Quantitation of 3,5,3′-Triiodothyronine (T3) Receptors by a Microcentrifuge Exchange Assay: Evidence for the Existence of a Nucleoplasmic Pool of Rat Liver T3Receptors*

Publisher Website
Google Scholar
Abstract
The total T3 receptor content of small aliquots of rat liver nuclear extracts was determined by incubation at a single, near-saturating concentration of [125I]T:) (3 nM) at 25 C for 2 h (exchange assay). Under these assay conditions the detectability of receptors occupied by endogenous T3 was only minimally reduced. Approximately 85% of the maximal binding capacity established by Scatchard analysis was determined by this technique. Compared to Scatchard analysis a 5-fold increase in the number of samples which could be assayed was realized without unacceptably high levels of background radioactivity. A nucleoplasmic pool of T3 receptors was obtained by three successive extractions of purified rat liver nuclei with a 0.15 M KC1 medium; a chromatin-bound pool was obtained by successive extractions of the residual chromatin with a 0.4 M KC1 medium. Approximately 41% (118 fmol/mgeq DNA) of the total extractable T3 receptor was recovered in the nucleoplasmic pool and 59% (170 fmol/mgeq DNA) in the chromatin-bound pool. The elution pattern of the six extracts was consistent with quantitative extractions of two distinct receptor pools rather than of successive, incomplete extractions of a single receptor pool. The intranuclear distribution of receptors labeled by in vivo administration of [125I]T3 was virtually identical to the distribution determined by in vitro exchange assay of the same nuclear extracts. Thirty minutes after the in vivo administration of 5-[3H] orotic acid, 74% of the extractable acid-soluble nucleotides were recovered in the isotonic nuclear extracts, thus supporting the reference to these extrac as the "cleoplasmicb" pool. The nonequivalence of the isotonic and hypertonic nuclear extracts was further substantiated by the demonstration of differences in their protein composition on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and by the presence of 30S ribonucleoprotein particles (informofers) exclusively in the isotonic extract after ultracentrifugation in 15–30% sucrose gradients. Using gel filtration chromatography the nucleoplasmic receptor was estimated to have a mol wt of 43,000–48,000 and a Stokes' radius of 32–34 Å. These values did not differ significantly from those of the chromatin-bound receptor. The equilibrium association constants for T3 binding (1.14–1.82 × 1010 M-1) and the degree of occupancy by T3 (20–30%) were similarly indistinguishable between the two receptors. In summary, using a microcentrifuge exchange assay technique for T3 receptor quantitation, evidenc was obtained for the existence of a nucleoplasmic pool of T3 receptors in addition to the chromatin-bound population. Although the two intranuclear pools were qualitatively and quantitatively distinct, their receptors were not distinguishable by several criteria.