Isolation and purification of an Fc epsilon receptor activated ion channel from the rat mast cell line RBL-2H3.
Derivatives of the antiallergic drug cromolyn [disodium 5,5'-[(2-hydroxy-1,3-propanediyl)-bis(oxy)]bis [4-oxo-(4H-1-benzopyran)-2- carboxylate]], which can be conjugated covalently at the propane 2-position to macromolecules and to insoluble matrices, were synthesized. Conjugates of these derivatives with macromolecules were examined for their binding to cells of the rat basophilic leukemia line RBL-2H3, which is widely employed as a model for immunologically induced mast cell degranulation. Only those drug-protein conjugates in which the cromolyn analogue with an amino group at the propane 2-carbon instead of the hydroxyl was linked to the carrier by glutaraldehyde were found to exhibit specific and saturable binding to these cells. Analysis of the binding data for these conjugates yielded an apparent binding constant of 3.8 +/- 0.2 X 10(8) M-1 and an apparent number of binding sites for the probe of 4000-8000 per cell. The conjugates found to bind specifically to the cells were also immobilized on agarose matrices and employed in an affinity-based isolation of the membrane component responsible for the observed binding. A single labeled polypeptide was eluted from these columns, onto which either whole cell lysates or solubilized purified plasma membranes of surface-radioiodinated RBL-2H3 cells had been adsorbed. This membrane protein appears on autoradiograms of nonreducing SDS-PAGE as a single broad band of approximately 110,000 daltons (Da) apparent molecular mass. On autoradiograms of reducing gels, the only band detected has an apparent mass of approximately 50,000 Da and appears narrower. Elution of the columns with the drug and disulfide-reducing agents or with the latter alone resulted in significantly higher yields of the 50-kDa polypeptide. Both the intact and reduced proteins bind strongly to immobilized concanavalin A and less so to immobilized wheat germ agglutinin, suggesting that the isolated intact protein is probably a dimer of two glycosylated subunits of similar molecular mass. Treatment of the reduced protein with endoglycosidase F leads to a decrease in its apparent molecular mass by approximately 12 kDa, suggesting that the extent of glycosylation of this polypeptide is approximately 25%. As shown in the following paper, the intact protein constitutes a Ca2+ channel that is activated upon IgE-Fc epsilon receptor aggregation.
Hemmerich
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Department of Chemical Immunology, Weizmann Institute of Science, Rehovot, Israel.
Pecht
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D000345:Affinity Labels; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D002458:Cell Fractionation; D003208:Concanavalin A / Q000378:metabolism; D004205:Cromolyn Sodium / Q000031:analogs & derivatives / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D006026:Glycoside Hydrolases / Q000378:metabolism; D006031:Glycosylation; D007073:Immunoglobulin E / Q000378:metabolism; D007473:Ion Channels / Q000276:immunology / Q000378:metabolism*; D046911:Macromolecular Substances; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase; D008407:Mast Cells / Q000032:analysis* / Q000276:immunology / Q000378:metabolism; D008565:Membrane Proteins / Q000276:immunology / Q000302:isolation & purification* / Q000378:metabolism; D008970:Molecular Weight; D051381:Rats; D011961:Receptors, Fc / Q000502:physiology*; D014407:Tumor Cells, Cultured; D014909:Wheat Germ Agglutinins / Q000378:metabolism
D000345:Affinity Labels; D000596:Amino Acids; D007473:Ion Channels; D046911:Macromolecular Substances; D008565:Membrane Proteins; D011961:Receptors, Fc; D014909:Wheat Germ Agglutinins; D003208:Concanavalin A; D007073:Immunoglobulin E; D006026:Glycoside Hydrolases; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase; D004205:Cromolyn Sodium