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 photoaffinity labeling with [I-125]T-4 and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Both the monomeric and polymeric forms were specifically 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 and reverse T-3 (rT(4)) gave the same inhibition as unlabeled L-T-4. Inhibitors of thyroid hormone binding to plasma proteins showed a different 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 quenching of tryptophan fluorescence by unlabeled L-T-4. (ApoA-II was not studied since it lacks tryptophan.) Since the self-association of apolipoproteins involves interaction between amphipathic alpha-helices, and since the polymeric forms show specific T-4 binding properties as in 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.