Identification of a region of UDP-galactose:N-acetylglucosamine beta 4-galactosyltransferase involved in UDP-galactose binding by differential labeling.
The location of regions in the primary structure of UDP-galactose:N-acetylglucosamine beta 4-galactosyl-transferase (GT) that are involved in binding UDP-galactose has been investigated by differential chemical modification with two different reagents in the presence and absence of UDP-galactose. Treatment with periodate-cleaved UDP and NaCNBH3 resulted in a loss of 80% of GT activity, which was largely prevented by UDP-galactose. Stoichiometry of labeling and peptide maps of the modified enzyme samples indicated partial labeling at many sites. A major site of reaction in the absence of UDP-galactose that was essentially unmodified in its presence was found to correspond to Lys341 in the cDNA sequence of GT. As a second approach, the reactivities of the amino groups of GT were compared in the presence and absence of saturating levels of UDP-galactose by trace acetylation with [3H]acetic anhydride. UDP-galactose binding was found to perturb the reactivities of a number of lysines in the C-terminal region of GT, the most pronounced effect being a reduction in the reactivity of Lys351. The two procedures thus identified a region between residues 341 and 351 as being associated with UDP-galactose binding. This region overlaps a small section in the sequence of GT that was previously noted to be similar to part of bovine alpha-1,3-galactosyltransferase (Joziasse, D. H., Shaper, J. H., Van den Eijnden, D. H., Van Tunen, A. J., and Shaper, N. L. (1989) J. Biol. Chem. 264, 14290-14297). Sequence comparisons indicate that extended regions at the C terminus of each enzyme encompassing this area may represent homologous UDP-galactose-binding domains.
Yadav
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Department of Biochemistry and Molecular Biology, University of Miami, Florida 33136-1015.
Brew
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D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002417:Cattle; D002851:Chromatography, High Pressure Liquid; D003126:Colostrum / Q000201:enzymology; D005260:Female; D007700:Kinetics; D007788:Lactose Synthase / Q000378:metabolism*; D008969:Molecular Sequence Data; D009237:N-Acetyllactosamine Synthase / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D010446:Peptide Fragments / Q000302:isolation & purification; D011247:Pregnancy; D011487:Protein Conformation; D012689:Sequence Homology, Nucleic Acid; D014530:Uridine Diphosphate / Q000494:pharmacology; D014531:Uridine Diphosphate Galactose / Q000378:metabolism*; D014539:Uridine Diphosphate Sugars / Q000378:metabolism*
D010446:Peptide Fragments; D014539:Uridine Diphosphate Sugars; D014531:Uridine Diphosphate Galactose; D014530:Uridine Diphosphate; D007788:Lactose Synthase; D009237:N-Acetyllactosamine Synthase