A Quantitative Immunostaining Method for the Measurement of UDP-galactose:lactosylceramide Galactosyltransferase for the Synthesis of Globotriaosylceramide in Rabbit Small Intestine and HeLa Cells
✍ Scribed by M. Mobassaleh; K. Mishra; G. Keusch
- Publisher
- Elsevier Science
- Year
- 1993
- Tongue
- English
- Weight
- 549 KB
- Volume
- 214
- Category
- Article
- ISSN
- 0003-2697
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✦ Synopsis
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 (\left(G_{3}\right)). We describe a quantitative more sensitive and specific method for the measurement of UDP-galactose:lactosylceramide galactosyltransferase activities in rabbit small intestine and HeLa cells which utilizes the specific binding of Shiga toxin to the product, (\mathbf{G b}{3}). Intestinal microsomal or HeLa cell sonicate preparations were incubated in the presence of lactosylceramide and (\left[{ }^{14} \mathrm{C}\right]) 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. (\mathbf{G b}{3}) 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 (\mathbf{G b}_{3}) 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. ( 1983 Academic Press, Inc.