Hans J. Cahnmann

Thyroxine Transport Proteins of Plasma. Molecular Properties and Biosynthesis

Publisher Summary This chapter discusses the molecular properties and biosynthesis of the thyroxine transport proteins of plasma. The purification of the complete amino acid sequence and the high-resolution X-ray diffraction model of pre-albumin (PA) have helped enhance the knowledge of the physiology of these proteins. Apart from thyroxine-binding globulin and PA, several other serum proteins interact with the thyroid hormones. However, both human and bovine serum albumin, in addition to several less active sites, possess one binding site for thyroxine with a rather high affinity constant. Despite its abundance in plasma, albumin transports only about 10% of the circulating thyroid hormone. Other proteins interacting with thyroxine include lipoproteins. In severe illnesses and in acute stress, the disappearance rate of PA from plasma is usually normal, which suggests that the low serum PA level in the patients is the result of a low rate of synthesis.

Gas chromatographic determination of iodinated compounds

The qualitative and quantitative gas chromatographic evaluation of a large variety of iodinated substances (iodoamino acids and their side chain and position analogs, intermediates in the synthesis or degradation of the thyroid hormones, and related compounds) is described. As little as a few hundred picograms can be determined accurately in most cases by means of an electron capture detector. Nonvolatile compounds have been converted to their volatile trimethylsilyl derivatives by treatment with N,O-bis(trimethylsilyl)acetamide at room temperature or at 80°. Various characteristics of these derivatives as well as precautions which must be taken for the determination of picogram and nanogram amounts are described. Mass spectrometry shows that the gas chromatographically determined derivatives of the iodoamino acids contain three trimethylsilyl groups.

HIGH-AFFINITY BINDING OF THYROID HORMONES TO NEUROBLASTOMA PLASMA MEMBRANES

The binding of thyroid hormones to isolated plasma membranes was studied in NB41A3 neuroblasts. Saturable binding of L-T3, D-T3 and L-T4 was observed. Binding was time-dependent, with equilibrium reached in less than 60 min and maximal binding occurring between pH 7.4 and 7. Saturation experiments demonstrated two classes of sites for L-T3: a high-affinity site with Ka 8.4 X 10(9) M-1 and a low-affinity site with Ka 7.3 X 10(6) M-1.L-T3 and D-T3 inhibited each others binding, L-T3 being several-times more potent. Affinity labeling of isolated membranes with bromoacetylated thyroid hormones disclosed stereospecific binding to SDS-PAGE bands with approximate molecular masses of 27 kDa (preferentially labeled by BrAc-L-T3), 32 kDa (preferentially labeled by BrAc-D-T3), and 48 and 87 kDa (preferentially labeled by BrAc-L-T4). Binding of BrAc-L-T3 to the 27 kDa band accounted for 3.4% of total binding, was selectively inhibited by excess L-T3, and may be involved in intracellular transport of L-T3.

Iodoamino acid distribution in thyroglobulin iodinated in vivo and in vitro

Abstract The amount and the distribution of the iodoamino acids in thyroglobulin iodinated in vivo were compared to those obtained by iodination in vitro . Normal bovine thyroglobulin (54 atoms I per molecule) and virtually iodine-free human goiter thyroglobulin (0.5 atom I per molecule) were iodinated with 100 and 200 moles 131 I 2 per molecule thyroglobulin at pH 9.0 and at pH 7.2, and then hydrolyzed with pronase. The distribution of the radioactivity among the various iodoamino acids, including 3,3′,5′-triiodothyronine, 3′,5′-diiodothyronine, and monoiodohistidine, in the pronase digest was determined by cation exchange chromatography and thin-layer chromatography. Native rat thyroglobulin, iodinated in vivo by equilibrium labeling, was analyzed in the same way. In equilibrium-labeled native thyroglobulin small amounts of 3,3′,5′-triiodothyronine, 3,3′- and 3′,5′-diiodothyronine, and monoiodohistidine were present in addition to mono- and diiodotyrosine, 3,3′,5-triiodothyronine, and thyroxine. In thyroglobulin which had been iodinated in vitro , considerably smaller amounts of the thyroid hormones thyroxine and 3,3′,5-triiodothyronine were present and the biologically inactive iodothyronines (3,3′,5′-triiodothyronine and 3′,5′-diiodothyronine) formed a proportionately higher percentage of the total iodothyronines. No labeled 3,3′-diiodothyronine could be detected in thyroglobulin iodinated in vitro . Monoiodohistidine, present in native thyroglobulin only in minute amounts, was found in considerably larger amounts after iodination in vitro . Thyroglobulin iodinated in vitro is less extensively hydrolyzed by pronase than native thyroglobulin which suggests structural differences between the two types of thyroglobulin. Although the various differences between thyroglobulin iodinated in vivo and in vitro may be partly due to species differences, they seem to arise mainly from differences in the mechanism of the two types of iodination.

Binding of thyroxine to human plasma low density lipoprotein through specific interaction with apolipoprotein B (apoB-100)

Human plasma low density lipoprotein (LDL), which binds 0.2% of plasma T4, was shown to interact with the hormone through its protein moiety, apolipoprotein B-100. LDL and LDL2, the major subfraction of LDL, were found to have 3 equivalent binding sites for T4 with Ka = 2.5 x 10(6) M-1. Photoaffinity labeling of LDL with inner ring-labeled [125I]T4, followed by SDS-PAGE or agarose-SDS-PAGE of the labeled products, revealed that apoB-100 and its proteolytic cleavage products, apoB-74 and apoB-26, bound [125I]T4. In the presence of 1 or 10 microM T4, labeling was decreased in 7 separate experiments by 40-53% or 65-86%, respectively, consistent with a Ka of approximately 10(6) M-1. Binding of T4 to apoB-100 associated with VLDL was also demonstrated by photoaffinity labeling. The observed thyroid hormone binding property of lipid-complexed apoB-100 and the knowledge that receptors for the apolipoprotein exist in various tissues suggest a possible physiological role in thyroid hormone transport.

NONENZYMIC SYNTHESIS OF THYROXINE RESIDUES IN THYROGLOBULIN.

Abstract Iodine-labeled 4-hydroxy-3,5-diiodophenylpyruvic acid was prepared and converted into its hydroperoxide (thyroxine precursor). Thyroglobulin was permitted to react with the hydroperoxide and the nature and amount of the iodothyronine residues formed in this model reaction for the biosynthesis of thyroxine were investigated. Iodine-rich bovine thyroglobulin (54 atoms I per molecule) and iodine-poor human thyroglobulin (6–10 atoms I per molecule) were used. After the coupling, the thyroglobulin was analyzed by chromatographic fractionation of its pronase digest. The analysis showed the formation of about 1 and 2 iodothyronine residues per molecule in the coupling reaction with iodine-poor and iodine-rich thyroglobulin, respectively. The hydroperoxide reacts with protein-bound diiodotyrosine and monoiodotyrosine. Hence both thyroxine and 3,3′,5′-triiodothyronine residues are formed. The molar ratio of newly formed thyroxine to 3,3′,5′-triiodothyronine residues was 4 in iodine-rich thyroglobulin (diiodotyrosine to monoiodotyrosine molar ratio of 0.9) and 0.4 in iodine-poor thyroglobulin (diiodotyrosine to monoiodotyrosine molar ratio of 0.2). Thus, the hydroperoxide reacts more efficiently with diiodotyrosine than with monoiodotyrosine residues. The amount of iodothyronine residues formed by coupling with the hydroperoxide (intermolecular reaction) was compared with that formed when thyroglobulin is iodinated to a comparable iodine level (intramolecular reaction). At high iodine levels both model reactions proceed with similar efficiencies, but at low iodine levels coupling with the hydroperoxide of 4-hydroxy-3,5-diiodophenylpyruvic acid is considerably more efficient.

Synthesis of [3,5-125I]triiodo-l-thyronine of high specific activity

Abstract Two methods for the synthesis of [3,5- 125 I]triiodo- l -thyronine of high specific activity are described. This triiodthyronine which carries the iodine label exclusively in the nonphenolic ring has not been available so far. Both methods start from [3,5- 125 I]diiodo- l -thyronine which is iodinated either with iodine in potassium iodide or with iodide and chloramine T. The concentration of the iodinating agent is critical in both methods and the pH of the reaction mixture must be high enough (∼11) to cause complete ionization of the phenolic group of the substrate. The triiodothyronine obtained in over 70% yield is purified by ion-exchange chromatography.

Molecular size of the nuclear thyroid hormone receptor

Among the previously reported putative nuclear thyroid hormone receptor forms having molecular masses of 56-59 kDa and 45-49 kDa, respectively, only the former can be the endogenous receptor. The latter must be a degradation product because it is virtually absent in rat liver nuclear extracts prepared in the presence of 20% glycerol and 5 mM Mg2+, which inhibit degradation. In the absence of glycerol, the receptor form of lower mass was present in large amounts in nuclear extracts. Sucrose could not replace glycerol as a protective agent, even in the presence of Mg2+. Thus, the endogenous nuclear thyroid hormone receptor appears to be labile under the experimental conditions used in preparing nuclear extracts. The molecular mass of the nuclear receptor was determined to be 57 kDa on the basis of SDS-polyacrylamide gel electrophoresis after photoaffinity labeling of nuclear proteins with (3,5-125I)-labeled thyroxine.

PHOTOCHROMISM OF 2,6-DINITRODIPHENYL ETHERS ENHANCED IN THE PRESENCE OF A HOST BIOMOLECULE*

Abstract— The photochromic behavior of 2,6‐dinitrodiphenyl ethers is described. In the course of a study for the photoaffinity labeling of the binding site for thyroxine on human prealbumin, it was found that the thyroxine analog 3‐[4‐(4‐hydroxy‐3,5‐diiodophenoxy)‐3,5‐dinitrophenyl]propionic acid (1), on irradiation with >350nm light in the presence of prealbumin, gave a pink species (λmax 508 nm). In the absence of prealbumin, no color formation was observed. Irradiation of 3‐[4‐(4‐hydroxyphenoxy‐3,5‐dinitrophenyl]propionic acid (2) and 3‐(4‐phenoxy‐3,5‐dinitrophenyl)propionic acid (3), analogs of 1, in acetonitrile, aqueous bicarbonate, or ethanol gave a colored species (λmax 500–520 nm depending on the solvent) in the absence of a host molecule. In the presence of β‐cyclodextrin, which may be considered a model for a binding site on a protein, photocoloration occurred at a faster rate than in the absence of ß‐cyclodextrin. For both 2 and 3, the colored species underwent thermal back reaction to the starting compound. The activation energy for the thermal back reaction was estimated to be about 40 kj/mol.

Model reactions for the biosynthesis of thyroxine. Part XVII. On the mechanism of the conversion of 4-hydroxy-3,5-di-iodophenylpyruvic acid into thyroxine

4-Hydroxy-3,5-di-iodobenzoylglyoxylic acid is a potential intermediate in the nonenzymic formation of thyroxine from 3,5-di-iodotyrosine and 4-hydroxy-3,5-di-iodophenylpyruvic acid; its ethyl ester has been synthesised. The lack of reactivity of this ester towards 3,5-di-iodotyrosine indicates that the diketo-acid is not an intermediate in the formation of thyroxine.