Isolation of a basophilic membrane protein binding the anti-allergic drug cromolyn.
The membrane protein component in basophils, responsible for the specific, Ca2+-dependent, binding of the anti-allergic drug cromolyn [disodium cromoglycate, DSCG; the disodium salt of 1,2 bis(2- carboxychromon -5- yloxy )-2-hydroxy propane] was isolated by two procedures based on affinity for the drug. In the first procedure, involving immunoprecipitation, rat basophilic leukemia cells (RBL-2H3), surface labeled by 125I were reacted with a polyvalent conjugate of DSCG and bovine serum albumin and then subjected to solubilization by the non-ionic detergent Nonidet P-40 (NP-40). From these lysates, precipitation was specifically attained by subsequent addition of rabbit anti-DSCG antibodies. In an SDS-polyacrylamide gel electrophoresis (SDS-PAGE), a single radioactive band was observed, having an apparent mol. wt. of 60 000 daltons. Competitive inhibition of the immunoprecipitation in the presence of free drug or excess of EDTA demonstrated the specificity of the isolation. Furthermore, this particular membrane component could not be isolated from several other cell types examined. The second isolation from several other cell types examined. The second isolation procedure employed affinity chromatography on DSCG immobilized on polyacryl- hydrazido agarose beads. The DSCG-binding protein was eluted from the affinity column with either DSCG or with EDTA and also migrated on SDS-PAGE as a single band of 60 000 mol. wt., similar to that obtained by the immunoprecipitation procedure. These and other results suggest that this newly isolated protein is the one responsible for the Ca2+-dependent binding of the drug to the basophil membrane.
D000818:Animals; D001491:Basophils / Q000378:metabolism*; D002352:Carrier Proteins / Q000302:isolation & purification / Q000378:metabolism*; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D002846:Chromatography, Affinity; D004205:Cromolyn Sodium / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D007942:Leukemia, Experimental / Q000378:metabolism*; D008565:Membrane Proteins; D008970:Molecular Weight; D051381:Rats
D002352:Carrier Proteins; D008565:Membrane Proteins; C039229:cromolyn binding protein, rat; D004205:Cromolyn Sodium