary enzymatic characterization of the purified sulfotransferase indicates a high degree of substrate specificity. Transfer of sulfate occurs to heparan sulfate, N-heparan sulfate, and N-desulfated heparin, but not to N-acetylated heparan sulfate, N-acetylated heparin, chondroitin, chondroitin sulfate, and tyrosine-containing tripeptides.
Department of Biochemistry, University of Massachusetts Medical Center, Worcester 01655.
E
E
Brandan
C B
CB
Hirschberg
D000818:Animals; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D006056:Golgi Apparatus / Q000201:enzymology; D008099:Liver / Q000201:enzymology*; D008566:Membranes / Q000201:enzymology; D051381:Rats; D015238:Sulfotransferases; D013466:Sulfurtransferases / Q000302:isolation & purification*
D013466:Sulfurtransferases; D015238:Sulfotransferases; C021350:heparitin sulfotransferase