Abstract
3,5,3'-Triiodo-L-thyronine (L-T3)-binding sites from rat and human red cells were characterized as to their distribution between the two surfaces of the membrane. Analysis of L-T3 binding to sealed right-side-out and inside-out vesicles from erythrocyte membrane revealed that high affinity L-T3-binding sites are located on the external side in rat erythrocytes and on the internal side in human red cells. These results were further confirmed by preincubation of intact red cells with p-chloromercuribenzoate, a slowly permeant reagent that interacts reversibly with SH groups of proteins. Following this treatment only the SH groups of L-T3 sites from rat erythrocytes were found to be blocked. Scatchard analysis of the binding data for rat right-side-out and human inside-out vesicles showed high affinity sites with Kd values of 0.2 x 10(-10) and 2 x 10(-10) M, respectively. The results suggest that the orientation of L-T3-binding sites in the erythrocyte membrane is species-dependent.
Highlights
3,5,3’-Triiodo-~-thyronin(~e-T&binding sitesfrom rat and human red cells werecharacterized as to their distribution betweenthetwosurfaces of the membrane
The results suggest that the orientation of ~ - T ~ - bindi nsgitesintheerythrocyte membrane is species-dependent
Much evidence has accumulated supporting the proposal that some of the biological effects of L - T ~are mediated through the interaction of the hormone with a non-histone chromatin-associated receptor [11, 12]
Summary
Rats-Blood was collected by heart puncture from male SpragueDawley rats (400-600 g body weight) using 100 mM Na2EDTA(1 ml/. Red blood cells were washed three or four times by sedimenting at 2500 rpm for 15min and resuspending p-Chloromercuribentoate (PCMB) Studies Intact erythrocytes (hematocrit of 10%)were treated at 4 “C with. PCMB as indicated in the figures to minimize diffusion of the organomercurial through the lipid bilayer [7]. After incubation for 30 min, the cells were pelleted and washed twice with 20 volumes of 138 mM NaCI, 10 mM sodium phosphate buffer, pH 7.4, to remove any noreacted PCMB. Ghosts and membrane vesicles were prepared as described. Intact vesicles were treated with PCMB as above
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