THYROID-HORMONE BINDING TO ISOLATED HUMAN APOLIPOPROTEINS A-II, C-I, C-II, AND C-III - HOMOLOGY IN THYROXINE-BINDING SITES

Thyroid hormone binding to lipid-free apolipoprotein (ape) A-II, C-I, C-II, and C-III isolated from human plasma was investigated by photoaf finity labeling with [I-125]T-4 and sodium dodecyl sulfate-polyacrylam ide gel electrophoresis. Both the monomeric and polymeric forms were s pecifically labeled. Inhibition by 10 mu M unlabeled L-T-4 was greater than or equal to 50%, suggesting affinity constants in the nM to mu M range; the least inhibition was seen with apoA-II. Unlabeled D-T-4 an d reverse T-3 (rT(4)) gave the same inhibition as unlabeled L-T-4. Inh ibitors of thyroid hormone binding to plasma proteins showed a differe nt inhibitor potency with each apolipoprotein and a pattern different from that seen with T-4 binding globulin (TBG) and transthyretin (TTR) . Also in contrast to TBG, where only unsaturated nonesterified fatty acids (NEFA) are effective inhibitors, both unsaturated and saturated NEFA as well as other lipids inhibited T-4 labeling. The flavonoid EMD 21388 was ineffective, confirming that it is a selective inhibitor of T-4 binding to TTR. T-4 binding to the apoCs was confirmed by the que nching of tryptophan fluorescence by unlabeled L-T-4. (ApoA-II was not studied since it lacks tryptophan.) Since the self-association of apo lipoproteins involves interaction between amphipathic alpha-helices, a nd since the polymeric forms show specific T-4 binding properties as i n the parent monomer, the T-4-binding domain appears to be outside the alpha-helical domain, as previously seen with apoA-I. The position of the T-4 binding sites of apoA-I, A-II, C-I, C-II, C-III, and E in the N-terminal, exon 3-coded regions (and in the exon 2-coded region of A -IV) is associated with amino acid homology, which is also shared with the T-4 binding domains of TBG, TTR, and serum albumin.