Human ovarian cancer, lymphoma spleen, and bovine milk GlcNAc:β1,4Gal/GalNAc transferases:: two molecular species in ovarian tumor and induction of GalNAcβ1,4Glc synthesis by α-lactalbumin

Affinity Gel-UDP was utilized to purify GlcNAc:beta1,4Gal/GalNAc transferases (Ts) from human lymphoma spleen, ovarian tumor, and ovarian cancer sera. Mn2+ was found to be an absolute requirement for activity. Two molecular species containing both beta1,4Gal/GalNAc-T activities were discernible when the purified ovarian tumor microsomal enzyme was subjected to Sephacryl S-100 HR column chromatography as well as native polyacylamide gel-electrophoresis. Acceptor specificity studies of the affinity-purified lymphoma spleen and ovarian tumor microsomal enzymes and the conventionally purified, as well as the cloned, bovine milk GlcNAc:beta1,4Gal-Ts using a number of synthetic acceptors showed that the beta (1,6)-linked GlcNAc moiety to alpha -GalNAc was the most efficient acceptor. As compared to the purified milk enzyme, the recombinant form exhibited sixfold GlcNAc:beta1,4 GalNAc-T activity and up to eightfold GlcNAc6SO(3)beta-:beta1,4Gal-T activity. Further, the recombinant enzyme catalyzed the transfer of GalNAc to the terminal beta -linked GlcNAc6SO(3) moiety. Alpha-lactalbumin (alpha -LA) inhibited up to 85%, the transfer of Gal to the GlcNAc moiety linked either to Man or GlcNAc. On the contrary, alpha -LA had no significant influence on the transfer of GalNAc to the above acceptors. alpha -LA had no appreciable effect on the recombinant enzyme, except for the transfer of Gal or GaINAc to Glc. Both alpha- and beta -glucosides, as well as alpha -N-acetylglucosaminide. did not serve as acceptors. (C) 2001 Elsevier Science Ltd. All rights reserved.