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TitleA new assay for determining ganglioside sialyltransferase activities lactosylceramide-2,3-sialyltransferase (SAT I) and monosialylganglioside-2,3- sialyltransferase (SAT IV)
Publication TypeJournal Article
Year of Publication2014
AuthorsSun, C.Q., Hubl U., Hoefakker P., Vasudevamurthy M.K., and Johnson K.D.
JournalPLoS ONE
Date Published2014
ISSN19326203 (ISSN)
Keywords4,4-difluoro-4-bora-3a,4a-diaza-s-indacene, Animal, Animals, antibody specificity, Antigens, CD, article, Boron compounds, boron derivative, boron dipyrromethene difluoride lactosylceramide, boron dipyrromethene difluoride monosialotetrahexosylganglioside, bovine, carbohydrate analysis, Carbohydrate Conformation, Carbohydrate Sequence, cattle, CDw17 antigen, Clinical Enzyme Tests, CMP-N-acetylneuraminic acid-monosialoganglioside sialyltransferase, comparative study, conformation, controlled study, enzyme activity, enzyme assay, enzyme specificity, enzymology, fluorescence analysis, ganglioside, Gangliosides, growth, development and aging, haematoside synthetase, heart muscle, high performance liquid chromatography, kidney, lactosylceramide, lactosylceramide 2,3 sialyltransferase, Lactosylceramides, leukocyte antigen, liver, metabolism, microsome, Microsomes, molecular genetics, Molecular Sequence Data, monosialylganglioside 2,3 sialyltransferase, Myocardium, organ size, Organ Specificity, procedures, Radiometry, reagent, sheep, Sialyltransferase, Sialyltransferases, sphingolipid, spleen, Substrate Specificity, unclassified drug, veterinary
AbstractA new assay for the determination of lactosylceramide-2,3-sialyltransferase (SAT I, EC and monosialoganglioside sialyltransferase (SAT IV, EC 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. © 2014 Sun et al.

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