A highly sensitive fluorometric assay for sialyltransferase activity using CMP-9-fluoresceinyl-NeuAc as donor.
This paper presents a very sensitive fluorometric assay for sialyltransferase activity based on the transfer of 5-acetamido-9-deoxy-9-fluoresceinylthioure-idoneuraminic acid onto distinct glycoproteins, thus allowing determination of acceptor specificities. Acceptor protein-bound fluorescence was quantified after gel filtration which separated fluorescent sialoglycoprotein from the fluorescence-labeled CMP-glycoside donor. Kinetic constants obtained for five different purified sialyltransferases indicated that CMP-9-fluoresceinyl-NeuAc was a suitable donor substrate for each enzyme, affording low Km values and Vmax values comparable in magnitude (15-100%) to that obtained with the parent CMP-NeuAc. Sensitivity was enhanced 200- to 1000-fold compared to the radiometric sialyltransferase assay as it is used routinely. The method was applied to determination of the kinetic properties of purified rat liver alpha 2,6-sialyltransferase with four separate glycoprotein acceptors differing in glycan structure, employing very small amounts of donor, acceptor, and enzyme, and to the study of sialyltransferase activity of the human promyelocytic cell line HL-60 toward three different acceptors.
Gross
H J
HJ
Institut für Biochemie II, Universität Heidelberg, Federal Republic of Germany.
Sticher
U
U
Brossmer
R
R
D000818:Animals; D002460:Cell Line; D002851:Chromatography, High Pressure Liquid; D002853:Chromatography, Liquid; D003569:Cytidine Monophosphate N-Acetylneuraminic Acid; D005470:Fluorometry; D005690:Galactose / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D008099:Liver / Q000201:enzymology* / Q000378:metabolism; D051381:Rats; D012680:Sensitivity and Specificity; D012794:Sialic Acids; D012799:Sialyltransferases / Q000032:analysis*; D013379:Substrate Specificity
D012794:Sialic Acids; D003569:Cytidine Monophosphate N-Acetylneuraminic Acid; D012799:Sialyltransferases; D005690:Galactose