Assay of sialyltransferase activity by reversed-phase ion-pair high-performance liquid chromatography.
Sialyltransferases (CMP-N-acetylneuraminic acid:glycoprotein sialyltransferases, EC 2.4.99.1) are involved in the transfer of a sialic acid moiety from CMP-N-acetylneuraminic acid (CMP-NeuAc) to an oligosaccharide side-chain of an acceptor, asialoglycoprotein (AGP), according to the following reaction: CMP-NeuAc + AGP----NeuAc-O-AGP + CMP. This enzyme occurs in elevated levels in the sera of patients with a wide variety of neoplastic diseases and its assay might be useful in monitoring treatment. Radioactive CMP-NeuAc has been used in assays and the radioactive sialylated product separated and counted by liquid scintillation spectrometry. This study shows that a simple, rapid, non-radiochemically based high-performance liquid chromatographic method developed for the analysis of CMP-sialic acid synthetase can be used for the quantitation of sialyltransferase activity by monitoring simultaneously the utilization of CMP-NeuAc and the release of CMP. We describe the application of this method to assay of commercially available sialyltransferase activity and to activities from synovial, ascites and gastric fluids.
Spiegel
L B
LB
Department of Chemistry, Youngstown State University, OH 44555.
Hadjimichael
J
J
Rossomando
E F
EF
D000818:Animals; D001201:Ascites / Q000201:enzymology; D002851:Chromatography, High Pressure Liquid; D003569:Cytidine Monophosphate N-Acetylneuraminic Acid / Q000032:analysis; D005750:Gastric Juice / Q000201:enzymology; D006801:Humans; D051379:Mice; D009961:Orosomucoid / Q000032:analysis; D051381:Rats; D012799:Sialyltransferases / Q000032:analysis*; D013582:Synovial Fluid / Q000201:enzymology; D000094802:beta-D-Galactoside alpha 2-6-Sialyltransferase
D009961:Orosomucoid; D003569:Cytidine Monophosphate N-Acetylneuraminic Acid; D012799:Sialyltransferases; D000094802:beta-D-Galactoside alpha 2-6-Sialyltransferase