A quantitative immunostaining method for the measurement of UDP-galactose:lactosylceramide galactosyltransferase for the synthesis of globotriaosylceramide in rabbit small intestine and HeLa cells.
Galactosyltransferase is required for the addition of galactose to lactosylceramide (galactose beta 1-4 glucose beta 1-1 ceramide), resulting in the synthesis of globotriaosylceramide (Gb3). We describe a quantitative more sensitive and specific method for the measurement of UDP-galactose:lactosylceramide galactosyltransferase activities in rabbit small intestine and HeLa cell which utilizes the specific binding of Shiga toxin to the product, Gb3. Intestinal microsomal or HeLa cell sonicate preparations were incubated in the presence of lactosylceramide and [14C]UDP-galactose. The lipid reaction products were extracted on C18 Bond-Elut columns, separated by high-performance thin-layer chromatography and exposed to Shiga toxin followed by polyclonal rabbit anti-Shiga toxin antibody and goat anti-rabbit IgG alkaline phosphatase conjugate. Gb3 was visualized with NBT and BCIP and quantitated by densitometry. These data were compared with a standard assay in which, following incubation and lipid extraction, radioactivity was measured by scintillation counting of the isolated lipids. There was a 22-fold increase in enzyme activity by the immunostaining method compared to the usual scintillation counting technique. This is attributable to the exclusion of radioactive lipids other than Gb3 in calculating enzyme activity and the correction for endogenous UDP-galactose. Thus, the immunostaining method provides increased accuracy, sensitivity, and specificity in the assay of galactosyltransferase activity.
Mobassaleh
M
M
Division of Pediatric Gastroenterology and Nutrition, New England Medical Center, Boston, Massachusetts 02111.
Mishra
K
K
Keusch
G T
GT
D000818:Animals; D002250:Carbon Radioisotopes; D002458:Cell Fractionation; D002851:Chromatography, High Pressure Liquid; D005700:Galactosyltransferases / Q000032:analysis* / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D007421:Intestine, Small / Q000201:enzymology*; D007700:Kinetics; D008861:Microsomes / Q000201:enzymology* / Q000648:ultrastructure; D011817:Rabbits; D011865:Radioisotope Dilution Technique; D012588:Scintillation Counting / Q000379:methods; D014281:Trihexosylceramides / Q000096:biosynthesis*
D002250:Carbon Radioisotopes; D014281:Trihexosylceramides; C018549:globotriaosylceramide; D005700:Galactosyltransferases; C045097:UDP-galactose-lactosylceramide alpha 1-4-galactosyltransferase