Purification and characterization of chondroitin 4-sulfotransferase from the culture medium of a rat chondrosarcoma cell line.
Chondroitin 4-sulfotransferase, which transfers sulfate from 3'-phosphoadenosine 5'-phosphosulfate to position 4 of N-acetylgalactosamine in chondroitin, was purified 1900-fold to apparent homogeneity with 6.1% yield from the serum-free culture medium of rat chondrosarcoma cells by affinity chromatography on heparin-Sepharose CL-6B, Matrex gel red A-agarose, 3',5'-ADP-agarose, and the second heparin-Sepharose CL-6B. SDS-polyacrylamide gel electrophoresis of the purified enzyme showed two protein bands. Molecular masses of these protein were 60 and 64 kDa under reducing conditions and 50 and 54 kDa under nonreducing conditions. Both the protein bands coeluted with chondroitin 4-sulfotransferase activity from Toyopearl HW-55 around the position of 50 kDa, indicating that the active form of chondroitin 4-sulfotransferase is a monomer. Dithiothreitol activated the purified chondroitin 4-sulfotransferase. The purified enzyme transferred sulfate to chondroitin and desulfated dermatan sulfate. Chondroitin sulfate A and chondroitin sulfate C were poor acceptors. Chondroitin sulfate E from squid cartilage, dermatan sulfate, heparan sulfate, and completely desulfated N-resulfated heparin hardly served as acceptors of the sulfotransferase. The transfer of sulfate to the desulfated dermatan sulfate occurred preferentially at position 4 of the N-acetylgalactosamine residues flanked with glucuronic acid residues on both reducing and nonreducing sides.
Yamauchi
S
S
Department of Life Science, Aichi University of Education, Aichi 448-8542, Japan.
Hirahara
Y
Y
Usui
H
H
Takeda
Y
Y
Hoshino
M
M
Fukuta
M
M
Kimura
J H
JH
Habuchi
O
O
D000818:Animals; D002813:Chondrosarcoma / Q000201:enzymology* / Q000473:pathology; D002853:Chromatography, Liquid; D003470:Culture Media; D016895:Culture Media, Serum-Free; D004591:Electrophoresis, Polyacrylamide Gel; D051381:Rats; D013379:Substrate Specificity; D015238:Sulfotransferases / Q000302:isolation & purification* / Q000378:metabolism; D014407:Tumor Cells, Cultured
D003470:Culture Media; D016895:Culture Media, Serum-Free; D015238:Sulfotransferases; C019893:chondroitin 4-sulfotransferase