PURIFICATION AND CHARACTERIZATION OF HEPARAN-SULFATE 2-SULFOTRANSFERASE FROM CULTURED CHINESE-HAMSTER OVARY CELLS

Heparan sulfate 2-sulfotransferase, which catalyzes the transfer of su lfate from adenosine 3'-phosphate 5'-phosphosulfate to position 2 of L -iduronic acid residue in heparan sulfate, was purified 51,700-fold to apparent homogeneity with a 6% yield from cultured Chinese hamster ov ary cells, The isolation procedure included a combination of affinity chromatography on heparin Sepharose CL-6B and 3',5'-ADP-agarose, which was repeated twice for each, and finally gel chromatography on Supero se 12, Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of th e purified enzyme showed two protein bands with molecular masses of 47 and 44 kDa, Both proteins appeared to be glycoproteins, because their molecular masses decreased after N-glycanase digestion, When complete ly desulfated and N-resulfated heparin and mouse ngelbreth-Holm-Swarm tumor heparan sulfate were used as accepters, the purified enzyme tran sferred sulfate to position 2 of L-iduronic acid residue but did not t ransfer sulfate to the amino group of glucosamine residue or to positi on 6 of N-sulfoglucosamine residue, Heparan sulfates from pig aorta an d bovine liver, however, were poor accepters, The enzyme showed no act ivities toward chondroitin, chondroitin sulfate, dermatan sulfate, and keratan sulfate, The optimal pH for the enzyme activity was around 5. 5. The enzyme activity was minimally affected by dithiothreitol and wa s stimulated strongly by protamine. The K-m value for adenosine 5'-pho sphate 5'-phosphosulfate was 0.20 mu M.