A New Assay for Determining Ganglioside Sialyltransferase Activities Lactosylceramide-2,3-Sialyltransferase (SAT I) and Monosialylganglioside-2,3-Sialyltransferase (SAT IV)

Abstract

A new assay for the determination of lactosylceramide-2,3-sialyltransferase (SAT I, EC 2.4.99.9) and monosialoganglioside sialyltransferase (SAT IV, EC 2.4.99.2) is described. The assay utilised the commercially available fluorophore labelled sphingolipids, boron dipyrromethene difluoride (BODIPY) lactosylceramide (LacCer), and BODIPY-monosialotetrahexosylganglioside (GM1) as the acceptor substrates, for SAT I and SAT IV, respectively. HPLC coupled with fluorescence detection was used to analyse product formation. The analysis was performed in a quick and automated fashion. The assay showed good linearity for both BODIPY sphingolipids with a quantitative detection limit of 0.05 pmol. The high sensitivity enabled the detection of SAT I and SAT IV activities as low as 0.001 μU, at least 200 fold lower than that of most radiometric assays. This new assay was applied to the screening of SAT I and SAT IV activities in ovine and bovine organs (liver, heart, kidney, and spleen). The results provided evidence that young animals, such as calves, start to produce ganglioside sialyltransferases as early as 7 days after parturition and that levels change during maturation. Among the organs tested from a bovine source, spleen had the highest specific ganglioside sialyltransferase activity. Due to the organ size, the greatest total ganglioside sialyltransferase activities (SAT I and SAT IV) were detected in the liver of both bovine and ovine origin.

Function and Structure Analysis of Glycolipid Microdomains

“…washing method is available and started to be used not only for the removal of the detergent after membrane microdomain fraction-ation, but also for other applications, including the recovery of glycolipids from glycosyltransferase, carbohydrate hydrolysis enzyme reactions, cell lysates, and removal of detergents from the reaction mix (33). In this article, analysis of glycolipid microdomains using fluorescence microscopy and mass spectrometry was described.…”

Quantifying Carbohydrate-Active Enzyme Activity with Glycoprotein Substrates Using Electrospray Ionization Mass Spectrometry and Center-of-Mass Monitoring

“…Moreover, the introduction of fluorophores (or chromophores) into the substrate can alter, in some cases substantially, CAZyme activity. 13 Separation techniques (e.g., high-performance liquid chromatography) coupled to mass spectrometry (MS) detection represent label-free approaches. However, such methods generally require quenching of the reaction and, as such, do not allow for continuous monitoring.…”

“…Spectroscopic techniques, such as fluorescence and luminescence, also offer high sensitivity but are not readily amenable to monitoring multiple substrates simultaneously. Moreover, the introduction of fluorophores (or chromophores) into the substrate can alter, in some cases substantially, CAZyme activity . Separation techniques (e.g., high-performance liquid chromatography) coupled to mass spectrometry (MS) detection represent label-free approaches.…”