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The use of galactosyltransferase to probe nitrocellulose-immobilized glycoproteins for nonreducing terminalN-acetylglucosamine residues

Authors
Journal
Analytical Biochemistry
0003-2697
Publisher
Elsevier
Publication Date
Volume
154
Issue
2
Identifiers
DOI: 10.1016/0003-2697(86)90015-1
Keywords
  • Carbohydrate Structure
  • Gel Electrophoresis Proteins
  • Glycoproteins
  • Cell Adhesion
  • Lectins
Disciplines
  • Biology

Abstract

Abstract We report the use of UDPgalactose: N-acetyl- d-glucosaminyl-glycopeptide 4-β- d-galactosyltransferase (EC 2.4.1.38), purified from bovine milk, to detect nonreducing terminal N-acetylglucosamine residues on glycoproteins immobilized on nitrocellulose by electrophoretic transfer from sodium dodecyl sulfate-polyacrylamide gels. Soluble galactosyltransferase incorporates radiolabeled galactose from the substrate UDP-[6- 3H]galactose into the appropriate immobilized acceptor with high specificity. Incorporation is proportional to substrate amount and is saturable with time. The kinetics of labeling are independent of substrate amount. Half-maximal incorporation occurs by 4 h and saturation occurs by 16 h. We have used galactosyltransferase as a probe (i) to verify the presence of nonreducing terminal N-acetylglucosamine residues in bovine rod outer segment membrane rhodopsin and in several glycoproteins in F9 murine teratocarcinoma cells and (ii) to detect previously reported endo-β- N-acetylglucosaminidase activity in a commercial preparation of endoglycosidase F.

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