Purification and characterization of heparan sulfate 2-sulfotransferase from cultured Chinese hamster ovary cells.
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.
Kobayashi
M
M
Institute for Molecular Science of Medicine, Aichi Medical University, Nagakute, Japan.
Habuchi
H
H
Habuchi
O
O
Saito
M
M
Kimata
K
K
D000244:Adenosine Diphosphate; D000818:Animals; D001011:Aorta; D016466:CHO Cells; D002236:Carbohydrate Conformation; D002240:Carbohydrate Sequence; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D006224:Cricetinae; D004187:Disaccharides / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D006497:Heparan Sulfate / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D051379:Mice; D008969:Molecular Sequence Data; D008970:Molecular Weight; D012685:Sepharose / Q000031:analogs & derivatives; D013379:Substrate Specificity; D015238:Sulfotransferases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D013552:Swine; D014407:Tumor Cells, Cultured
D000244:Adenosine Diphosphate; D004187:Disaccharides; D006497:Heparan Sulfate; D012685:Sepharose; D015238:Sulfotransferases; C037405:heparin-sepharose; C021350:heparitin sulfotransferase