ANALYSIS OF GLYCOSPHINGOLIPID GLYCOSYLTRANSFERASE PRODUCTS ON TLC PLATES BY COMBINED STORAGE PHOSPHOR AND IMMUNOSTAINING TECHNIQUES

Measurement of glycosyltransferase activity in whole cell extracts is often complicated by the fact that several enzymes in an homogenate ar e capable of using the same nucleotide sugar donor, thereby generating a range of products from both an exogenous and any endogenous accepto rs. We report the use of a novel combination of techniques to simultan eously 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-[C-14]Gal to a reactio n mixture containing an homogenate from a human leukemia cell line, TH P-1. After the C-14-labeled products were separated on a TLC plate, st orage phosphor technology and immunostaining (with carbohydrate sequen ce-specific monoclonal antibodies) were used sequentially on the same plate to simultaneously identify and quantify each of the glycosyltran sferase products. This method allows product identification and quanti fication in the femtomole range. Thus, low levels of endogenous accept ers were easily detected. We have used a similar method with UDP-[H-3] Gal to obtain glycosyltransferase product profiles from several human leukemia/lymphoma cell lines and subsequently identify two galactosylt ransferase activities in these cell lines: UDP-Gal:Gal beta 1-4Glc bet a 1-1Cer alpha 1,4galactosyltransferase; and UDP-Gal:GlcNAc beta 1-3Ga l beta 1-4Glc beta 1-1Cer beta 1,4galactosyltransferase. In addition t o product characterization, this method was used with reaction mixture s at different pH to demonstrate the usefulness of the method for char acterizing multiple enzyme activities simultaneously. (C) 1994 Academi c Press, Inc.