Affinity labeling of the carbohydrate binding site of the lectin discoidin I using a photoactivatable radioiodinated monosaccharide.
N-(4-Azidosalicyl)galactosamine (GalNASA), a photoactivatable, radioiodinatable analogue of N-acetylgalactosamine (GalNAc), has been prepared and characterized. We have used this reagent for labeling of the carbohydrate binding site of discoidin I, an endogenous lectin produced by Dictyostelium discoideum. GalNASA behaved as a ligand for discoidin I, as judged by its ability to compete in an assay measuring the carbohydrate binding activity of discoidin I. In this assay, it exhibited a Ki,app of 800 microM, comparable to that of GalNAc. The Ki,app of GalNASA decreased to 40 microM upon prior photolysis with ultraviolet light. In contrast, N-(4-azidosalicyl)ethanolamine produced no inhibition of carbohydrate binding regardless of photolysis. Covalent labeling of discoidin I with 125I-GalNASA was entirely dependent upon ultraviolet light. A portion of the labeling, representing 40-60% of the total, was sensitive to reagents which were known to inhibit carbohydrate binding by discoidin I, including GalNAc, asialofetuin, and ethylenediaminetetraacetic acid. N-Acetylglucosamine, which is not a ligand of discoidin I, was without effect. As a control, no carbohydrate-sensitive labeling was observed upon incubation of 125I-GalNASA with bovine serum albumin. The carbohydrate-sensitive fraction of discoidin I photolabeling with 125I-GalNASA exhibited a Kd of 15-40 microM, in agreement with the Ki,app of prephotolyzed GalNASA observed in the carbohydrate binding assay. Some labeling occurred if 125I-GalNASA was photolyzed prior to incubation with discoidin I, suggesting the involvement of long-lived species in the labeling reaction. Partial proteolytic digestion of photolabeled discoidin I revealed specific fragments whose labeling was completely blocked by GalNAc.(ABSTRACT TRUNCATED AT 250 WORDS)
Kohnken
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Cell Biology Group, Worcester Foundation for Experimental Biology, Shrewsbury, Massachusetts 01545.
Berger
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D000345:Affinity Labels / Q000138:chemical synthesis*; D001386:Azides / Q000138:chemical synthesis* / Q000378:metabolism; D004023:Dictyostelium / Q000378:metabolism; D000070821:Discoidins; D005656:Fungal Proteins / Q000302:isolation & purification / Q000378:metabolism*; D005688:Galactosamine / Q000031:analogs & derivatives* / Q000138:chemical synthesis / Q000378:metabolism; D007202:Indicators and Reagents; D007700:Kinetics; D037102:Lectins / Q000378:metabolism*; D009005:Monosaccharides; D015800:Protozoan Proteins; D012710:Serum Albumin, Bovine / Q000378:metabolism
D000345:Affinity Labels; D001386:Azides; D000070821:Discoidins; D005656:Fungal Proteins; D007202:Indicators and Reagents; D037102:Lectins; D009005:Monosaccharides; D015800:Protozoan Proteins; C055415:N-(4-azidosalicyl)galactosamine; D012710:Serum Albumin, Bovine; D005688:Galactosamine