Biosynthesis of heparin/heparan sulfate. Identification of a 70-kDa protein catalyzing both the D-glucuronosyl- and the N-acetyl-D-glucosaminyltransferase reactions.
The D-glucuronosyl- (GlcA) and N-acetyl-D-glucosaminyl- (GlcNAc) transferase reactions involved in heparin/heparan sulfate biosynthesis were assayed, measuring transfer of radiolabeled GlcA or GlcNAc monosaccharide units from the corresponding UDP-sugars to the appropriate oligosaccharide acceptors. The assays were applied to enzyme purification from bovine serum. The two activities remained inseparable through a series of different chromatographic steps, resulting in approximately -2000-fold purification. Further purification was achieved by chromatofocusing, which showed an isoelectric point of pH approximately -7.0, similar for both activities. SDS-polyacrylamide gel electrophoresis (PAGE) of subfractions from the chromatofocusing procedure revealed an approximately 70-kDa protein in amounts reflecting enzyme activity. SDS-PAGE followed by extraction of gel segments and renaturation of proteins showed that the GlcA- and GlcNAc-transferase activities were both recovered from the same single segment, corresponding to the 70-kDa component. It is proposed that the two glycosyltransferase reactions are catalyzed by the same Golgi enzyme (see also Lidholt, K., Weinke, J. L., Kiser, C. S., Lugemwa, F. N., Bame, K. J., Cheifetz, S., Massagué, J., Lindahl, U., and Esko, J. D. (1992) Proc. Natl. Acad. Sci. U.S.A. 89, 2267-2271).
Lind
T
T
Department of Medical and Physiological Chemistry, Uppsala University, Sweden.
Lindahl
U
U
Lidholt
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K
D000818:Animals; D002240:Carbohydrate Sequence; D002384:Catalysis; D002417:Cattle; D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D014453:Glucuronosyltransferase / Q000097:blood / Q000302:isolation & purification / Q000378:metabolism*; D006493:Heparin / Q000096:biosynthesis*; D006497:Heparan Sulfate / Q000096:biosynthesis*; D008969:Molecular Sequence Data; D017351:N-Acetylglucosaminyltransferases / Q000097:blood / Q000302:isolation & purification / Q000378:metabolism*
D014453:Glucuronosyltransferase; D006493:Heparin; D006497:Heparan Sulfate; D017351:N-Acetylglucosaminyltransferases; C037649:N-acetyllactosaminide beta-1,6-N-acetylglucosaminyltransferase