Effect of calcium ion on triiodothyronine binding to kidney outer mitochondrial membrane in vitro.

Abstract

The effect of Ca ion on 3,5,3''-triiodothyronine (T3) binding to rat kidney outer mitochondrial membranes was examined in vitro. The outer mitochondrial membranes were prepared by using a discontinuous sucrose density gradient centrifugation. The membrane fraction, which is enriched with monoamine oxidase activity, contains specific binding sites for T3. Scatchard analysis of T3 binding to outer mitochondrial membranes gave a Ka of 0.53 .times. 1010 M-1. The binding of [125I]-T3 to the membranes was inhibited by the addition of CaCl2 (0.25 .times. 10-4-2.5 .times. 10-3 M); 50% inhibition was obtained by 0.75 .times. 10-4 M CaCl2 in the presence of 0.1 mM EGTA [ethyleneglycol bis(.beta.-aminoethylether) N,N,N'',N''-tetraacetic acid]. When outer mitochondrial membranes were solubilized with Triton X-100, 4 main T3 binding activities were isolated by a gel filtration study. The binding of [125I]-T3 to the solubilized T3 receptors derived from outer mitochondrial membranes was not strongly inhibited by calcium. When outer mitochondrial membranes were preincubated in the presence of 1 mM Ca, the number of T3 binding sites in the membranes was decreased, and this was associated with an increase in the number of T3 binding sites in the supernatants of the incubation mixture. Scatchard analysis showed that the number of T3 binding sites in the membranes is decreased by Ca ion without any change in the association constant. In studies with gel filtration of receptors which are released by Ca2+ from outer mitochondrial membranes, 3 main T3 binding activities were isolated. Mg2+, Mn2+, Zn2+ and Cu2+ did not affect T3 binding to outer mitochondrial membranes. Ca ion regulates T3 binding to the outer mitochondrial membrane through the release of T3 receptors from the membranes.