PURIFICATION AND CHARACTERIZATION OF HEPARAN-SULFATE 6-SULFOTRANSFERASE FROM THE CULTURE-MEDIUM OF CHINESE-HAMSTER OVARY CELLS

Heparan sulfate 6-sulfotransferase, which catalyzes the transfer of su lfate from S'-phosphoadenylyl sulfate to position 6 of N-sulfoglucosam ine in heparan sulfate, was purified 10,700-fold to apparent homogenei ty with a 40% yield from the serum-free culture medium of Chinese hams ter ovary cells. The isolation procedure included affinity chromatogra phy of the first heparin-Sepharose CL-GB column (stepwise elution), 3' ,5'-ADP-agarose, and the second heparin-Sepharose CL-6B column (gradie nt elution). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified enzyme showed two protein bands with molecular masses of 52 and 45 kDa. Both proteins appeared to be glycoproteins, because their molecular masses decreased after N-glycanase digestion. When co mpletely desulfated and N-resulfated heparin was used as acceptor, the purified enzyme transferred sulfate to position 6 of N-sulfoglucosami ne residue but did not transfer sulfate to the amino group of glucosam ine residue or to position 2 of the iduronic acid residue. Heparan sul fate was also sulfated by the purified enzyme at position 6 of N-sulfo glucosamine residue. Chondroitin and chondroitin sulfate did not serve as accepters. The optimal pH for enzyme activity was around 6.3. The enzyme activity was inhibited by dithiothreitol and was stimulated str ongly by protamine. The K-m, value for adenosine S'-phosphate 5'-phosp hosulfate was 0.44 mu M.