Assay and characterization of carbohydrate binding by the lectin discoidin I immobilized on nitrocellulose.
Discoidin I is the most abundant galactose binding lectin produced by the cellular slime mold Dictyostelium discoideum and has been implicated in cell-substratum adhesion. We have developed an assay of carbohydrate binding activity utilizing binding of 125I-asialofetuin to discoidin I, or to other lectins, immobilized on nitrocellulose. Among the proteins examined, only lectins exhibited the ability to bind asialofetuin. Specificity of asialofetuin binding was demonstrated by competition with monosaccharides, which inhibited binding consistent with the known sugar specificity of the lectins examined. Experiments with fetuin and derivatives differing in their oligosaccharide structure indicated a requirement for terminal galactosyl residues for probe binding to discoidin I. We have used this assay to characterize the carbohydrate binding behavior of discoidin I. The extent of asialofetuin binding to discoidin I was dependent on the concentrations of both lectin and ligand. Interpretation of equilibrium binding data suggested that, under saturating conditions, 1 mol of oligosaccharide was bound per mole discoidin I monomer. Furthermore, discoidin I in solution and discoidin I on nitrocellulose were equally effective at competing for soluble asialofetuin, suggesting that immobilization had no effect on the carbohydrate binding behavior of discoidin I. Binding was strongly inhibited by ethylenediaminetetraacetic acid; both Ca2+ and Mn2+ could overcome that inhibition, but Mg2+ could not. Preincubation of discoidin I at 60 degrees C stimulated asialofetuin binding 2-fold by increasing the affinity, while preincubation at higher temperatures resulted in a complete loss of activity.(ABSTRACT TRUNCATED AT 250 WORDS)
D001212:Asialoglycoproteins; D001665:Binding Sites; D001667:Binding, Competitive; D050260:Carbohydrate Metabolism; D002412:Cations / Q000494:pharmacology; D003101:Collodion / Q000494:pharmacology*; D000070821:Discoidins; D004492:Edetic Acid / Q000494:pharmacology; D060748:Fetuins; D005656:Fungal Proteins / Q000037:antagonists & inhibitors / Q000302:isolation & purification / Q000378:metabolism*; D007457:Iodine Radioisotopes; D037102:Lectins / Q000037:antagonists & inhibitors / Q000302:isolation & purification / Q000378:metabolism*; D008722:Methods; D009005:Monosaccharides / Q000494:pharmacology; D011485:Protein Binding; D015800:Protozoan Proteins; D013696:Temperature; D014357:Trypsin / Q000494:pharmacology; D000509:alpha-Fetoproteins / Q000494:pharmacology
D001212:Asialoglycoproteins; D002412:Cations; D000070821:Discoidins; D060748:Fetuins; D005656:Fungal Proteins; D007457:Iodine Radioisotopes; D037102:Lectins; D009005:Monosaccharides; D015800:Protozoan Proteins; D000509:alpha-Fetoproteins; C017029:asialofetuin; D003101:Collodion; D004492:Edetic Acid; D014357:Trypsin