Photoaffinity labeling of GDP-fucose:nLcOse4Cer alpha 1----3-fucosyltransferase from human small cell lung carcinoma NCI-H69 cells with the GDP-fucose analog GDP-hexanolaminyl-4-azidosalicylic acid.

Abstract

An iodinatable photoactive analog of GDP-fucose, GDP-hexanolaminyl-4-azidosalicylic acid, has been prepared and applied to studies of the previously described alpha 1----3-fucosyltransferase from NCI-H69 cells (Holmes, E. H., Ostrander, G. K., and Hakomori, S. (1985) J. Biol. Chem. 260, 7619-7627). The NCI-H69 cell alpha 1----3-fucosyltransferase was obtained from a 0.2% Triton X-100-solubilized enzyme fraction after affinity purification on a GDP-hexanolamine-Sepharose column and gel filtration through a fast protein liquid chromatography Superose 12 column. Increasing concentrations of the photoaffinity reagent were found to result in loss of up to 35% of the original enzyme activity at under 100 microM final concentrations. The inactivation was photolysis dependent and could be prevented by the addition of GDP-fucose prior to photolysis. The photoprobe behaved as a competitive inhibitor with respect to GDP-fucose with a Ki of 23 microM, identical to that of GDP. Photoincorporation of 125I-labeled GDP-hexanolaminyl-4-azidosalicylic acid into the enzyme fraction labeled a slow migrating protein band in a native polyacrylamide gel which corresponded to enzyme activity. Inclusion of GDP-fucose prevented photolabeling of this band. Sodium dodecyl sulfate gel electrophoresis of the photolabeled, GDP-fucose-protected band yielded a 125I-labeled protein band that migrated at Mr 45,000, most probably corresponding to an alpha 1----3-fucosyltransferase protein subunit. These studies suggest photoaffinity labeling using nucleotide affinity ligands linked to photoactivatable, heterobifunctional cross-linking reagents may be generally applicable to photoaffinity labeling glycosyltransferase enzyme proteins.

• Authors: E H Holmes
• Journal: The Journal of biological chemistry (J Biol Chem) Vol. 265 Issue 22 Pg. 13150-6 (Aug 05 1990) ISSN: 0021-9258 [Print] United States
• PMID: 2165495 (Publication Type: Journal Article, Research Support, U.S. Gov't, P.H.S.)

Chemical References

• Affinity Labels
• Azides
• Guanine Nucleotides
• GDP-hexanolaminyl-4-azidosalicylic acid
• Guanosine Diphosphate
• Fucosyltransferases
• Hexosyltransferases
• galactoside 3-fucosyltransferase

Topics

• Affinity Labels (metabolism)
• Azides (chemical synthesis, metabolism, pharmacology)
• Carcinoma, Small Cell
• Cell Line
• Chromatography, Affinity
• Chromatography, Gel
• Electrophoresis, Polyacrylamide Gel
• Fucosyltransferases (antagonists & inhibitors, isolation & purification, metabolism)
• Guanine Nucleotides (metabolism)
• Guanosine Diphosphate (analogs & derivatives, chemical synthesis, metabolism, pharmacology)
• Hexosyltransferases (metabolism)
• Humans
• Kinetics
• Lung Neoplasms
• Photolysis
• Tumor Cells, Cultured (enzymology)