Purification and characterization of chondroitin 4-sulfotransferase from the culture medium of a rat chondrosarcoma cell line

Chondroitin I-sulfotransferase, which transfers sulfate from 3'-phosphoaden osine 5'-phosphosulfate to position 4 of N-acetylgalactosamine in chondroit in, was purified 1900-fold to apparent homogeneity with 6.1% yield from the serum-free culture medium of rat chondrosarcoma cells by affinity chromato graphy on heparin-Sephharose CL-GB, Matrex gel red A-agarose, 3',5'-ADP-aga rose, and the second heparin-Sepharose CL-GB. SDS-polyacrylamide gel electr ophoresis of the purified enzyme showed two protein bands. Molecular masses of these protein were 60 and 64 kDa under reducing conditions and 50 and 5 4 kDa under nonreducing conditions. Both the protein bands coeluted with ch ondroitin 4-sulfotransferase activity from Toyopearl HW-55 around the posit ion of 50 kDa, indicating that the active form of chondroitin Li-sulfotrans ferase is a monomer. Dithiothreitol activated the purified chondroitin 4-su lfotransferase. The purified enzyme transferred sulfate to chondroitin and desulfated dermatan sulfate. Chondroitin sulfate A and chondroitin sulfate C were poor accepters. Chondroitin sulfate E from squid cartilage, dermatan sulfate, heparan sulfate, and completely desulfated N-resulfated heparin h ardly served as accepters 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 o n both reducing and nonreducing sides.