Milton Tabachnick

Thyroxine-protein interactions: II. The binding of thyroxine and its analogues to human serum albumin☆

Abstract The relative affinities of various thyroxine analogues for albumin have been determined. Estimations of relative binding affinities were based on a comparative study of the abilities of the analogues to displace thyroxine from binding sites on albumin. The results indicate that the substituents on thyroxine most directly involved in binding are: (1) the phenolic hydroxyl group; (2) iodine atoms in both the 3′- and 5′-positions of the thyronine molecule; (3) the diphenyl ether group. The absence of any one of these substituents from the molecule leads to an appreciable reduction in relative binding affinity. With regard to the alanine side chain of thyroxine, it appears that neither a free carboxylate ion nor a free α-amino group are required for binding. The results of experiments on the effect of d -thyroxine on the binding of l -thyroxine indicate that binding is not stereospecific. The constants for the binding of tetraiodothyropropionic acid by albumin have been determined.

Further characterization of human thyroxine-binding globulin.

Abstract Thyroxine-binding globulin has been purified from human blood plasma by a three-step procedure consisting of affinity chromatography on thyroxine-Sepharose, chromatography on DEAE-Sephadex A-50, and preparative electrophoresis in polyacrylamide gel. By ultracentrifugal analysis the molecular weight of thyroxine-binding globulin was found to be 60 700 compared to an estimated value of 65 000 by sodium dodecylsulfate-polyacrylamide gel electrophoresis. Thyroxine-binding globulin contained about 13% carbohydrate which included per mole of protein, 2 residues glucose, 6 residues galactose, 12 residues mannose, 12 residues glucosamine, and 6 residues sialic acid. Analysis by gas-liquid chromatography revealed the presence of long-chain fatty acids associated with thyroxine-binding globulin. A second form of thyroxine-binding globulin which moved appreciably slower electrophoretically than thyroxine-binding globulin was also isolated. The slower moving thyroxine-binding globulin had a low sialic acid content, 0.6 residue per mole, and is apparently produced in significant quantity by desialylation of thyroxine-binding globulin during the purification procedure. Slow thyroxine-binding globulin had a molecular weight in the vicinity of 57 500 as determined by sodium dodecylsulfate-polyacrylamide gel electrophoresis. No evidence was found for a subunit structure in either thyroxine-binding globulin or the slower moving form of thyroxine-binding globulin.

Uptake of triiodothyronine and thyroxine by isolated rabbit adipocytes

The prevailing view regarding uptake of triiodothyronine (T3) and thyroxine (T4) by tissues is that cellular uptake is largely dependent upon the concentration of free hormone in blood and extracellular fluids [1,2]. It is also assumed that thyroid hormone reaches the cell surface as the free unbound molecule and enters sensitive cells freely by passive diffusion. Although a great deal of information exists on the interaction of T 3 and T4 with the proteins of blood plasma, little knowledge is available on uptake of the hormone by cells. Adipose tissue may be considered a target tissue for thyroid hormone, based on the fact that triiodothyronine enhances lipolysis in adipose tissue or free cells, especially in the presence of catecholamines [3 -6] . A diminished lipolytic response to norepinephrine has also been found in adipose tissue of hypothyroid human beings [7]. In addition, adipose tissue or fat cells from hyperthyroid rats show an increased rate of oxygen consumption [5,6]. Since a relatively homogeneous preparation of fat cells can be obtained readily from adipose tissue [8], these cells can serve as a model system for an investigation of thyroid hormone transport into cells. We have studied the uptake of T3 and T4 by intact rabbit adipocytes. The results indicate the existence in fat cells of high affinity, low capacity binding sites, with similar affinities for triiodothyronine and thyroxine. Since triiodothyronine was taken up by the lipid layer of lysed cells to a much greater extent than by intact cells, it is suggested that a barrier to free diffusion of thyroid hormone exists in the fat cell. 2. Materials and methods

Thyroxine-protein interactions: III. Effect of fatty acids, 2,4-dinitrophenol and other anionic compounds on the binding of thyroxine by human serum albumin☆

Abstract The effect of various anionic compounds on the binding of thyroxine by human serum albumin has been studied. The results show that anions such as 2,4-dinitrophenol, salicylate, and fatty acids are capable of displacing thyroxine from binding sites on albumin. The compounds investigated exhibit the following order of effectiveness in reducing thyroxine binding by albumin: oleate > linoleate > dodecyl sulfate > palmitate > 2,4-dinitrophenol > laurate > octanoate > salicylate.

In vivo demonstration of the presence of specific saturable binding sites for triiodothyronine in nuclei of tadpole liver

Abstract The binding of l -triiodothyronine (T3) by subcellular fractions of tadpole liver has been studied following in vivo administration of [125I]T3. A steady state between T3 in the nuclei and T3 in the extranuclear fraction of liver was reached 2 hr after intraperitoneal injection of a low dose of [125I]T3 into animals maintained at 25°. Saturable binding sites for T3 were not detectable in mitochondrial and microsomal fractions but were definitely present in nuclei and possibly cytosol. As determined by a loading experiment, the maximum binding capacity of the T3 binding sites in nuclei was 350 fmol/mg DNA or about 2700 molecules of T3 per nucleus. Injection of a high dose of an unlabeled iodothyronine simultaneously with a tracer dose of [125I]T3 indicated that 3′-isopropyldiiodothyronine competed most effectively for the nuclear T3 binding sites followed closely by triiodothyronine and thyroxine, while 3,5-diiodothyronine had the least competitive effect. Analysis of subnuclear fractions for distribution of radioactivity, after in vivo labeling of nuclei with a small dose of [125I]T3, indicated that 63% of the hormone was associated with chromatin. Extraction of nuclei with 0.8 M KCl removed about 50% of the macromolecular bound [125I]T3. Chromatographic analysis of the 0.8 M KCl extract on Sephadex G-200 indicated a molecular weight in the range 70–80,000 for the T3 binding macromolecule(s) in tadpole liver nuclei.

Effect of long-chain fatty acids on the binding of thyroxine and triiodothyronine to human thyroxine-binding globulin.

The effect of long-chain fatty acids on the binding of thyroxine to highly purified human thyroxine-binding globulin has been studied by equilibrium dialysis performed at pH 7.4 and 37 degrees C. At a fixed molar ratio of 2000:1 of fatty acid to thyroxine-binding globulin, the degree of binding inhibition based on the percent change in nK value relative to the control as determined from Scatchard plots was: palmitic, 0%; stearic, 0%; oleic, 76%; linoleic, 69%; and linolenic, 61%. At a 500:1 molar ratio of oleic acid to thyroxine-binding globulin, equivalent to 0.125 mM free fatty acid in serum, thyroxine binding was inhibited by 18%, increasing to 93% at a 4500:1 molar ratio. At molar ratios of oleic acid to thyroxine-binding globulin of 1000:1, 2000:1 and 4000:1, the degree of inhibition of triiodothyronine binding was 24%, 41% and 76%, respectively. The results indicate that the unsaturated long-chain fatty acids are potent inhibitors of thyroxine binding to thyroxine-binding globulin, whereas the saturated fatty acids have little or no effect on thyroxine binding.

Thyroxine-protein interactions: VI. Structural requirements for binding of benzene derivatives to thyroxine-binding sites on human serum albumin☆

Structural requirements for binding to thyroxine-binding sites on human serum albumin were studied by measuring the relative abilities of various substituted benzene derivatives, such as phenols and benzoates, to displace thyroxine from albumin in equilibrium dialysis experiments performed at pH 7.4 and 30 °. Relatively strong binding of benzene derivatives to thyroxine-binding sites on albumin depended upon two structural requirements. These were (1) a requirement for an anionic group, and (2) the presence on the phenyl ring of at least one highly polarizable substituent such as an iodine atom in the case of benzoates, or the presence of two highly polarizable substituents in the case of phenols. The effect of different substituents on the binding of phenols was in the order: alkyl group (CH3-, or (CH3)2C-) < NO2, Cl < Br, I. The diphenyl ether structure of thyroxine was not an obligatory requirement for strong binding since compounds with a single benzene ring were bound as tightly as thyroxine as shown by the competition experiments and confirmed by fluorescence quenching. Bulky halogen atoms ortho to the carboxylate group caused large reductions in relative binding affinities of benzoates. This ortho effect indicated that coplanarity of the carboxylate anion with the phenyl ring was required for optimal binding. Based on the steric requirement for coplanarity, a model for binding to the primary thyroxine-binding site was suggested which visualized the outer diiodophenolate portion of thyroxine as fitting into a cleft in a predominantly hydrophobic region of albumin.

TISSUE DISTRIBUTION OF INJECTED 125I-LABELED PORCINE RELAXIN: ORGAN UPTAKE, WHOLE-BODY AUTORADIOGRAPHY, AND RENAL CONCENTRATION OF RADIOMETABOLITES

It is well established that many polypeptide hormones such as glucagon, ACTH, TSH and LH bind to specific receptors located on the membranes of target cells. Receptor binding is accompanied by activation of adenyl cyclase, generating cyclic 3’, 5’-AMP(cAMP) from ATP. The increase in the intracellular concentration of this so-called second messenger, CAMP, initiates a physiological response by activating enzymes, altering membrane permeability, etc., etc. The specificity of the actions of hormones that function through this mechanism depend both upon the presence of proper membrane receptors on a given type of cell and upon the CAMP-responsive systems available within that cell. It has recently become of interest to determine if the ovarian polypeptide hormone relaxin likewise exerts its effects on the pubic symphysis, cervix and uterus via a receptor binding, CAMP-generating mechanism. Indeed, there are several reports of “specific” in vitro binding of IT-relaxin to homogenates of guinea pig and mouse pubic symphysis,’ segments or membrane-rich preparations of rat uteri,’, and to fibroblasts cultured from mouse pubic symphysis or human skin.3 It is claimed that intravenously injected 1251-labeled relaxin binds specifically to the uterus in rats.4 Moreover, relaxin treatment is reported to increase CAMP levels in mouse pubic symphysis and in rat uterus and cervix.‘, 6 . 7 The foregoing studies not withstanding, our own preliminary efforts to demonstrate specific binding of lz51-labeled porcine relaxin to membrane-rich preparations of rodent uteri have yielded consistently negative data. Therefore, we have turned to alternative methods for demonstrating the affinity of relaxin for specific target tissues. Other workers have successfully employed the technique of whole body autoradiography to detect sites where skeletal growth

Effect of temperature on the transport of triiodothyronine (T3) into liver nuclei of Rana catesbeiana tadpoles in vivo

Abstract To determine if inhibition of triiodothyronine (T 3 )-induced anuran metamorphosis at low temperature can be attributed to the failure of operation of a temperature-sensitive system for transport of T 3 into the nucleus as has been postulated for thyroxiine ( M. D. Griswold, M. S. Fischer, and P. P. Cohen, 1972 , Proc. Nat. Acad. Sci. 69, 1486–1489), the effect of temperature on the uptake of T 3 by liver nuclei of Rana catesbeiana tadpoles has been studied in vivo . A low dose (2 pmol/g body wt) or a high dose (300 pmol/g body wt) of [ 125 I]T 3 was injected intraperitoneally into tadpoles and uptake of [ 125 I]T 3 by whole liver and liver nuclei was determined in animals maintained at 25 or 5°. Transfer of T 3 from cytoplasm into nucleus was definitely slower at low temperature when a low dose of T 3 was administered. However, transport of T 3 into the nucleus is not blocked at 50° but takes place whether a low or high dose of the hormone is injected. For example, after injection of 2 pmol of [ 125 I]T 3 /g body wt, the level of nuclear-bound [ 125 I]T 3 at 5° in 24 hr approached to within 70% of the maximum which had been reached in 2 hr at 25°. When the high dose (300 pmol of [ 125 I]T 3 /g body wt) was injected, there was no apparent major inhibition of nuclear uptake of T 3 at the lower temperature since the level of nuclear-bound T 3 at 5° was about the some as that at 25° from 3 to 24 hr. Since there is appreciable nuclear uptake of T 3 at 5° it appears that some mechanism other than a block in transport of T 3 into the nucleus is involved in causing the inhibition of T 3 -induced metamorphosis that occurs at low temperature.

Modification of thyroxine-binding globulin with p-iodophenylsulfonyl (pipsyl) chloride and effect on thyroxine binding activity

Thyroxine-binding globulin (TBG) is the major transport protein of the thyroid hormones in human plasma. As part of a study of the chemistry of the thyroxine binding site on TBG, we have been investigating a series of protein modifying reagents for their effect on thyroxine binding. Among the reagents assayed, p-iodophenylsulfonyl (pipsyl) chloride proved to be of interest, since relatively low levels of protein modification with this reagent resulted in concomitant decreases in thyroxine binding activity. The present communication describes the results of an investigation involving the effect of derivatization of TBG with pipsyl chloride on the thyroxine binding activity of the protein. The results indicate that there is a direct relationship between the degree of pipsylation and the percent decrease in thyroxine binding activity at relatively low levels of pipsyl group incorporated per mole protein. Analyses of hydrolyzates of pipsylated TBG preparations modified with [35S]pipsyl chloride, indicate that the principal group derivatized in TBG is an e-amino group of a specific lysyl residue probably located near the thyroxine binding site.