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Characterization of the Target Cell Receptor for IgE

III. Properties of the Receptor Isolated from Rat Basophilic Leukemia Cells by Affinity Chromatography¹

MRC Group in Allergy Research, Department of Immunology, University of Manitoba, Winnipeg, Manitoba, Canada R3E 0W3

Abstract

The receptor for IgE from surface-labeled and Nonidet P-40- (NP-40) solubilized rat basophilic leukemia (RBL) cells was isolated with the aid of IgE coupled to Sepharose, by using KSCN as the eluting agent. The IgE-rebinding capacity of this eluate was only 12 to 19% suggesting that some alteration of the receptor had taken place. Upon sucrose density gradient centrifugation and gel filtration the receptor exhibited considerable size heterogeneity, most likely as a consequence of detergent binding. Detergent binding could also be implied from isopycnic centrifugation which yielded a density for receptor which was below that of free IgE. When these experiments were repeated in the presence of SDS, the size heterogeneity disappeared and the density was found to be 1.28 g/ml which is in the normal range for proteins. Analysis of the receptor eluate by SDS polyacrylamide gel electrophoresis on 5% gels indicated a single component with a slightly slower mobility than that of receptor coprecipitated with IgE. When the same receptor eluate was analyzed on 10% gels, two components, with apparent m.w. of 45,000 daltons and 55,000 daltons were seen. Coprecipitated receptor again exhibited only a single peak with an identical relative mobility to that of the 45,000-dalton component in KSCN eluate. The binding of both components to IgE-Sepharose could be inhibited by free IgE; identical concentrations of a purified fraction of rat IgG had no effect. Although complete inhibition of binding was seen only with the lower m.w. component, the inhibition data still suggest some specificity in the interaction of the 55,000 dalton component with IgE.

Footnotes

¹ Presented, in part at the 60th Annual Meeting of the Federation of American Societies for Experimental Biology, Anaheim, California, in April, 1976.

² Recipient of a Medical Research Council of Canada, Post-doctoral Fellowship.

³ Present address: Department of Medicine, Medical College of Virginia, Richmond, Virginia.

⁴ To whom reprint requests should be sent.