Heparan sulfate 2-sulfotransferase, which catalyzes the transfer of
sulfate 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 ovary 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
Superose 12 .
Sodium dodecyl sulfate-
polyacrylamide gel electrophoresis of the 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 completely desulfated and N-resulfated
heparin and mouse Engelbreth-Holm-Swarm
tumor heparan sulfate were used as acceptors, the purified
enzyme transferred
sulfate to position 2 of L-
iduronic acid residue but did not transfer
sulfate to the amino group of
glucosamine residue or to position 6 of N-sulfoglucosamine residue. Heparan
sulfates from pig aorta and bovine liver, however, were poor acceptors. The
enzyme showed no activities 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 was stimulated strongly by
protamine. The Km value for
adenosine 3'-phosphate 5'-phosphosulfate was 0.20 microM.