Odette Michel

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.

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 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.

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.

Influence de la thyroïdectomie sur les mécanismes des 11β et 21-hydroxylations dans la corticosurrénale de rat

Summary Oxygen consumption rate of rat adrenal cortex mitochondria with malate-isocitrate as substrate, was unchanged after thyroidectomy in the presence or absence of ADP. Respiratory chain enzymes located in the inner mitochondrial membrane had similar activities in normal and thyroidectomized rats. When ADP was replaced by deoxycorticosterone, 11β-hydroxylation rate was markedly lowered after thyroidectomy, probably on account of a decrease of NADPH biosynthesis via mitochondrial malic enzyme. Among the outer membrane enzymes, it was not possible to detect any kynurenine hydroxylase activity whereas thyroidectomy decreased rotenone-insensitive cytochrome c reductase and had no effect on monoamine oxydase activity. The 21-hydroxylation rate of 17α-hydroxyprogesterone was slowed with adrenal cortex microsomes isolated from thyroidectomized animals, probably as a consequence of a lower utilization of added NADPH. On the other hand, microsomal NADPH cytochrome c reductase exhibited a decreased activity after thyroidectomy.

Sur le métabolisme du sulfoconjugué de la 3,5,3′-triiodothyronine chez le rat

Abstract 1. 1. The comparative metabolism of 3,5,3′-triiodothyronine (T 3 ) and of its sulfoconjugate (ST 3 ) have been studied in the thyroidectomized Rat. The former is degraded more rapidly than the latter, which the cells concentrate much less intensely. 2. 2. The different aspects of ST 3 metabolism, among others the formation in liver of the glycuroconjugate of T 3 from it, the excretion of iodide after its injection, the fact that it exists in plasma but not in urine, all lead to the supposition that it is partially hydrolyzed by some arylsulfatases in tissues and that its hormone moiety is deiodinated. 3. 3. ST 3 probably plays the part of a storage form of T 3 which the cells can use after hydrolysis of the ester bond.

Oxidative phosphorylation reactions and cholesterol hydroxylation mechanisms in human term placental mitochondria

Abstract A simple and rapid method is described for the preparation of mitochondria of human term placenta. Phosphorylative oxidation properties were determined by polarography. Respiratory control values were higher than 3, showing that mitochondria are tightly coupled, but ADP/O ratios were lower than theoretical values due to ATPase activity. The activity of several specific mitochondrial marker enzymes was measured. In the outer membrane, rotenone-insensitive NADH-cytochrome c reductase and monoamine oxidase exhibited activities of 331 nmol of cytochrome c reduced/min/mg protein and 26.7 nmol of tyramine consumed/min/mg protein respectively. No kynurenine hydroxylase activity was detected. In the inner membrane, activities of succinate cytochrome c reductase and cytochrome oxidase were 15.3 nmol of cytochrome c reduced/min/mg protein and 21.6 nmol O2 utilized/min/mg protein respectively. Glutamate, malate and succinate oxidases had low activities of about 1 to 3 nmol O2 used/min/mg protein. In the matrix, glutamate dehydrogenase activity was very slight. Mitochondrial proteins were analyzed by polyacrylamide gel electrophoresis in the presence of phenol, urea and acetic arid. Mitochondria contained at least 20 components with mol. wt. ranging from 15,000 to 150,000. A component which probably contained cytochrome P450 subunits was characterized with a mol. wt. of 52,000. The concentration of the phosphorylating respiratory chain cytochromes was determined; the latter were in a similar range to that reported for adrenal cortex mitochondria, but their concentration was lower. The content of cytochrome P450 was lower than in other endocrine tissues. Oxygen consumption rate was stimulated by addition of (20α)-hydroxycholesterol and (22R)-hydroxycholesterol but not by (22S)-hydroxycholesterol. The placental cholesterol hydroxylating mechanism appeared to follow a pathway similar to that of adrenal cortex mitochondria.

Electrophoretic analysis and cytochrome P450 contents of bovine adrenal cortex mitochondria and of their subfractions

Abstract Adrenal cortex mitochondria were fractionated into outer membrane and inner membrane. This second membrane was further separated into water-soluble proteins, KCl extractable proteins, “structural proteins” and structured cytochromes. Cytochrome P 450located in the inner membrane was found only in the structured cytochrome fraction. The protein components of all the fractions were analyzed by sodium dodecyl sulphate polyacrylamide gel electrophoresis. Mitochondria contain at least 20 components ranging in molecular weight from 10,000–120,000 daltons. The outer membrane is different from the inner membrane both in number of components and size distribution. In structured cytochrome proteins a component of 52,000 molecular weight represents about 25% of the whole protein contents. This corresponds to the subunit of cytochrome P450. Proteins synthesized in isolated adrenal cortex mitochondria in the presence of radioactive leucine reveal six groups of labelled polypeptides when subjected to polyacrylamide gel electrophoresis. The molecular weights of these proteins ranged from 10,000–54,000 daltons. In the fraction of structured cytochromes a labelled component having a molecular weight 52,000 daltons was found. It is thought that this component may correspond to the subunit of cytochrome P 450 and be coded partly by mitochondrial nucleic acids.

Effect of thyroidectomy on pregnenolone and progesterone biosynthesis in rat adrenal cortex

The effects of thyroidectomy on pregnenolone and progesterone biosynthesis were investigated in rat adrenal cortex mitochondria and microsomes. The sequential hydroxylations of cholesterol, and the latters side-chain cleavage, which constitute the limiting step in steroidogenesis, were studied by measuring oxygen consumption rates in the presence of cholesterol derivatives. The addition of (20R)-hydroxycholesterol, (22R)-hydroxycholesterol or (20R,22R)-dihydroxycholesterol stimulated these rates, but addition of cholesterol or (22S)-hydroxycholesterol had little effect. Thyroidectomy significantly reduced oxygen consumption rates in the presence of the sterols by about 30%. Oxygen uptake was small in the presence of respiratory inhibitors; the addition of sterols raised this uptake but subsequent thyroidectomy did not change it. Rat adrenal cortex mitochondria and microsomes converted pregnenolone ino progesterone through 3 beta ol dehydrogenase/delta 4-5 isomerase, in two different successive steps. Values for enzyme activities were 0.18, 0.26 and 0.81 nmol progesterone/min/mg protein for the overall complex, the 3 beta ol dehydrogenase and the delta 4-5 isomerase respectively. All enzyme activities were unchanged by thyroidectomy. Similar results were obtained for corresponding microsomal activities whose values were in the same range. For both microsomes and mitochondria, the dehydrogenase reaction was the limiting step in the enzyme reaction leading to progesterone formation from pregnenolone. The limiting step in corticosteroidogenesis leading to prenenolone formation by an NADPH-dependent step was slowed down by thyroidectomy, probably because the reaction that transfers energy from NADH to NADP was inhibited. The enzyme complex leading to progesterone which involves NAD+ as cofactor, was unchanged by thyroidectomy. Thyroid hormones may therefore affect the availability of the energy mechanisms connected with the proton motive force, since thyroidectomy reduces both the phosphorylative oxidation and energy-dependent hydroxylation reactions involved in steroidogenesis.