HUMAN-MELANOMA AND CHINESE-HAMSTER OVARY CELLS GALACTOSYLATE N-ALKYL-BETA-GLUCOSIDES USING UDP GAL-GLCNAC BETA-1,4 GALACTOSYLTRANSFERASE

We previously showed that human melanoma, CHO and other cells can conv ert beta-xylosides into structural analogs of ganglioside G(M3). We ha ve investigated several potential accepters including a series of n-al kyl-beta-D-glucosides (n = 6-9). All were labeled with H-3-galactose w hen incubated with human melanoma cells. Octyl-beta-D-glucoside (Glc b eta Octyl) was the best acceptor, whereas neither octyl-alpha-D-glucos ide nor N-octanoyl-methylglucamine (MEGA 8) were labeled. Analysis of the products by a combination of chromatographic methods and specific enzyme digestions showed that the accepters first received a single Ga l beta 1,4 residue followed by an alpha 2,3 linked sialic acid. Synthe sis of these products did not affect cell viability, adherence, protei n biosynthesis, or incorporation of radio-labeled precursors into glyc oprotein, glycolipid or proteoglycans. To determine which beta 1,4 gal actosyl transferase synthesized Gal beta 1,4Glc beta Octyl, we analyze d similar incubations using CHO cells and a mutant CHO line (CHO 761) which lacks GAG-core specific beta 1,4 galactosyltransferase. The muta nt cells showed the same level of incorporation as the control, elimin ating this enzyme as a candidate. Thermal inactivation kinetics using melanoma cell microsomes and rat liver Golgi to galactosylate Glc beta Octyl showed the same half-life as UDP-Gal:GlcNAc beta 1,4 galactosyl transferase, whereas LacCer synthase was inactivated at a much faster rate. We show that Glc beta Octyl is a substrate for purified bovine m ilk UDP-Gal:GlcNAc beta 1,4 galactosyltransferase Furthermore, the gal actosylation of Glc beta Octyl by CHO cell microsomes can be competiti vely inhibited by GlcNAc or GlcNAc beta MU. These results indicate tha t UDP-Gal:GlcNAc beta 1,4 galactosyltransferase is the enzyme used for the synthesis of the alkyl lactosides when cells or rat liver Golgi a re incubated with alkyl beta glucosides.