Purified labeled IgE receptor from rat basophilic leukemia (RBL-1) cells obtained as described in the preceding paper was characterized in regard to binding activity and specificity in several assay systems, stability, and apparent molecular size on Sepharose CL-6B. Based on comparative binding studies with IgE-Sepharose and control protein Sepharose conjugates, the highly purified receptor preparations were 68 to 78% active. Estimates of activity based on differences in receptor migration on Sepharose CL-6B in the presence and absence of soluble IgE were 70 to 80%. Comparable estimates of receptor activity were obtained with an ammonium sulfate precipitation assay, in which crude and purified soluble receptor preparations were similarly affected by dilution in their ability to bind 125I-IgE. The IgE-receptor interaction was inhibited by rat IgE but not by human IgE, rat IgG, heat-inactivated rat IgE, or several other proteins, in accord with known specificity characteristics of receptor on intact RBL-1 cells. These results suggest that the receptor is not grossly altered in its binding properties by the purification. Highly purified preparations of receptor were rapidly inactivated at 37°C but were stable for at least a month at 4°C in solution at neutral pH and contained only one radioactive band when analyzed in sodium dodecyl sulfate-polyacrylamide gels (M(r)=45,000 to 50,000 under denaturing conditions). Upon chromatography of highly purified receptor on Sepharose CL-6B, two peaks of radioactivity corresponding to estimated molecular weights of 40,000 and 170,000 were obtained. Both of the peaks bound to IgE-Sepharose. When the receptor was preincubated with soluble IgE, the two original peaks were markedly diminished and there was a new radioactive peak at M(r)≃460,000. We belive that the most likely interpretation of these observations is that the solubilized purified receptor exists in monomeric and multimeric forms, both capable of binding IgE.
|Number of pages||7|
|Journal||Journal of Biological Chemistry|
|State||Published - Dec 1 1979|