Separation of two thyrotropin binding components from porcine thyroid tissue by affinity chromatography: characterization of high and low affinity sites.
Two distinct thyrotropin (TSH) binding species have been separated from solubilized porcine thyroid membranes. Membranes was solubilized with 1% Triton X-100, and the supernatant was recovered by centrifugation at 105,000 X g. Scatchard analysis of thyrotropin binding to solubilized membranes (SM) yielded a nonlinear plot with Kd values for the high and low affinity components similar to those of intact membranes. Chromatography of the SM preparation on concanavalin A-Sepharose 4B resulted in the retention of 10-20% of the binding activity. Upon elution of the column, a peak of binding material (5-7% of total activity) was eluted at 0.3 M alpha-methyl-D-mannoside. This concanavalin A (Con A) bound fraction exhibited a linear Scatchard plot with a Kd value similar to that of the high affinity component of the SM. The protein fraction that did not bind to Con A (Con A unbound) also exhibited a linear Scatchard plot, but with affinity similar to that of the low affinity component of SM. Discontinuous sucrose density gradient ultracentrifugation revealed the presence of two major binding peaks in the solubilized membrane preparation. The slowly sedimenting peak corresponded to that seen in the Con A bound fraction, whereas the rapidly sedimenting peak corresponded to that of the Con A unbound fraction. Sepharose 6B chromatography indicated that in the case of the Con A unbound fraction, a single peak of specific binding activity was eluted in the void volume, and in the case of the Con A bound fraction, one major peak with an approximate Stokes radius of 67 A and several other minor peaks were eluted. These results demonstrate the physical separation of two distinct TSH binding species from thyroid membranes and provide further support for the model of multiple classes of binding sites.
D000818:Animals; D002417:Cattle; D002499:Centrifugation, Density Gradient; D002846:Chromatography, Affinity / Q000379:methods*; D002850:Chromatography, Gel; D003208:Concanavalin A / Q000378:metabolism; D011956:Receptors, Cell Surface / Q000302:isolation & purification*; D013961:Thyroid Gland / Q000378:metabolism*; D013972:Thyrotropin / Q000378:metabolism*
D011956:Receptors, Cell Surface; D003208:Concanavalin A; D013972:Thyrotropin