A new assay for determining ganglioside sialyltransferase activities lactosylceramide-2,3-sialyltransferase (SAT I) and monosialylganglioside-2,3-sialyltransferase (SAT IV).
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.
Callaghan Innovation Research Ltd, Lower Hutt, New Zealand.
Cynthia Q
CQ
Sun
Callaghan Innovation Research Ltd, Lower Hutt, New Zealand.
Ulrike
U
Hubl
Callaghan Innovation Research Ltd, Lower Hutt, New Zealand.
Petra
P
Hoefakker
Callaghan Innovation Research Ltd, Lower Hutt, New Zealand.
Madhusudan K
MK
Vasudevamurthy
Callaghan Innovation Research Ltd, Lower Hutt, New Zealand.
Keryn D
KD
Johnson
D000818:Animals; D015703:Antigens, CD; D001896:Boron Compounds; D002236:Carbohydrate Conformation; D002240:Carbohydrate Sequence; D002417:Cattle / Q000254:growth & development / Q000378:metabolism*; D004796:Clinical Enzyme Tests / Q000379:methods* / Q000662:veterinary; D005732:Gangliosides; D007668:Kidney / Q000201:enzymology; D007790:Lactosylceramides; D008099:Liver / Q000201:enzymology; D008861:Microsomes / Q000201:enzymology; D008969:Molecular Sequence Data; D009206:Myocardium / Q000201:enzymology; D009928:Organ Specificity; D012756:Sheep / Q000254:growth & development / Q000378:metabolism*; D012799:Sialyltransferases / Q000032:analysis*; D013154:Spleen / Q000201:enzymology; D013379:Substrate Specificity
C095489:4,4-difluoro-4-bora-3a,4a-diaza-s-indacene; D015703:Antigens, CD; D001896:Boron Compounds; D005732:Gangliosides; D007790:Lactosylceramides; C009744:CDw17 antigen; D012799:Sialyltransferases; C019916:CMP-N-acetylneuraminic acid-monosialoganglioside sialyltransferase; C021343:haematoside synthetase