Glycosyltransferase assays utilizing N-acetyllactosamine acceptor immobilized on a cellulose membrane.
Solid-phase assays for measuring the activity of four different glycosyltransferase enzymes that utilize N-acetyllactosamine as an acceptor are reported. These enzymes are alpha1,3-galactosyltransferase (E.C. 2.4.1.151), alpha1,3-fucosyltransferase (E.C. 2.4.1.65), alpha2,6-(N)-sialyltransferase (E.C. 2.4.99.1), and alpha2,3-(N)-sialyltransferase (E.C. 2.4.99.5). The acceptor is immobilized on a cellulose membrane in two different ways, through either an amine-cleavable linker or a photolinker. Incubation with a glycosyltransferase and nucleotide donor sugar resulted in the transfer of a monosaccharide from the donor to immobilized N-acetyllactosamine. For galactosyltransferase, transfer was confirmed by mass spectrometry of the products cleaved from the membrane surface after amine treatment or irradiation. When radioactive donors were utilized, the transfer of radioactive sugars could be monitored by autoradiography. Alternatively the transfer of radioactive sugar onto the membranes could be measured by scintillation counting of the products after cleavage from the membrane. Cytidine 5(')-monophosphate-sialic acid carrying a fluorescent tag in the saccharide was also successfully utilized in this assay system. Fluorescent product on the membrane surface was detected by imaging. Glycosyltransferase assays with these versatile membranes have the potential to be adapted for high-throughput screening.
Jobron
Laurence
L
Jerini AG, Invalidenstr. 130, 10115 Berlin, Germany.
Sujino
Keiko
K
Hummel
Gerd
G
Palcic
Monica M
MM
D000606:Amino Sugars / Q000737:chemistry*; D000818:Animals; D015374:Biosensing Techniques; D002482:Cellulose; D003432:Cross-Linking Reagents; D016695:Glycosyltransferases / Q000032:analysis* / Q000378:metabolism; D006801:Humans; D008567:Membranes, Artificial; D009005:Monosaccharides / Q000032:analysis
D000606:Amino Sugars; D003432:Cross-Linking Reagents; D008567:Membranes, Artificial; D009005:Monosaccharides; C000458:N-acetyllactosamine; D002482:Cellulose; D016695:Glycosyltransferases