Ladislav Korcek

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

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.

Proliferation of arterial smooth muscle cells incubated in serum from brain-stimulated rats

Platelet deficient serum prepared from rats subjected to acute electrical stimulation of the lateral hypothalamus demonstrated mitogenic activity when added to incubating media supporting growth of homologous arterial smooth muscle cells in vitro. This activity did not appear to be related to the presence of platelet-derived growth factor, hyperlipidemic lipoproteins or increased amounts of insulin. Plasma arginine vasopressin concentration was elevated in these animals, but further investigation is required to determine if this elevation is causally related. Since hypothalamic stimulation is also associated with severe endothelial injury in vivo, the mitogenic activity of the blood of such animals could induce proliferation of SMC which have migrated into the arterial intima. Such features have been observed in chronically stimulated animals and may be of relevance for the role of neural factors in atherogenesis.

Effect of hypothalamic stimulation on the endothelial morphology of the aorta in the conscious squirrel monkey

The role of neurogenic factors in the development of atherosclerosis has not previously been studied in detail. In recent years evidence has accumulated to implicate endothelial injury as a primary stimulus for the proliferation of myo-intimal cells resulting in the formation of the early morphologic lesion. In the present investigation, the effect on aortic endothelial morphology of repetitive electrical stimulation of the lateral hypothalamus in the conscious, unrestrained squirrel monkey, maintained on a cholesterol-free low-fat diet, has been studied. Stimulation was performed with a self-powered, miniaturized electronic stimulator connected to indwelling electrodes. Implanted nonstimulated animals served as controls. Endothelial injury in the form of cell degeneration, denudation, with plasma insudation and partial junctional separation were observed electron-microscopically in stimulated animals compared with controls. These alterations were found to be independent of hypercholesterolemia and/or hypertension. Possible pathways for the induction of injury in this neurogenic model are: (1) direct, through neural circuits from the brain to the vessel wall, and (2) indirect, by elaboration of angiopathic substances inside or outside of the CNS, released into the circulation and transported to the vessel wall where they exert their effects. Reversibility of the endothelial injury progression to established lesions and mechanisms involved remain to be determined in further investigations.

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.

Binding of 125I-labeled p-iodobenzoate to human serum albumin: Interaction with the primary thyroxine binding site☆

Abstract The competitive effect of thyroxine on the interaction of 125 I-labeled p -iodobenzoate with human serum albumin was studied by equilibrium dialysis. At 30 °C and pH 7.4 in 0.06 m potassium phosphate buffer containing 0.7 m m EDTA, p -iodobenzoate is bound at a single binding site on human serum albumin with K = 1.8 × 10 6 m −1 , while thyroxine is bound to the protein at a single strong binding site, K 1 = 1.4 × 10 6 m −1 , plus a second set of several weaker binding sites. The calculated value of the association constant for thyroxine binding based on its competitive effect was less than, but in the same range as the value of K a as determined directly. Excellent agreement between the calculated competitive binding constant and K a for thyroxine was obtained when correction was made for interaction of thyroxine with the secondary set of binding sites. The results indicate that the p -iodobenzoate binding site is identical to the primary strong thyroxine binding site on human serum albumin.

Effect of Long-Chain Fatty Acids on the Binding of T4 and T3 to Human Thyroxine-Binding Globulin (TBG)

Long-chain free fatty acids (FFA) such as oleic and linoleic acids have been shown to inhibit the binding of T4 to binding proteins in serum (1–3). In a recent report, Chopra and coworkers (4) have presented evidence indicating tht circulating FFA, especially oleic acid, makes an important contribution to thyroid hormone binding inhibitor (THBI) activity found in sera of certain patients with nonthyroidal illnesses (NTI). Although oleic acid and linoleic acid are known to inhibit T4 binding to albumin (5), the effect of FFA on T4 binding to TBG, the major thyroid hormone-binding protein in serum, has not been studied directly. In the present study, we compared the effect of different FFA on the binding of T4 to pure TBG.

Ventricular fibrillation and the Brattleboro rat

Despite considerable evidence from both clinical and animal experiments, the role of beta-adrenergic stimulation in the development of life-threatening cardiac arrhythmias is not well understood. Recent studies indicate that the posterior pituitary hormone, arginine vasopressin, may have a modulatory effect on cardiovascular sympathetic tone, especially with respect to blood pressure and heart rate. Employing the homozygous Brattleboro rat, an animal genetically deficient in the synthesis of arginine vasopressin, the following report offers evidence that the neuropeptide may also act on central pathways to suppress arrhythmogenic activity elicited by beta-adrenoceptor stimulation.