Characterization and membrane organization of beta 1----3- and beta 1----4-galactosyltransferases from human colonic adenocarcinoma cell lines Colo 205 and SW403: basis for preferential synthesis of type 1 chain lacto-series carbohydrate structures.
Evidence indicates that activation of a beta 1----3N-acetylglucosaminyltransferase is responsible for accumulation of large quantities of lacto-series tumor-associated antigens in human colonic adenocarcinomas. Expression of type 1 and 2 core chain derivatives characterize human colonic adenocarcinomas, whereas normal adult colonic epithelial cells express detectable quantities of only type 1 chain derivatives. The basis for preferential synthesis of type 1 chain lacto-series carbohydrate structures characteristic of normal colonic mucosa and human colonic adenocarcinoma Colo 205 cells has been studied. The beta 1----3- and beta 1----4galactosyltransferase enzymes associated with synthesis of type 1 and 2 core chain structures, respectively, have been separated from a Triton X-100 solubilized membrane fraction of Colo 205 cells by chromatography on an alpha-lactalbumin-Sepharose column and their properties studied. Optimal transfer of beta 1----3-linked galactose to acceptor Lc3 occurred in the presence of 0.1% Triton CF-54 with Triton X-100 providing 75% of maximal activity. The enzyme was active over a broad pH range from 6.5 to 7.5 and had a near absolute requirement for Mn2+. The Km values for donor UDPgalactose and acceptor Lc3 were determined to be 48 and 13 microM, respectively. In contrast, the beta 1----4galactosyltransferase required taurodeoxycholate for maximal activity and the Km for Lc3 was found to be 20-fold higher than that for the beta 1----3-specific enzyme under the same assay conditions. Studies with membrane-bound beta 1----3- and beta 1----4galactosyltransferases as found in Golgi-rich membrane fractions of SW403 and Colo 205 adenocarcinoma cells showed that preferential synthesis of type 1 chain structures occurs under conditions similar to those in vivo for biosynthesis of lacto-series core chains. The results suggest that both the higher affinity of the beta 1----3galactosyltransferase for acceptor Lc3 and the membrane organizational features result in preferential synthesis of type 1 chain structures.
Holmes
E H
EH
Pacific Northwest Research Foundation, Seattle, Washington 98122.
D000230:Adenocarcinoma / Q000201:enzymology*; D002021:Buffers; D002236:Carbohydrate Conformation; D002458:Cell Fractionation; D002460:Cell Line; D002462:Cell Membrane / Q000201:enzymology; D002499:Centrifugation, Density Gradient; D002845:Chromatography / Q000379:methods; D003110:Colonic Neoplasms / Q000201:enzymology*; D003902:Detergents; D005700:Galactosyltransferases / Q000276:immunology / Q000302:isolation & purification*; D006017:Glycolipids / Q000032:analysis; D006801:Humans; D007425:Intracellular Membranes / Q000201:enzymology; D007785:Lactose / Q000378:metabolism*; D013379:Substrate Specificity; D014407:Tumor Cells, Cultured
D002021:Buffers; D003902:Detergents; D006017:Glycolipids; D005700:Galactosyltransferases; C053372:glucosaminylgalactosylglucosylceramide beta-galactosyltransferase; D007785:Lactose