The interaction of N-acetylglucosamine and an affinity-label analogue with alpha-lactalbumin and lactose synthetase.
We have attempted to detect binding of N-acetylglucosamine (NAG) to alpha-lactalbumin, the B protein of lactose synthetase, under conditions in which binding of NAG to lysozyme, a protein to which alpha-lactalbumin has a significant sequence homology, is observed. Using 1H nuclear magnetic resonance spectroscopy, uv difference spectroscopy, competition of NAG with N-methylnicotinamide chloride, and fluorescence spectroscopy, no binding was detected. The synthesis of a NAG analogue, N-diazoacetyl-glucosamine (diazoNAG), was carried out, and the molecule was demonstrated to be an active galactose acceptor in the lactose synthetase reaction. Use of this molecule in photochemical labeling experiments resulted in a large amount of nonspecific labeling of alpha-lactalbumin, lactose synthetase A protein, ribonuclease, and lysozyme, but competition experiments in the presence of an excess of NAG revealed some specific labeling in the case of A protein and lysozyme, but not with alpha-lactalbumin or a ribonuclease control. Thus, it is highly questionable that a NAG binding site is retained in alpha-lactalbumin; furthermore, it appears that the galacyosyl acceptor makes significant contacts with the A protein rather than alpha-lactalbumin in the lactose synthetase complex.
D000117:Acetylglucosamine / Q000378:metabolism*; D000345:Affinity Labels; D001665:Binding Sites; D005944:Glucosamine / Q000031:analogs & derivatives*; D007768:Lactalbumin / Q000378:metabolism*; D007788:Lactose Synthase / Q000378:metabolism*; D009682:Magnetic Resonance Spectroscopy; D009113:Muramidase; D011485:Protein Binding; D012260:Ribonucleases
D000345:Affinity Labels; D007768:Lactalbumin; D007788:Lactose Synthase; D012260:Ribonucleases; D009113:Muramidase; D005944:Glucosamine; D000117:Acetylglucosamine