An assay method for ganglioside synthase using anion-exchange chromatography.
A rapid procedure which is based on combined ion-exchange chromatography and solubility was established for determination of the activity of ganglioside synthases and cerebroside sulfotransferase. The procedure consists of selective elution of radiolabeled reaction products (acidic glycolipids) freed from labeled precursors and breakdown products on a DEAE-Sephadex column and of direct radioassay of the products in the eluate. Monosialogangliosides were eluted from the column with 40 mM ammonium acetate (AcONH4) in methanol, cerebroside sulfate with 90 mM AcONH4 in methanol, and disialogangliosides with 40 mM AcONH4 in isopropanol/n-hexane/water (55/20/19, v/v/v). The established procedure is simple, reproducible, and economical. Using rat Golgi membrane as enzyme source the recovery rate of the products was over 95%.
Kawano
M
M
Biochemistry Laboratory, Hokkaido University School of Medicine, Sapporo, Japan.
Honke
K
K
Tachi
M
M
Gasa
S
S
Makita
A
A
D000818:Animals; D002852:Chromatography, Ion Exchange; D005700:Galactosyltransferases / Q000032:analysis*; D005732:Gangliosides / Q000096:biosynthesis*; D006017:Glycolipids / Q000096:biosynthesis; D006056:Golgi Apparatus / Q000378:metabolism; D051381:Rats; D011919:Rats, Inbred Strains; D012799:Sialyltransferases / Q000032:analysis*; D015238:Sulfotransferases / Q000032:analysis*; D013466:Sulfurtransferases / Q000032:analysis
D005732:Gangliosides; D006017:Glycolipids; D005700:Galactosyltransferases; D012799:Sialyltransferases; D013466:Sulfurtransferases; D015238:Sulfotransferases; C020556:galactosylceramide sulfotransferase