Limitations in the conventional analysis of the interaction of triiodothyronine with solubilized nuclear receptor sites. Inapparent binding of triiodothyronine to nonspecific binding sites

S Seelig, H L Schwartz, J H Oppenheimer
1981 Journal of Biological Chemistry  
Minnesota 55455 The binding of triiodothyronine (T3) to solubilized hepatic nuclear receptors at 0°C has frequently been assumed to behave as a reversible bimolecular process since a plateau uptake is reached in 48 h and the Scatchard plot is linear. In order to test this assumption further, we have examined in a more detailed fashion the kinetics of this interaction. The dissociation rate constant ( k -1) derived from an analysis of the association of [126rJTs with its receptor was 0.059/h
more » ... tor was 0.059/h (tl,z for dissociation of 11.7 h). In contrast, k-l, determined from the rate of dissociation of T3 either after the addition of excess unlabeled T3 or after dilution of [126rfl?3 *receptor complex, was 0.0087/h (t1,z of dissociation of 80 h). This difference raised the possibility that a reversible bimolecular model may not have been appropriate for the system under study. One of the underlying assumptions which we tested was the concept that unbound hormone as determined in a resin or Sephadex assay was indeed "free" and available for reaction with receptor in solution. Tracer [lZ6I]T3 and solubilized receptors were allowed to interact for 48 h, at which time approximately 30% of the T1 was "free." From 48 to 96 h, no additional ligand was bound. Fresh receptor added at 48 h failed to increase the quantity of [lZ6rJT3 bound. Paper chromatography showed that in excess of 95% of the "'1 was in the form of T3 molecule. We therefore inferred that the hormone bound by the test resin was not free in solution but rather bound to a site or sites other than the receptor. These inapparent sites were considered to be nonspecific and of unlimited capacity. A mathematical model was constructed to represent the irreversible partitioning of T3 between the limited capacity specific sites and the unlimited capacity inapparent nonspecific sites as a function of time and total T3 concentration. T h i s model predicted the attainment of a plateau region in the uptake curve by specific receptors and a nonlinear function which could be used to fit the experimental points employed in the conventional Scatchard plot. Thus, the attainment of a plateau value in the association curve and an apparently linear Scatchard plot are clearly an insufficient basis for the assumption of a bimolecular reversible process. These considerations may be applicable to other systems and emphasize the potential dangers of "kinetic mimicry." A substantial body of evidence suggests that thyroid hormone action is initiated by an interaction of the hormone with specific nuclear non-histone protein receptor (1). For this reason, quantitation of the number of nuclear sites and their affinity under various pathophysiological circumstances has become a matter of general interest. Three methods are currently used to assess nuclear T31 binding: (a) in vivo isotopic displacement techniques in the whole animal (2), ( b ) in vitro displacement techniques using isolated nuclei (3), and (c) in vitro competition experiments with solubilized nuclear receptor sites which are generally carried out at 0°C (4-7). The latter method is particularly attractive for making such measurements since it requires relatively small amounts of starting material and achieves a relatively high "signal to noise" ratio. The broad range of biological applications of this method has prompted us to re-examine the kinetics of the interaction of TS with the solubilized nuclear receptor sites. In a recent preliminary study, we obtained results which exposed major inconsistencies in the basic kinetic model which we and others had previously assumed (4, 7) . Additional experiments were, therefore, carried out to clarify this problem. The results of these studies have led us to propose a revised kinetic analysis. Our studies were interpreted to indicate that T3 added to the solubilized nuclear preparations at 0°C binds both to the specific receptor sites and, unexpectedly, to nonspecific sites which are not apparent in the conventional reversible bimolecular kinetic analysis. Our studies suggest that within the time span of the experiment T3 interacts with both sets of sites in an essentially irreversible manner. These findings cast doubt on the suitability of standard Scatchard analysis of the system under study. A new model for studying the competitive interaction of tracer and nonradioactive T3 with the receptor sites in solubilized nuclear preparations at 0°C was therefore developed. EXPERIMENTAL PROCEDURES Treatment of Animals and Preparation of Nuclei-Male Sprague-Dawley rats weighing 150-200 g, obtained from Taconic Farms, Germantown, NY, were surgically thyroidectomized by the supplier and given 100 pCi of 1311 after 1 week of a low iodine diet. Animals were considered to be adequately hypothyroid only after growth retardation could be demonstrated, some 3-4 weeks after 1311 administration. Hepatic nuclei were obtained by centrifugation through 2.4 M sucrose as previously described (8). The nuclei were washed with 0.14 M NaCl, 3 m~ MgC12 (1-2 ml/g of tissue) and harvested by centrifugation at 1,ooO X g for 10 min. Recovery of nuclei as judged by measurement of DNA ranged from 60-70%. The protein/DNA ratio was approximately 2.5. Solubilization of Nuclear Receptor-The technique of Silva et al. The abbreviation used is: Ts, triiodothyronine. 154
pmid:6257704 fatcat:htgz7rwxmjdfzfacsuht4se7di