Separation and characterization of chondroitin 6-sulfotransferase and chondroitin 4-sulfotransferase from chick embryo cartilage.
Two distinct sulfotransferases (chondroitin 6-sulfotransferase and chondroitin 4-sulfotransferase), which catalyzed transfer of sulfate to position 6 and position 4 of acetylgalactosamine residues of chondroitin, were extracted from epiphyseal cartilage of 14-day-old chick embryos and separated by gel chromatography on Sephacryl S-200 in the presence of 3 M guanidine-HCl. When the enzyme solutions containing 3 M guanidine-HCl were dialyzed against 0.02 M Tris-HCl, pH 7.2, containing 10% glycerol, chondroitin 4-sulfotransferase became almost insoluble, whereas chondroitin 6-sulfotransferase remained soluble. Endogenous acceptors for sulfate transfer were completely removed from both enzyme preparations. Addition of basic proteins and polyamines as well as Mn2+ to the incubation medium caused a stimulation of both sulfotransferases; the stimulation of chondroitin 6-sulfotransferase with these cations was higher than that of chondroitin 4-sulfotransferase. The Km values for 3'-phosphoadenylyl sulfate of both enzymes were much smaller in the presence of protamine or spermine than in the presence of Mn2+. The two sulfotransferases differed in the requirement for sulfhydryl compounds; in the absence of sulfhydryl compounds, the activity of chondroitin 4-sulfotransferase was very low, whereas the activity of chondroitin 6-sulfotransferase was essentially unaffected. These observations indicate that at least two sulfotransferases are involved in the biosynthesis of chondroitin sulfate, and suggest that the production of the isomers of chondroitin sulfate in chondrocytes is affected by various factors such as the intracellular concentration of sulfhydryl compounds and basic substances.
D000818:Animals; D002356:Cartilage / Q000201:enzymology*; D002642:Chick Embryo; D002854:Chromatography, Paper; D004789:Enzyme Activation; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008345:Manganese / Q000494:pharmacology; D011073:Polyamines / Q000494:pharmacology; D015238:Sulfotransferases / Q000302:isolation & purification*; D013466:Sulfurtransferases; D000097811:Carbohydrate Sulfotransferases
D008345:Manganese; D011073:Polyamines; D015238:Sulfotransferases; D013466:Sulfurtransferases; D000097811:Carbohydrate Sulfotransferases; C019893:chondroitin 4-sulfotransferase