Kinetic study of human beta-1,4-galactosyltransferase expressed in E. coli.
Recombinant beta-1,4-galactosyltransferase which synthesizes the Gal beta 1-->4GlcNAc group of glycoprotein sugar chains was obtained as a soluble form from Escherichia coli by transfection of the human cDNA lacking the transmembrane segment. Kinetic study revealed that the soluble transferase has the same apparent Km values toward sugar nucleotide and sugar acceptors as those of mouse membrane-bound beta-1,4-galactosyltransferase previously characterized [Nakazawa et al. (1991) Eur. J. Biochem. 196, 363-368]. However, the Vmax value of this transferase was low when compared to that of the mammalian transferase, probably due to the instability of the transferase caused by the lack of protein glycosylation. The soluble transferase was purified from the E. coli lysates almost to homogeneity by chromatography on DEAE-Sepharose and alpha-lactalbumin-Sepharose columns. Using this purified transferase, the acceptor specificity of the transferase has been studied. The results showed that the transferase has apparent Km values of 170, 190, and 830 microM for agalacto-poly-N-acetyllactosamine, lacto-N-triose II, and lacto-N-triaosylceramide, respectively, but has apparently no activity toward glucosylceramide. These results suggest that the beta-1,4-galactosyltransferase may be involved in the synthesis of poly-N-acetyllactosamine, lacto-N-neotetraose, and probably lacto-N-neotetraosylceramide in addition to the formation of the Gal beta 1-->4GlcNAc group of glycoprotein sugar chains and lactose.
Nakazawa
K
K
Department of Microbiology, Keio University School of Medicine, Tokyo.
Furukawa
K
K
Narimatsu
H
H
Kobata
A
A
D002240:Carbohydrate Sequence; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D006023:Glycoproteins / Q000096:biosynthesis / Q000737:chemistry; D006028:Glycosphingolipids / Q000737:chemistry; D006801:Humans; D066298:In Vitro Techniques; D007700:Kinetics; D008969:Molecular Sequence Data; D009237:N-Acetyllactosamine Synthase / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D009844:Oligosaccharides / Q000737:chemistry; D011134:Polysaccharides / Q000737:chemistry; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D013379:Substrate Specificity
D006023:Glycoproteins; D006028:Glycosphingolipids; D009844:Oligosaccharides; D011134:Polysaccharides; D011994:Recombinant Proteins; C037199:poly-N-acetyllactosamine; D009237:N-Acetyllactosamine Synthase