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 sulfate from 3'-phosphoadenylyl sulfate to position 6 of N-sulfoglucosamine in heparan sulfate, was purified 10,700-fold to apparent homogeneity with a 40% yield from the serum-free culture medium of Chinese hamster ovary cells. The isolation procedure included affinity chromatography of the first heparin-Sepharose CL-6B column (stepwise elution), 3',5'-ADP-agarose, and the second heparin-Sepharose CL-6B column (gradient 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 completely desulfated and N-resulfated heparin was used as acceptor, the purified enzyme transferred sulfate to position 6 of N-sulfoglucosamine residue but did not transfer sulfate to the amino group of glucosamine residue or to position 2 of the iduronic acid residue. Heparan sulfate was also sulfated by the purified enzyme at position 6 of N-sulfoglucosamine residue. Chondroitin and chondroitin sulfate did not serve as acceptors. The optimal pH for enzyme activity was around 6.3. The enzyme activity was inhibited by dithiothreitol and was stimulated strongly by protamine. The Km value for adenosine 3'-phosphate 5'-phosphosulfate was 0.44 microM.
Habuchi
H
H
Institute for Molecular Science of Medicine, Aichi Medical University, Japan.
Habuchi
O
O
Kimata
K
K
D000818:Animals; D016466:CHO Cells; D002240:Carbohydrate Sequence; D002846:Chromatography, Affinity; D002851:Chromatography, High Pressure Liquid; D006224:Cricetinae; D003412:Cricetulus; D016895:Culture Media, Serum-Free; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D013379:Substrate Specificity; D015238:Sulfotransferases / Q000302:isolation & purification* / Q000378:metabolism
D016895:Culture Media, Serum-Free; D015238:Sulfotransferases; C021350:heparitin sulfotransferase