Study of binding protein-ligand interaction by ammonium sulfate-assisted adsorption on cellulose esters filters.
A technique for the study of neutral carbohydrate binding protein-ligand interaction is described in this report. It is based on filtration on cellulose esters filters of a mixture of the binding protein and the radioactive ligand, following a treatment of this mixture with ammonium sulfate; the technique is described for the galactose binding protein and for the maltose binding protein of Escherichia coli. For the galactose binding protein, an ammonium sulfate concentration far below that required for precipitation of the protein is sufficient to promote an almost complete retention of the protein on the filters. Furthermore, the addition of ammonium sulfate does not modify the amount of preexisting binding protein-ligand complex, and, in much less than one second, leads to a conformation of the protein-ligand complex which does not allow further ligand binding or dissociation. Hence, the technique is not only very useful for the detection of binding proteins in crude extracts and during purification procedures, it is also of value in the determination of the kinetic parameters of protein-ligand interactions.
D018528:ATP-Binding Cassette Transporters; D000327:Adsorption; D000645:Ammonium Sulfate / Q000494:pharmacology; D001426:Bacterial Proteins / Q000378:metabolism*; D002135:Calcium-Binding Proteins; D002352:Carrier Proteins / Q000378:metabolism*; D002482:Cellulose / Q000031:analogs & derivatives; D002633:Chemotaxis; D004926:Escherichia coli / Q000378:metabolism*; D029968:Escherichia coli Proteins; D005690:Galactose / Q000378:metabolism; D007700:Kinetics; D008024:Ligands; D008320:Maltose / Q000378:metabolism; D058619:Maltose-Binding Proteins; D009004:Monosaccharide Transport Proteins; D033902:Periplasmic Binding Proteins
D018528:ATP-Binding Cassette Transporters; D001426:Bacterial Proteins; D002135:Calcium-Binding Proteins; D002352:Carrier Proteins; D029968:Escherichia coli Proteins; D008024:Ligands; D058619:Maltose-Binding Proteins; D009004:Monosaccharide Transport Proteins; D033902:Periplasmic Binding Proteins; C023550:galactose-binding protein; C461599:maltose transport system, E coli; D008320:Maltose; D002482:Cellulose; D000645:Ammonium Sulfate; D005690:Galactose