Synthesis and characterization of a carbene-generating biotinylated N-acetylglucosamine for photoaffinity labeling of beta-(1-->4)-galactosyltransferase.
A photoreactive N-acetylglucosamine derivative, N-[2-[2-[2-(2-biotinylaminoethoxy)-ethoxy]ethoxy]-4-[3-(trifluo rom ethyl)-3-H-diazirin-3-yl]benzoyl]-N4-[2-(acetylamino)-deoxy-beta-D -glucopyranosyl]-L-aspartamide (BDGA), was synthesized as a carbene-generating biotinylated probe for UDP-galactose:N-acetylglucosamine beta-(1-->4)-galactosyltransferase (GalT). The photoaffinity labeling experiments of bovine GalT with BDGA under various condition were examined based on the quantitative chemiluminescent detection of the biotinyl residue which was photochemically introduced into in GalT protein. A progressive decrease in the yield of specific photolabeling was observed upon lowering the incubation temperature from 37 degrees C to 20 degrees C or 4 degree C. The amount of photoincorporation was also decreased when UMP was not included in the incubation mixture. Using a crude protein mixture of recombinant human GalT, a band corresponding to the glutathione S-transferase fusion GalT protein was also specifically visualized. Furthermore, combine use of BDGA photolabeling with an immobilized avidin was found to be effective for the selective retrieval of photolabeled GalT from a reaction mixture containing a large amount of unlabeled GalT protein. The results obtained clearly demonstrate that the covalent biotinylation using the carbene-generating photoaffinity reagent BDGA would be useful for the analysis of acceptor substrate binding sites within the GalT protein.
Hatanka
Y
Y
Research Institute for Wakan-yaku, Toyama Medical and Pharmaceutical University, Japan.
Hashimoto
M
M
Nishihara
S
S
Narimatsu
H
H
Kanaoka
Y
Y
D000117:Acetylglucosamine / Q000031:analogs & derivatives*; D000345:Affinity Labels / Q000138:chemical synthesis*; D001224:Aspartic Acid / Q000031:analogs & derivatives; D001391:Azo Compounds / Q000138:chemical synthesis / Q000378:metabolism; D001710:Biotin / Q000031:analogs & derivatives*; D004591:Electrophoresis, Polyacrylamide Gel; D005700:Galactosyltransferases / Q000378:metabolism*; D006801:Humans; D006838:Hydrocarbons; D007700:Kinetics; D008163:Luminescent Measurements; D009682:Magnetic Resonance Spectroscopy; D013058:Mass Spectrometry; D008697:Methane / Q000031:analogs & derivatives / Q000378:metabolism; D015394:Molecular Structure; D010782:Photolysis; D011994:Recombinant Proteins / Q000378:metabolism; D013053:Spectrophotometry
D000345:Affinity Labels; D001391:Azo Compounds; D006838:Hydrocarbons; D011994:Recombinant Proteins; C030011:carbene; D001224:Aspartic Acid; D001710:Biotin; D005700:Galactosyltransferases; D008697:Methane; D000117:Acetylglucosamine