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 acceptors 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:Gal beta 1-4Glc beta 1-1Cer alpha 1,4galactosyltransferase; and
UDP-Gal:GlcNAc beta 1--3Gal beta 1--4Glc beta 1--1Cer beta 1,4galactosyltransferase. 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.