Characterization of the binding of thyroxine to high density lipoproteins and apolipoproteins A-I.

We studied binding of T4 to the lipid-complexed apolipoproteins (apo) of high density lipoproteins (HDL), the major lipoprotein carrier of thyroid hormones in human plasma, and to lipid-free apoA-I. HDL isolated from fresh normal plasma by ultracentrifugation (density, 1.063-1.210 g/mL) was photoaffinity labeled with [3,5-(125)I]T4 and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Two bands corresponding to apoA-I (28.3K) and apoC-II or apoC-III (8.6-9.2K) were seen, and their radioactivity decreased by 50-60% when labeled in the presence of 1 mumol/L T4. Photoaffinity labeling of isolated apoA-I also was demonstrated and was decreased 74% by 1 mumol/L T4, suggesting a higher affinity of the lipid-free protein for T4. T4 binding of isolated apoA-I was optimal at pH 7-8, reached a maximum after 1 h at 23 C, and decreased after incubation at 37 C. Scatchard analysis revealed a single T4-binding site with a Ka of 7.5 x 10(7) L/mol at 23 C, pH 8.2. The potency of T4 analogs as inhibitors of T4 binding to isolated apoA-I was L-T4 = D-T4 = triiodothyroacetic acid = L-rT3 much greater than L-T3 much greater than L-thyronine. The binding of T4 to apoA-I was reduced by known inhibitors of T4 binding to serum proteins (diclofenac = mefenamic acid = furosemide = 8-anilinonaphthalene sulfonic acid much greater than dilantin greater than heparin greater than barbital) and by lipids (unsaturated fatty acids greater than cholesterol = cholesterol esters = phospholipids greater than saturated fatty acids = diglycerides = triglycerides). We conclude that the binding of T4 to HDL is mediated by a specific interaction of the hormone with apoA-I and with apoC-II and/or apoC-III. Since the lipid constituents of HDL inhibit T4 binding to apoA-I, the HDL subfraction in plasma that carries most of the HDL-bound T4 should be one with a low lipid content.