Purification, photoaffinity labeling, and characterization of a single enzyme for 6-sulfation of both chondroitin sulfate and keratan sulfate.
A soluble sulfotransferase that could 6-sulfate both chondroitin sulfate and corneal keratan sulfate was purified 27,500-fold using a sequence of affinity chromatographic steps with heparin-Sepharose, wheat germ agglutinin-agarose, and 3',5'-ADP-agarose. The essentially pure enzyme had a specific activity 40 times greater than the most purified chondroitin 6-sulfotransferase previously reported and exhibited a single sharp Coomassie Blue-stained and a heavy silver-stained protein band of 75 kDa on SDS-polyacrylamide gel electrophoresis. Chromatography of the purified enzyme on Sephacryl demonstrated a size of 150 kDa, which indicated that the native enzyme exists as a dimer. In addition to 6-sulfation of nonsulfated GalNAc, the purified serum enzyme had the ability to sulfate GalNAc 4-sulfate residues to give GalNAc 4,6-disulfate residues. The purified enzyme exhibited a Km of 40 microM for adenosine 3'-phosphate 5'-phosphosulfate when either chondroitin sulfate or corneal keratan sulfate were used as the acceptors. Use of both chondroitin sulfate and keratan sulfate in the same experiment demonstrated mutual competition, establishing that the sulfation of these substrates is by the same enzyme. Photoaffinity labeling of the purified enzyme with 2-azidoadenosine 3',5'-di[5'-32P]phosphate occurred only with the 75-kDa protein, confirming that this is the chondroitin 6-sulfotransferase/keratan sulfotransferase.
Sugumaran
G
G
Edith Nourse Rogers Memorial Veterans Hospital, Bedford, Massachusetts 01730, USA.
Katsman
M
M
Drake
R R
RR
D000255:Adenosine Triphosphate / Q000378:metabolism; D000345:Affinity Labels; D000818:Animals; D002645:Chickens; D002809:Chondroitin Sulfates / Q000378:metabolism*; D007632:Keratan Sulfate / Q000378:metabolism*; D008970:Molecular Weight; D010777:Photochemistry; D013379:Substrate Specificity; D013431:Sulfates / Q000378:metabolism; D015238:Sulfotransferases / Q000302:isolation & purification* / Q000378:metabolism
D000345:Affinity Labels; D013431:Sulfates; D000255:Adenosine Triphosphate; D002809:Chondroitin Sulfates; D007632:Keratan Sulfate; D015238:Sulfotransferases