Purification and characterization of fetal bovine serum beta-N-acetyl-D-galactosaminyltransferase and beta-D-glucuronyltransferase involved in chondroitin sulfate biosynthesis.
beta-N-Acetylgalactosaminyltransferase II and beta-glucuronyltransferase II, involved in chondroitin sulfate biosynthesis, transfer an N-acetylgalactosamine (GalNAc) and glucuronic acid (GlcA) residue, respectively, through beta-linkages to an acceptor chondroitin oligosaccharide derived from the repeating disaccharide region of chondroitin sulfate. They were copurified from fetal bovine serum approximately 2500-fold and 850-fold, respectively, by sequential chromatographies on Red A-agarose, phenyl-Sepharose, S-Sepharose and wheat germ agglutinin-agarose. Identical and inseparable chromatographic profiles of both glycosyltransferase activities obtained through the above chromatographic steps and gel filtration suggest that the purified enzyme activities are tightly coupled, which could imply a single enzyme with dual transferase activities; beta-N-acetylgalactosaminyltransferase and beta-glucuronyltransferase, reminiscent of the heparan sulfate polymerase reaction. However, when a polymerization reaction was performed in vitro with the purified serum enzyme preparation under the polymerization conditions recently developed for the chondroitin-synthesizing system, derived from human melanoma cells, each monosaccharide transfer took place, but no polymerization occurred. These results may suggest that the purified serum enzyme preparation contains both beta-N-acetylgalactosaminyltransferase II and beta-glucuronyltransferase II activities on a single polypeptide or on the respective polypeptides forming an enzyme complex, but is different from that obtained from melanoma cells in that it transfers a single GalNAc or GlcA residue but does not polymerize chondroitin.
Tsuchida
K
K
Department of Biochemistry, Kobe Pharmaceutical University, Japan.
Lind
T
T
Kitagawa
H
H
Lindahl
U
U
Sugahara
K
K
Lidholt
K
K
D000818:Animals; D002417:Cattle; D002807:Chondroitin / Q000378:metabolism; D002809:Chondroitin Sulfates / Q000096:biosynthesis*; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D004795:Enzyme Stability; D005312:Fetal Blood / Q000201:enzymology*; D014453:Glucuronosyltransferase / Q000097:blood*; D009097:Multienzyme Complexes / Q000737:chemistry; D017350:N-Acetylgalactosaminyltransferases / Q000097:blood*
D009097:Multienzyme Complexes; D002807:Chondroitin; D002809:Chondroitin Sulfates; D017350:N-Acetylgalactosaminyltransferases; D014453:Glucuronosyltransferase