Measurement of beta-galactosyltransferase activity in cell extracts with an ELISA-based assay.
An enzyme-linked immunosorbent assay (ELISA)-based glycosyltransferase assay has been used to measure UDP-Gal:N-acetylglucosamine beta-1,4-galactosyl-transferase (EC 2.4.1.38) activity in detergent extracts of chinese hamster ovary (CHO) cells. LEC11 cells (a mutant of the CHO cell line, Pro -5), which are known to express a complex array of carbohydrate structures, were used to develop the assay for use with whole cell extracts. A detergent-solubilized preparation of the enzyme from whole cells was used to convert the substrate, lactotriglycosylceramide, to the product, neolactotetraglycosylceramide. The monoclonal antibody, 1B2, which specifically binds to the Gal beta 1-4GlcNAc epitope, was used in an ELISA to identify and quantify the product. The enzyme activity in the preparations was found to be similar to that obtained by conventional radioactive assay methods. The beta-galactosyltransferase found in LEC11 cell detergent extracts exhibited an absolute requirement for the nucleotide sugar and MnCl2. The activity of the enzyme was also strictly dependent on the presence of exogenous glycolipid acceptor. When Triton X-114 was used to solubilize the LEC11 beta-galactosyltransferase, activity was found in both the hydrophilic and the hydrophobic phases, suggesting the presence of two forms of the enzyme. The ELISA-based assay was used to compare beta-1,4-galactosyltransferase activity in detergent extracts of four CHO cell lines: Pro-5, Lec1, LEC11, and LEC12 and in detergent-solubilized microsomes from human leukemia cells. The results from this study demonstrate the utility of the ELISA-based assay for measuring glycosyltransferase activity in detergent-solubilized whole cells and microsome preparations.
Stults
C L
CL
Department of Chemistry and Biochemistry, San Francisco State University, California 94132.
Macher
B A
BA
D000818:Animals; D002240:Carbohydrate Sequence; D002460:Cell Line; D004797:Enzyme-Linked Immunosorbent Assay; D005732:Gangliosides / Q000032:analysis; D006028:Glycosphingolipids / Q000302:isolation & purification; D006801:Humans; D007700:Kinetics; D007788:Lactose Synthase / Q000032:analysis*; D015464:Leukemia, Myelogenous, Chronic, BCR-ABL Positive / Q000201:enzymology; D008861:Microsomes / Q000201:enzymology; D008969:Molecular Sequence Data; D009154:Mutation; D009237:N-Acetyllactosamine Synthase / Q000032:analysis* / Q000378:metabolism
D005732:Gangliosides; D006028:Glycosphingolipids; D007788:Lactose Synthase; D009237:N-Acetyllactosamine Synthase