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.
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.
Holmes
E H
EH
Pacific Northwest Research Foundation, Seattle, Washington 98122.
D000345:Affinity Labels / Q000378:metabolism*; D001386:Azides / Q000138:chemical synthesis / Q000378:metabolism* / Q000494:pharmacology; D018288:Carcinoma, Small Cell; D002460:Cell Line; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D005647:Fucosyltransferases / Q000037:antagonists & inhibitors / Q000302:isolation & purification / Q000378:metabolism*; D006150:Guanine Nucleotides / Q000378:metabolism*; D006153:Guanosine Diphosphate / Q000031:analogs & derivatives / Q000138:chemical synthesis / Q000378:metabolism* / Q000494:pharmacology; D006602:Hexosyltransferases / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D008175:Lung Neoplasms; D010782:Photolysis; D014407:Tumor Cells, Cultured / Q000201:enzymology*
D000345:Affinity Labels; D001386:Azides; D006150:Guanine Nucleotides; C065105:GDP-hexanolaminyl-4-azidosalicylic acid; D006153:Guanosine Diphosphate; D005647:Fucosyltransferases; D006602:Hexosyltransferases; C031692:galactoside 3-fucosyltransferase