Raymond Michel

Natural and artificial iodoproteins.

Publisher Summary This chapter deals with biochemical and physiological activity of iodoproteins as related to their composition. It emphasizes the action of iodine on proteins, which are concerned with the process of halogenation. The mechanism of the formation of thyroxine within the framework of the chemistry of proteins is also presented along with the description of iodinated scleroproteins. The thyroid gland in the vertebrates is relatively rich in iodine. The iodoproteins extracted from the thyroid and the skeletal tissues are of different types. All the natural iodinated amino acids identified in the living organisms are essentially derivatives of L-tyrosine. In context to artificial iodoproteins, the most extensively iodinated derivatives have halogen fixed on nitrogen atom 1. Also, only the cyclic or heterocyclic amino acids are likely to form iododerivatives, which are stable in the iodinated proteins. The structure of the protein can influence both the ease of iodination of the tyrosine residues and the subsequent condensation of diiodotyrosine to thyroxine, whether in nature or in vitro .

Sur la deshalogénation enzymatique des iodotyrosine par le corps thyroïde et sur son rôle physiologique. II.

1. 1. Slices of thyroid gland immersed in an isotonic solution of monoiodotyrosine, diiodotyrosine, or thyroxine marked with 131I in very weak concentrations dehalogenize the first two amino acids, but not thyroxine. The liver, intestines and kidney behave in the same way generally less intensely, while cardiac muscle and spleen do not act on the three amino acids. 2. 2. Dehalogenation of the iodotyrosines is realised by a specific deiodase. It carries out the formation of monoiodotyrosine when the diiododerivative is the substrate. The action of deiodase is not bound to some cellular structures, and the enzyme has been extracted from the thyroid. 3. 3. The iodides formed in contact with slices of gland acting on diiodotyrosine reenter into the thyroxinogenese cycle and give birth to some iodoamino acids. These have been observed whilst constituents of thyroglobuline in the slices of thyroid glands operated the dehalogenization. 4. 4. The physiological signification of these facts is discussed, with reference to: a. the secretion of thyroxine and the total or nearly total absence of iodotyrosines in the circulating blood, and b. the eventuality of a recuperation in situ of the iodides liberated by thyroid dehalogenization of these amino acids.

Étude radiochromatographique des étapes de l'ioduration de la tyrosine et de l'histadine

1. 1. The halogenation of tyrosine and histidine with marked iodine has been studied by radiochromatography of the products formed through the action of 0.4 to 15.0 atoms iodine on one molecule of the amino-acid. The formation of monoiodotyrosine and of monoiodohistidine are independent stages of the reaction, just like that of the dihalogen derivatives. Lis conclusion, according to which the rate of the first process determines that of the second, does not appear to be tenable. 2. 2. Besides the lesser reactivity of histidine towards iodine, already mentioned, it has been established that an excess of reagent breaks the imidazole ring of this amino-acid under conditions where diiodotyrosine is stable. 3. 3. The chromatographic separation of 131I-marked mono- and diiodotyrosine and -histidine, obtained by direct reaction on the amino-acids, makes possible the preparation of small quantities of these substances.

On the peripheral metabolism of thyroid hormones.

The synthesis of radioactive labeled iodothyronines with high specific activity has permitted the administration of physiological doses to experimental animals and the use of such doses has enabled research work on the metabolism of thyroid hormones to progress considerably. The three chief metabolic pathways known for the iodothyronines are dehalogenation, oxidation of the alanine chain, and esterification of the phenolic group. Deiodination proceeds in all tissues and does not seem specific for a particular halogen atom of the The presence of iodothyropyruvic and iodothyroacetic acids in some organs and body f l u i d P apparently is connected with two processes: (1) oxidative deamination, giving a keto acid, of which (2) the oxidative decarboxylation leads to the corresponding acetic derivaThe presence of these substances in skeletal muscle as well as in liver and kidney, demonstrated by the enzymatic studies of Lardy’s group8s9 and in vitro studies on tissue slices,*o shows that the oxidative degradation of thyroid hormones is common to all tissues. The conjugation process can also be considered a general one, since Flock et al.”J2 detected the glucuronides of triiodothyronine (T3) and of thyroxine (T4) in plasma and urine after the administration of these hormones to hepatectomized dogs. However, the formation of g1~curonide’~J~ takes place chiefly in the liver, and the oxidation of the alanine chain occurs chiefly in the kidney; most studies on the metabolism of the thyroid hormones have been devoted to their fate in these organs. The nature of the metabolites formed by the whole tissues is not well defined. Even the fate of the iodothyroacetic acids is not known, except for the fact that they are deiodinated, as are their precursors.6J6 Our purpose here is not to cover all aspects of the metabolism of iodothyronines, but to discuss only certain matters actually being investigated. We shall consider first the initial phase of cellular metabolism: the penetration of iodothyronines into the cells. A survey of the available data on hepatic transformations of the hormones then will be presented in order to define the origin of some iodinated compounds existing in body fluids. Thus, this communication is concerned with two main subjects: (1) the kinetics of penetration of thyroid hormones from the plasma to lymph and to the cells, and (2) the nature of iodinated constituents in the plasma and in the lymph as reflecting partially the hepatic and general metabolism of iodothyronines.

Sur l'élimination biliaire de la triiodothyronine et de la thyroxine et sur leur glycuroconjugaison hépatique

1. 1. Biliary excretion of triiodothyronine and of thyroxine has been studied within 24 hours on fistulized rats injected with physiological (1–9 μg) or pharmacological (100–1800 μg) doses of these substances. It shows important differences in its speed and characters, according to the nature and the dose of injected product. Triiodothyronine is excreted in very high proportion (up to 70%) and only in a small part as glycuroconjugate, when administered in physiological doses. In the same conditions, thyroxine is excreted in a much smaller proportion, but chiefly as a glycuroconjugate. On higher doses, detoxication of both substances proceeds by the formation of combinations of this type. 2. 2. Glycuroconjugates of triiodothyronine and of thyroxine diffuse into plasma when bile duct is ligated after injection of each free amino acid. They have not been found in the blood of animals with normal entero-hepatic circulation. On the other hand, a new iodinated unknown substance (S) has been detected in the plasma of all animals injected with physiological doses of triiodothyronine as of thyroxine; substance S appears to be a metabolite common to both. 3. 3. Triiodothyronine disappears from plasma at a higher speed than thyroxine injected in the same amount, in normal as in bile duct ligated rats, owing to its faster fixation and use by the cells. 4. 4. Biliary excretion of triiodothyronine and thyroxine and of their conjugates plays an important role not only in their entero-hepatic circulation, but also in the regulation of their blood level. The mechanism of the hepatic behaviour of the two active products of thyroid secretion is discussed from the actual knowledge of their transport by plasma proteins and on their metabolism in cells.

Sur la formation de la thyroxine et de ses précurseurs dans les iodoprotéines. III

1. 1. The formation of mono-iodotyrosine, of di-iodotyrosine and of thyroxine, and the disappearance of tyrosine from casein and thyroglobulin under the action of increasing quantities of iodine have been studied under various conditions. 2. 2. The iodine takes part in substitution reactions, particularly intense in ammoniacal medium, and in oxidation reactions, more important at 7.8 pH 7.8 and in the presence of sodium bicarbonate. The former are concerned preferentially with tyrosine, but also with other amino acids (histidine, tryptophan) and to extent that increases with the quantity of halogen. Iodination of a protein is confined specifically to tyrosine only at very low concentration of halogen. 3. 3. Mono-iodotyrosine is abundantly present in the products of weak iodination, but it is progressively transformed into di-iodotyrosine under the action of greater quantities of iodine, and at the same time part of the di-substitution products gives rise to thyroxine. Thyroxine formation is a function not only of the tyrosine content of the protein but also of the position of this amino acid in the molecule. An important fraction of the di-iodotyrosine appears incapable of giving rise to thyroxine. 4. 4. Oxidation processes alter the thyroxine during the halogenation of proteins, especially in a bicarbonate medium at pH 7.8; similarly, they decompose part of the tyrosine and its substitution products. The existence of an optimum yield of thyroxine in the presence of 6–7 atoms of iodine per molecule of tyrosine illustrates the importance of such reactions when the halogen is present in excess. 5. 5. When the same quantities of halogen react with a given quantity of tyrosine casein is iodinated more easily and more intensely than thyroglobulin. Nevertheless, both give rise to thyroxine in quantities corresponding closely to the proportions of their tyrosine total contents. It thus appears that thyroglobulin has no special aptitude for the formation of thyroxine by chemical iodination.

Sur la sulfoconjugaison hépatique de la 3,5,3′-triiodo-L-thyronine et la présence d'un ester sulfurique de cette hormone dans la bile et la plasma

Abstract 1. 1. After injecting physiological doses of 3,5,3′-triiodo- L -thyronine (T 3 ) into thyroidectomized rats, the sulphuric estcr of this hormone (ST 3 ) is found in the bile. ST 3 was identified by paper chromatography (identity of the R F values of the natural and the synthetic compound) and by establishing the presence of SO 4 = and T 3 after hydrolysis. By varying the experimental conditions, it was possible to obtain ST 3 labelled either with 131 I or with both 35 S and 131 I. Excretion of ST 3 in the bile occurs very soon after the injection of T 3 : it may be observed within 1 h. 2. 2. After injection of T 3 , ST 3 is found in the plasma of the thyroidectomized rat. It must be regarded as a normal constituent of the plasma, its presence there being —at least partly—due to intestinal resorption of ST 3 from the bile. 3. 3. The physiological role of ST 3 differs from that of the glycuronides of T 3 , a substance that participates in an enterohepatic circulation of T 3 , because it is hydrolysed in the intestine and the T 3 liberated is then resorbed. ST 3 , a metabolic product of T 3 , circulates normally in the plasma; it may be a form in which the hormone is transported to the receptors, and the possibility that it is the reserve form of T 3 deserves consideration.

Specificite de L'Iodotyrosine desiodase des microsomes thyroïdiens et hepatiques

Abstract The deiodinating activity of thyroid and liver microsomes was studied with 19 compounds including 3-iodo- l -tyrosine and 3:5-diiodo- l -tyrosine and their analogs. While l -iodotyrosines were almost completely dehalogenated, d -iodotyrosines, α-methyl- dl -iodotyrosines and 3:5-diiodo-4-hydroxyphenyl- dl -lactic acid were poor substrates for the deiodinase. All the other compounds tested remained unchanged including 3:5-diiodo-4-hydroxyphenyl-α guanidyl propionic acid, 3:5-diiodo-4-hydroxyphenyl propionic acid and 3:5-diiodotyramine. The phenol group played a major role since 3-iodo-5-nitro- l -tyrosine and 3-iodo- l -phenylalanine were not decomposed. In conclusion, thyroid and liver deiodinase are highly specific enzymes. It appears probable that the enzymatic site requires the carboxyl, amino and phenol groups of the substrate to exert its activity.Abstract The deiodinating activity of thyroid and liver microsomes was studied with 19 compounds including 3-iodo- l -tyrosine and 3:5-diiodo- l -tyrosine and their analogs. While l -iodotyrosines were almost completely dehalogenated, d -iodotyrosines, α-methyl- dl -iodotyrosines and 3:5-diiodo-4-hydroxyphenyl- dl -lactic acid were poor substrates for the deiodinase. All the other compounds tested remained unchanged including 3:5-diiodo-4-hydroxyphenyl-α guanidyl propionic acid, 3:5-diiodo-4-hydroxyphenyl propionic acid and 3:5-diiodotyramine. The phenol group played a major role since 3-iodo-5-nitro- l -tyrosine and 3-iodo- l -phenylalanine were not decomposed. In conclusion, thyroid and liver deiodinase are highly specific enzymes. It appears probable that the enzymatic site requires the carboxyl, amino and phenol groups of the substrate to exert its activity.

Effects of short- and long-term thyroidectomy on mitochondrial and nuclear activity in adult rat brain

We compared subcellular activities in brain and liver at various times after thyroidectomy. Male S.D. rats were used on days 5, 10 or 60 after surgery. Mitochondrial properties were estimated by determining the respective activities of oxidative phosphorylation, succinate oxidase, succinate and beta-hydroxybutyrate cytochrome c reductase and alpha-glycerophosphate dehydrogenase. Nuclear activity was estimated by measuring the RNA polymerase I activity. In brain, RNA polymerase I activity already declined at 5 days after thyroidectomy, whereas mitochondrial respiratory enzymes decreased significantly only after 60 days. In liver, nuclear RNA polymerase I and mitochondrial enzyme activities were observed to drop simultaneously by the 5th day after thyroid removal. On the other hand, daily T3 s.c. injections, 0.25 microgram/100 g B.W., were given for 10 days to rats immediately after thyroidectomy (10 days Tx) or to chronically hypothyroid rats (60 days Hth). Hormonal treatment either maintained or restored subcellular activities to their normal level, both in brain and liver. These data suggest that the metabolic properties of brain mitochondria are sensitive to thyroid hormones, but that the brain needs less iodothyronines than other organs. The fast reduction of RNA polymerase I by thyroidectomy and its subsequent restoration by T3 suggest that the nuclear activity greatly depends on thyroid status.

Spécificité des hormones thyroïdiennes et de l'hormone de croissance sur les propriétés et la protéinogenèse des particules subcellulaires: I. Rats thyroïdectomisés

Summary Protein biosynthesis is controlled by growth hormone (GH) and iodothyronines, moreover the latter regulate metabolic activity. Growth hormone had little incidence on oxidative phosphorylation. Respiratory control index (RC) and P/O ratios with various respiratory substrates were about the same with liver mitochondria isolated from thyroidectomized animals treated or not with GH. Respiration of thyroidectomized rat liver mitochondria with succinate or β-hydroxybutyrate was lowered in the controlled state, but whereas the P/O ratios were about the same for normal and operated animals, the respiratory control index was increased with β-hydroxybutyrate in hypothyroid rats. It appears that the thyroid hormones affected the three phosphorylation sites differently. Thyroidectomy decreased the biological decay of L-leucine (U-14C). By a double isotope procedure the ratio of 3H- and 14C-leucine was strongly suggestive of a slower protein turnover rate of whole mitochondria and especially inner mitochondrial membrane; differences were much less significant with other subcellular hepatic fractions (nuclei, microsomes, outer mitochondrial membrane and cell sap). These results suggest an action of thyroid hormones on the mitochondrial genome which controls the biosynthesis of some proteins of the inner mitochondrial membrane. The uncoupling effect of iodothyronines may be considered as physiologic and seems linked to the turnover rate of some protein components of the inner mitochondrial membrane.