ISSN:
0173-0835
Schlagwort(e):
Chemistry
;
Biochemistry and Biotechnology
Quelle:
Wiley InterScience Backfile Collection 1832-2000
Thema:
Biologie
,
Chemie und Pharmazie
Notizen:
This paper reviews a technique for detecting non-reducing terminal N-acetyl-glucosamine residues on glycoproteins in a mixture of proteins resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immobilized on nitrocellulose by electrophoretic transfer. Detection is accomplished by fluorography after enzymatic modification with galactosyltransferase (UDP-galactose: N-acetyl-D-glucosaminyl-glycopeptide 4-β-D-galactosyltransferase, E.C. 2.4.1.38), which transfers radiolabeled galactose from its nucleotide sugar substrate, UDP-[6-3H]-galactose, into any immobilized glycoprotein with a non-reducing terminal 2-acetamido-2-deoxy-D-glucose (GlcNAc) acceptor site. The kinetics of radiolabeling are independent of the amount of immobilized glycoprotein substrate. Half-maximal incorporation of [6-3H]galactose occurs by 5 h and saturation by 16 h. We have used enzyme blotting with galactosyltransferase: (i) to detect non-reducing terminal GlcNAc residues on isolated glycoproteins, (ii) to detect, in combination with endo-β-N-acetylglucosaminidase F digestion, Asn-linked oligosaccharides on glycoproteins, and (iii) to identify a subset of glycoproteins with non-reducing terminal N-acetylglucosamine residues in both F9 and PYS murine teratocarcinoma cell extracts, two of which appear to be unique to PYS cells. Because the family of glycosyltransferases recognizes a large variety of non-reducing terminal monosaccharide residues, this technique, termed enzyme blotting, could be applied to the analysis of any non-reducing terminal monosaccharide. This method could be used to obtain initial information about the carbohydrate structures of a particular glycoprotein without any prior purification steps. This technique may also be potentially useful in elucidating the functional contribution of non-reducing terminal monosaccharide residues in intercellular recognition and adhesion.
Zusätzliches Material:
3 Ill.
Materialart:
Digitale Medien
URL:
http://dx.doi.org/10.1002/elps.1150080910
Permalink