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Melatonin binding proteins identified in the rat brain by affinity labeling.
1Department of Biochemistry, George S. Wise Faculty of Life Sciences, Tel Aviv University, Israel.
FEBS Letters
|August 19, 1991
Summary
Researchers identified melatonin binding proteins in rat brain synaptosomes using N-Bromoacetyl-2-iodo-5-methoxytryptamine (BIM). BIM labeled three specific polypeptides, confirming them as melatonin binding proteins.
Area of Science:
- Neuroscience
- Biochemistry
- Molecular Biology
Background:
- Melatonin is a crucial hormone regulating circadian rhythms and other physiological processes.
- Identifying specific melatonin binding proteins is essential for understanding its diverse biological functions.
- Rat brain synaptosomes are a key model for studying neurotransmitter and hormone interactions in the central nervous system.
Purpose of the Study:
- To synthesize and utilize a novel melatonin analog, N-Bromoacetyl-2-iodo-5-methoxytryptamine (BIM), as a chemical probe.
- To identify and characterize melatonin binding proteins within rat brain synaptosomes.
- To elucidate the molecular targets of melatonin action in the brain.
Main Methods:
- Preparation of N-Bromoacetyl-2-iodo-5-methoxytryptamine (BIM) and its radiolabeled form ([125I]BIM).
- Incubation of rat brain synaptosomes with BIM and [125I]BIM to assess binding and incorporation.
- Analysis of protein labeling using SDS-PAGE and molecular weight determination.
- Competitive binding assays using BIM, 2-iodomelatonin, melatonin, and other related compounds.
Main Results:
- N-Bromoacetyl-2-iodo-5-methoxytryptamine (BIM) demonstrated time- and concentration-dependent irreversible inhibition of 2-[125I]iodomelatonin binding.
- [125I]BIM was covalently incorporated into synaptosomal proteins, indicating specific binding.
- The incorporation of [125I]BIM was specifically inhibited by BIM, 2-iodomelatonin, and melatonin, but not by 5-methoxytryptamine or N-acetyl serotonin.
- [125I]BIM covalently labeled three polypeptides with molecular weights of 92, 55, and 45 kDa, with labeling significantly reduced by melatonin.
Conclusions:
- The 92, 55, and 45 kDa polypeptides identified in rat brain synaptosomes are confirmed as melatonin binding proteins.
- N-Bromoacetyl-2-iodo-5-methoxytryptamine (BIM) serves as an effective photoaffinity label for identifying melatonin receptors.
- These findings provide a molecular basis for understanding melatonin's signaling pathways in the brain.