Effects of adenosine triphosphate and alkaline phosphatase on solubilized 3,5,3'-triiodothyronine-binding activity.

The T3-binding activity of salt-extractable nuclear proteins from rat liver was affected when ATP (2-10 mM; pH 8.0) was added concomitantly with T3 in the incubation medium. Scatchard analysis revealed that the equilibrium association constant was significantly reduced [5 mM ATP, 0.3 +/- 0.1 (+/- SE) 10(10) M-1; control, 1.1 +/- 0.15 X 10(10) M-1], but the maximum binding capacity remained unchanged. Similar values of inhibition were obtained when unbound receptors were preincubated with ATP. ATP achieved its maximal effect after 45 min of incubation at 30 C. Dilution experiments indicated that the effect of ATP was reversible. The inhibiting potency of nucleoside triphosphates at pH 8.0 was in the following order: ATP = CTP greater than GTP, whereas UTP had no effect. Nonhydrolyzable analogs of ATP were also inhibitory, and HPLC fractionation showed an approximately 98% recovery of ATP after incubation with nuclear extract. The adenine ring with at least two phosphates was essential, since ADP was as potent as ATP, whereas AMP had no effect. When the pH of the incubation medium was lowered to 7.3, the T3-binding activity was inhibited by ATP in the 0.1-1 mM range. Magnesium (3 mM) greatly increases the ATP effect at pH 7.3, but not at pH 8. The T3-binding activity was also drastically reduced when calf intestine alkaline phosphatase was added concomitantly in the incubation medium. Eight micrograms per ml enzyme were necessary to inhibit the T3 specific binding by 50% (30 C for 45 min). Scatchard analysis showed that the receptor affinity for T3 was decreased (control, 1.1 +/- 0.02 x 10(10) M-1; alkaline phosphatase, 0.41 +/- 0.03 x 10(10) M-1; n = 6), whereas the maximum binding capacity remained unchanged. Incubations performed with increasing concentrations of beta-mercaphoethanol (2.5, 5, 10, and 25 mM) revealed that the phosphatase inhibitory effect is thiol dependent. The inhibition was maximal at 2.5 mM and progressively decreased at 5 and 10 mM. No inhibition occurred at 25 mM. When a saturating concentration of T3 was employed, the specific binding was decreased at low thiol concentrations. These observations show that the nuclear T3 receptors may be modulated by ATP/ADP and phosphorylation/dephosphorylation processes. It is proposed that in vitro dephosphorylation leads to rapid oxydation of sulfhydryl groups which are essential for optimum T3 binding.