Characterization of a murine beta 1-4 galactosyltransferase expressed in COS-1 cells.
We inserted a full-length murine cDNA, which had been isolated from F9 embryonal carcinoma cells by using a bovine lactose synthetase A protein cDNA as a probe, in a mammalian expression vector (pCMGT1) and expressed it in COS-1 cells to characterize the pCMGT1-directed enzyme. The galactosyltransferase activity toward asialo-agalacto-transferrin (AsAg-Tf) in the pCMGT1-transfected cells was approximately eightfold higher than that in mock- or non-transfected cells. In contrast, no difference was observed in the specific activity of galactose transfer between pCMGT1-transfected cells and mock- or non-transfected cells when asialo-ovine submaxillary mucin were used as an acceptor. Since almost all [3H]galactose incorporated into the AsAg-Tf was released by digestion with streptococcal beta-galactosidase, most of the linkage created by this enzyme was in the Gal beta 1-4GlcNAc group. The acceptor specificity of the pCMGT1-directed enzyme was changed from N-acetylglucosamine to glucose by adding alpha-lactalbumin in the reaction mixture. Alpha-Lactalbumin also partially inhibited the galactose transfer to AsAg-Tf. The kinetic study revealed that the apparent Km values of the pCMGT1-directed enzyme for N-acetylglucosamine, AsAg-Tf and UDP-Gal are 2 mM, 60 microM and 24 microM, respectively. These results indicated that the murine cDNA isolated from F9 cells encodes an active enzyme which catalyzes not only the lactose synthesis but also the transfer of galactose to N-acetylglucosamine residues of Asn-linked sugar chains of glycoproteins in a beta 1-4 linkage.
Nakazawa
K
K
Department of Microbiology, Keio University School of Medicine, Tokyo, Japan.
Furukawa
K
K
Kobata
A
A
Narimatsu
H
H
D000818:Animals; D002460:Cell Line; D004247:DNA / Q000235:genetics; D004789:Enzyme Activation; D015971:Gene Expression Regulation, Enzymologic; D007768:Lactalbumin / Q000494:pharmacology; D007788:Lactose Synthase / Q000235:genetics / Q000378:metabolism; D008345:Manganese; D012313:RNA / Q000235:genetics; D013379:Substrate Specificity; D014162:Transfection; D001616:beta-Galactosidase / Q000494:pharmacology; D006022:beta-N-Acetylglucosaminylglycopeptide beta-1,4-Galactosyltransferase / Q000235:genetics / Q000378:metabolism*
D008345:Manganese; D012313:RNA; D004247:DNA; D007768:Lactalbumin; D007788:Lactose Synthase; D006022:beta-N-Acetylglucosaminylglycopeptide beta-1,4-Galactosyltransferase; D001616:beta-Galactosidase