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Nuclear reverse T3 binding sites: an artefact of isolation?
Acta Endocrinologica
|April 1, 1985
Summary
Rat liver nuclear extracts degrade reverse triiodothyronine (rT3) due to contamination by microsomal enzymes. This deiodination activity mimics the iodothyronine 5'-deiodinase, suggesting enzymatic processes rather than direct nuclear binding.
Area of Science:
- Biochemistry
- Endocrinology
- Molecular Biology
Background:
- Thyroid hormone metabolism is crucial for physiological regulation.
- Reverse triiodothyronine (rT3) is a key metabolite with distinct biological roles.
- Accurate assessment of nuclear receptor interactions requires understanding potential enzymatic interference.
Purpose of the Study:
- To investigate the nature of [125I]rT3 binding to rat liver nuclear extracts.
- To characterize the deiodination process observed in nuclear preparations.
- To determine if the observed activity represents true nuclear binding or enzymatic degradation.
Main Methods:
- Preparation of rat liver nuclear extracts using sucrose gradients.
- Incubation of [125I]rT3 with nuclear extracts and analysis of degradation products.
- Characterization of the deiodination activity including heat inactivation, substrate specificity, and inhibitor effects.
- Comparison of observed activity with known microsomal iodothyronine 5 ahydro-deiodinase characteristics.
Main Results:
- [125I]rT3 binding was abolished by washing nuclei with high-concentration sucrose.
- Degradation of rT3 into 3,3 ahydro-T2 and iodide (I-) was observed in nuclear extracts.
- The deiodination process exhibited enzymatic properties: heat inactivation, equimolar production of I- and 3,3 ahydro-T2, stimulation by sulfhydryl compounds, and inhibition by propylthiouracil.
- These characteristics closely resembled the microsomal iodothyronine 5 ahydro-deiodinase, operating via a ping-pong mechanism.
Conclusions:
- The observed deiodination of rT3 in hepatic nuclear extracts is primarily due to contamination by microsomal iodothyronine 5 ahydro-deiodinase.
- The initial 'binding' of [125I]rT3 was likely an artifact of this enzymatic deiodination and subsequent release of 125I-.
- While contamination is the main explanation, a nucleus-associated deiodinase activity cannot be entirely ruled out due to the use of Triton X-100 during nuclear preparation.