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Effect of calcium ion on triiodothyronine binding to kidney outer mitochondrial membrane in vitro
Abstract:
The effect of calcium 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, contained specific binding sites for T3. Scatchard analysis of T3 binding to outer mitochondrial membranes gave an association constant (Ka) of 0.53 X 10(10)M-1. The binding of [125I]-T3 to the membranes was inhibited by the addition of CaCl2(0.25 X 10(-4)--2.5 X 10(-3)M). 50% inhibition was obtained by 0.75 X 10(-4)M CaCl2 in the presence of 0.1 mM EGTA. When outer mitochondrial membranes were solubilized with Triton X-100, four main T3 binding activities were isolated by a gel filtration study. On the other hand, 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 calcium, 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 calcium ion without any change in the association constant. In studies with gel filtration of receptors which are released by Ca2+ from outer mitochondrial membranes, three main T3 binding activities were isolated. Mg2+, Mn2+, Zn2+ and Cu2+ did not affect T3 binding to outer mitochondrial membranes. The results indicate that calcium ion regulates T3 binding to the outer mitochondrial membrane through the release of T3 receptors from the membranes.
Insights
Calcium ions regulate thyroid hormone (T3) binding to kidney outer mitochondrial membranes by releasing T3 receptors. This mechanism decreases T3 binding sites within membranes, impacting thyroid hormone regulation.
Area of Science:
- Biochemistry
- Cell Biology
- Endocrinology
Background:
- Thyroid hormones, like 3,5,3'-triiodothyronine (T3), play crucial roles in cellular metabolism.
- Mitochondrial membranes are key sites for cellular energy production and hormone interaction.
- The specific binding sites and regulation of T3 within kidney outer mitochondrial membranes are not fully understood.
Purpose of the Study:
- To investigate the in vitro effect of calcium ions on T3 binding to rat kidney outer mitochondrial membranes.
- To elucidate the mechanism by which calcium influences T3 receptor interactions with these membranes.
Main Methods:
- Preparation of rat kidney outer mitochondrial membranes using sucrose density gradient centrifugation.
- In vitro binding assays using radiolabeled [125I]-T3.
- Scatchard analysis to determine binding affinity and site number.
- Solubilization of membranes with Triton X-100 and gel filtration for receptor isolation.
- Pre-incubation experiments with calcium ions and subsequent analysis of membrane and supernatant fractions.
Main Results:
- Specific T3 binding sites were identified in the outer mitochondrial membrane fraction.
- Calcium chloride (CaCl2) inhibited T3 binding to intact outer mitochondrial membranes in a dose-dependent manner.
- Calcium pre-incubation decreased T3 binding sites in membranes and increased them in the supernatant, without altering the association constant.
- Solubilized T3 receptors showed less inhibition by calcium, and gel filtration revealed T3 binding activities released by Ca2+.
Conclusions:
- Calcium ions regulate T3 binding to the outer mitochondrial membrane.
- This regulation occurs through the calcium-induced release of T3 receptors from the membrane.
- The findings suggest a novel mechanism for modulating thyroid hormone action at the mitochondrial level.