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Characterization of the chicken brain melatonin-binding protein using iodinated and tritiated ligands. 1994

D J Kennaway, and H M Hugel, and S A Rowe
Department of Obstetrics and Gynaecology, University of Adelaide, South Australia.

The melatonin-binding protein in chicken brain membranes was characterized using both [125I]-2-iodomelatonin and [3H]-melatonin as radioligands. Saturation studies conducted at 25 degrees C revealed a single class of binding site with dissociation constants of 24 +/- 4.8 pM (n = 7) and 125 +/- 21 pM (n = 6) for the iodinated and tritiated ligands, respectively. Calculation of the affinity constant using data from kinetic experiments gave values of 2.2 +/- 0.4 pM and 135 +/- 15 pM for the iodinated and tritiated ligands, respectively. Competition studies showed that the rank order of inhibition of binding by melatonin analogues was similar for both radioligands (2-iodomelatonin > melatonin > 2,3-dihydromelatonin > N-acetyl-5-methoxykynurenamine > N-acetylserotonin > 5-methoxytryptamine). The calculation of Ki, which depends upon the affinity constant, was 22 +/- 4.9 pM and 129 +/- 21 pM for 2-iodomelatonin and melatonin, respectively, when the affinity constant derived from the [125I]-2-iodomelatonin saturation experiments was used, but 4.9 +/- 1.5 pM and 33 +/- 5.5 pM when the kinetically derived constant was used. When [3H]-melatonin was used, the Ki for melatonin was 72 +/- 8 pM and 20 +/- 4.6 pM for 2-iodomelatonin and melatonin. Binding of [125I]-2-iodomelatonin to the membranes was partially reversible at 25 degrees C in contrast to the complete reversibility of [3H]-melatonin. Examination of the effects of temperature on binding indicated that at 37 degrees C both association and dissociation of both ligands were accelerated. Closer examination showed that at 37 degrees C there was a loss of approximately 40% of the [125I]-2-iodomelatonin binding sites and little influence upon the affinity of binding with time. By contrast, when [3H]-melatonin was used, the affinity decreased fourfold, with only a slight change in the number of sites. If membranes were incubated at 37 degrees C and then switched 25 degrees C, binding increased, emphasizing the fact that the binding sites were not destroyed. Whereas there appears to be little doubt that 2-iodomelatonin is a biologically active melatonin agonist, the binding of the radioactive form of this agonist to the putative melatonin receptor binding site is quite different from that of the endogenous ligand. This may have serious consequences in studies where receptor content is determined following physiological or pharmacological interventions.

UI MeSH Term Description Entries
D007457 Iodine Radioisotopes Unstable isotopes of iodine that decay or disintegrate emitting radiation. I atoms with atomic weights 117-139, except I 127, are radioactive iodine isotopes. Radioisotopes, Iodine
D008024 Ligands A molecule that binds to another molecule, used especially to refer to a small molecule that binds specifically to a larger molecule, e.g., an antigen binding to an antibody, a hormone or neurotransmitter binding to a receptor, or a substrate or allosteric effector binding to an enzyme. Ligands are also molecules that donate or accept a pair of electrons to form a coordinate covalent bond with the central metal atom of a coordination complex. (From Dorland, 27th ed) Ligand
D008550 Melatonin A biogenic amine that is found in animals and plants. In mammals, melatonin is produced by the PINEAL GLAND. Its secretion increases in darkness and decreases during exposure to light. Melatonin is implicated in the regulation of SLEEP, mood, and REPRODUCTION. Melatonin is also an effective antioxidant.
D011869 Radioligand Assay Quantitative determination of receptor (binding) proteins in body fluids or tissue using radioactively labeled binding reagents (e.g., antibodies, intracellular receptors, plasma binders). Protein-Binding Radioassay,Radioreceptor Assay,Assay, Radioligand,Assay, Radioreceptor,Assays, Radioligand,Assays, Radioreceptor,Protein Binding Radioassay,Protein-Binding Radioassays,Radioassay, Protein-Binding,Radioassays, Protein-Binding,Radioligand Assays,Radioreceptor Assays
D011956 Receptors, Cell Surface Cell surface proteins that bind signalling molecules external to the cell with high affinity and convert this extracellular event into one or more intracellular signals that alter the behavior of the target cell (From Alberts, Molecular Biology of the Cell, 2nd ed, pp693-5). Cell surface receptors, unlike enzymes, do not chemically alter their ligands. Cell Surface Receptor,Cell Surface Receptors,Hormone Receptors, Cell Surface,Receptors, Endogenous Substances,Cell Surface Hormone Receptors,Endogenous Substances Receptors,Receptor, Cell Surface,Surface Receptor, Cell
D001921 Brain The part of CENTRAL NERVOUS SYSTEM that is contained within the skull (CRANIUM). Arising from the NEURAL TUBE, the embryonic brain is comprised of three major parts including PROSENCEPHALON (the forebrain); MESENCEPHALON (the midbrain); and RHOMBENCEPHALON (the hindbrain). The developed brain consists of CEREBRUM; CEREBELLUM; and other structures in the BRAIN STEM. Encephalon
D002645 Chickens Common name for the species Gallus gallus, the domestic fowl, in the family Phasianidae, order GALLIFORMES. It is descended from the red jungle fowl of SOUTHEAST ASIA. Gallus gallus,Gallus domesticus,Gallus gallus domesticus,Chicken
D000818 Animals Unicellular or multicellular, heterotrophic organisms, that have sensation and the power of voluntary movement. Under the older five kingdom paradigm, Animalia was one of the kingdoms. Under the modern three domain model, Animalia represents one of the many groups in the domain EUKARYOTA. Animal,Metazoa,Animalia
D001667 Binding, Competitive The interaction of two or more substrates or ligands with the same binding site. The displacement of one by the other is used in quantitative and selective affinity measurements. Competitive Binding
D013574 Synaptosomes Pinched-off nerve endings and their contents of vesicles and cytoplasm together with the attached subsynaptic area of the membrane of the post-synaptic cell. They are largely artificial structures produced by fractionation after selective centrifugation of nervous tissue homogenates. Synaptosome

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