Analysis of Glycosphingolipid Glycosyltransferase Products on TLC Plates by Combined Storage Phosphor and Immunostaining Techniques

Abstract

Measurement of glycosyltransferase activity in whole cell extracts is often complicated by the fact that several enzymes in an homogenate are capable of using the same nucleotide sugar donor, thereby generating a range of products from both an exogenous and any endogenous acceptors. We report the use of a novel combination of techniques to simultaneously identify and quantify the products generated from a whole cell extract in a single experiment. Several radiolabeled glycosphingolipid products were generated by the addition of UDP-[ 14C]Gal to a reaction mixture containing an homogenate from a human leukemia cell line, THP-1. After the 14C-labeled products were separated on a TLC plate, storage phosphor technology and immunostaining (with carbohydrate sequence-specific monoclonal antibodies) were used sequentially on the same plate to simultaneously identify and quantify each of the glycosyltransferase products. This method allows product identification and quantification in the femtomole range. Thus, low levels of endogenous accepters were easily detected. We have used a similar method with UDP-[ 3H]Gal to obtain glycosyltransferase product profiles from several human leukemia/lymphoma cell lines and subsequently identify two galactosyltransferase activities in these cell lines: UDP-Gal:Galβ1-4Glcβ1-1Cer α1,4galactosyltransferase; and UDP-Gal:GlcNAcβ1-3Galβ1-4Glcβ1-1Cer β1,4galactosyltransferase. In addition to product characterization, this method was used with reaction mixtures at different pH to demonstrate the usefulness of the method for characterizing multiple enzyme activities simultaneously.