S8). with an equilibrium dissociation constant (KD) of 17 nmand a T1AM/apoB-100 stoichiometry of 1 1:1. NCRW0005-F05 Competition binding assays demonstrate that this binding site is usually highly selective for T1AM. Intracellular T1AM uptake is usually significantly enhanced by apoB-100-made up of lipoprotein particles. Modest enhancements to apoB-100 cellular uptake and secretion by T1AM were observed; however, multidose T1AM treatment did not affect lipid or lipoprotein inventoryin vivo. Thus, it appears that apoB-100 serves as a carrier of circulating T1AM and affords a novel mechanism by which T1AM gains access to cells. Keywords:Lipid Transport, Lipoprotein, Serum, Thyroid, Thyroid Hormone == Introduction == 3-Iodothyronamine (T1AM)2is a naturally occurring derivative of thyroid hormone that has biological actions unique from those of the predominant secreted form of thyroid hormone, thyroxine (T4), and its peripherally derived deiodination product 3,5,3-triiodothyronine (T3), the endogenous ligand for thyroid hormone receptors (1). T1AM has rapid short term effects, including induction of hypothermia, bradycardia, and hyperglycemia in mice (25). In addition, T1AM administration rapidly triggers NCRW0005-F05 a shift in fuel usage toward lipids and away from carbohydrates in both mice and Siberian hamsters (6). It has been suggested that T1AM may be generatedin vivoby enzymatic deiodination and decarboxylation of T4, but this has not yet been exhibited experimentally (7). T1AM has been NCRW0005-F05 detected in rodent brain, heart, and liver tissues and also in blood circulation of mice, guinea pigs, and Siberian hamsters (2,6,8). Quantitative analysis of T1AM levels in rat using liquid chromatography coupled to tandem mass spectrometry (LC/MS/MS) revealed that tissue concentrations of T1AM are substantially higher than serum concentrations, and in certain tissues, such as the liver, T1AM is present at significantly higher levels than T4and T3(9). However, human sera analyzed with a recently developed highly selective T1AM immunoassay exhibited T1AM levels comparable with those of total circulating T4(10). Thyroid hormones are present in circulation largely bound to carrier proteins. More than 99% of circulating T4is usually bound to serum proteins, including thyroxine-binding protein, and transthyretin (1113). T3binds to the same proteins, although with lower affinity. Because of the chemical similarities and potential biosynthetic origins of T1AM from thyroid hormones, and because of the presence of T1AM in both serum and tissues, we investigated whether T1AM, like thyroid hormones, was also bound to carrier proteins in serum. == EXPERIMENTAL PROCEDURES == == == == == == Materials == l-Thyroxine (T4), 3,3,5-triiodo-l-thyronine (T3), 3,3,5- triiodo-l-thyronine (rT3), 3,5-diiodo-l-thyronine (3,5-T2), andl-thyronine (T0) were obtained from Sigma, and 3-iodo-l-thyronine (T1) was from Toronto Research Chemicals Inc. (Canada). T1AM and other thyronamines such as 3,3,5,5-tetraiodothyronamine (T4AM), 3,3,5-triiodothyronamine (rT3AM), 3,5,3-triiodothyronamine (T3AM), 3,5-diiodothyronamine (3,5-T2AM), 3,3-diiodothyronamine (3,3-T2AM), and thyronamine (T0AM) were synthesized according to the literature (14). Anhydrous dimethylformamide (DMF) was obtained by passing through two columns of activated molecular sieves. Final compounds were characterized by1H NMR and mass spectrometry. == Determination of T1AM Binding to Serum Protein == 500 l of normal pooled serum (human and rat serum from Innovative Research, Novi, MI; mouse serum from Millipore, Billerica, MA) was incubated with tracer quantities of [125I]T1AM for 24 h at 4 C in the presence or absence of extra unlabeled T1AM (50 m). Bound and free [125I]T1AM was separated by filtering through 3K Amicon ultracentrifugal filters (Fisher) at 3000 rpm on a table top centrifuge (Beckman, GS-6R centrifuge) followed by repeated washing with Tris-HCl buffer (0.1m(pH 7.4)) at 4 C. After excessive washing, the bound [125I]T1AM was measured by gamma counting (Packard Gamma Cobra II D5005). A control experiment was done in a similar manner by using 500 l of buffer instead of sera. For the concentration-dependent binding experiment, 80 l of normal pooled human serum was incubated with different concentrations of [125I]T1AM (from 0.1 nmto 10 m), in 0.1mTris-HCl buffer (pH 7.4) for 24 h at 4 C in the presence or absence of excess unlabeled T1AM (50 m). Bound and free T1AM were separated by incubation with 0.5 ml of a charcoal (0.5%)/dextran (0.05%) suspension in Tris-HCl buffer for 15 min Rabbit polyclonal to ZC3H12A at 4 C with gentle shaking. After centrifugation for 15 min at 4000 gthe supernatant was utilized for the measurement of radioactivity by gamma counting. Specific binding of [125I]T1AM in this experiment, and all other experiments reported in this paper, was determined by subtracting nonspecific binding from total binding. == Preparation of Affinity Chromatography Supports == All solvents (distilled water, buffer) used in this synthesis were de-gassed with argon. Synthesis of T1AM made up of activated disulfide (compound3) and tyramine made up of activated disulfide (compound7,supplemental Fig. S1) are explained in thesupplemental material. Compounds3and7were both immobilized at thiol-Sepharose 4B (Sigma) according to the following process: 1 g of freeze-dried activated thiol-Sepharose 4B was suspended in distilled.