Jacob Bouhnik

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.

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 the protein content of rat adrenal cortex mitochondria and microsomes.

Abstract The effects of thyroidectomy on the protein content and properties of adrenorcortical mitochondria and microsomes were investigated in the rat. Thyroidectomy caused a drop in the cytochrome P450 concentration in both paniculate fractions. Polyacrylamidc get electrophoresis of mitochondrial and microsomal proteins showed a reduction in the protein constituent with a molecular weight of 52,000 daltons. This constituent corresponded to the cytochrome P450 subunit. Isolated adrenal cortex mitochondria from thyroidectomized rats incorporated 30% less leucine than normal mitochondria. Electrophoresis of labelled proteins showed no change in radioactivity distribution. In microsomes, the rate of 21-hydroxylase activity dropped by 30% after thyroidectomy. Although these effects were similar to those observed after dexamethasone treatment, there was no doubt that they were caused by the absence of thyroid hormones, since no reduction was found in the plasma ACTH concentration in thyroidectomized animals. The drop in the content of cytochrome P450 in adrenal cortex mitochondria and microsomes could explain the diminished hydroxylating activity of these organelles.

Effets de la thyroïdectomie du rat sur la structure et les fonctions des mitochondries des muscles squelettiques

Summary Mitochondria used in the present study were isolated from skeletal muscle of normal and thyroidectomized rats. The preparations were controlled by electron microscopy. It was not possible to find any morphological change induced by thyroidectomy, nevertheless, some differences appeared in the cytochrome contents which were slightly decreased. Oxygen consumption rates of thyroidectomized rat mitochondria were decreased when the particles were maintained in states 3 and 4 in the presence of various substrates, but the P/O ratios were not modified. The activities of mitochondrial enzymes were in general slightly affected by thyroidectomy except for glycerol-1-phosphate cytochrome c reductase and NADH rotenone sensitive cytochrome c reductase which were decreased and for glutamate dehydrogenase activity which was increased. The tRNA nucleotidyltransferase activity found in the mitochondrial matrix was not influenced by the absence of thyroid secretion. Normal rat muscle mitochondria incorporate 14C-leucine with an artificial ATP-generating system or with a respiratory substrate. The amino acid incorporation was decreased by thyroidectomy. Muscle mitochondria analyzed by polyacrylamide gel electrophoresis contained more than 30 protein components with MW ranging from 10.000 to 135.000. Thyroidectomy lowered the amount of a fraction of about 54.000 MW. It is not impossible that all the data observed in the absence of thyroid secretion are in relation with changes induced in the mitochondrial genome as previously shown in mitochondria isolated from liver of thyroidectomized rats.

Ultrastructural localization of wheat germ agglutinin binding sites on the membranes of rat liver mitochondria

Using ferritin-conjugated wheat germ agglutinin it was demonstrated that the cytoplasmic side of the outer membrane of isolated rat liver mitochondria was almost completely devoid of N -acetylglucosamine-like residues. However, by incubating digitonin-derived mitoplasts, these carbohydrate moieties were found to be heavily exposed on the outer side of the entire surface of the inner mitochondrial membrane, and probably on the inner side of the outer membrane. These ultrastructural findings confirm the presence of carbohydrates on the mitochondrial membranes and agree with a general asymmetrical structure of the biomembranes which corresponds to that of the plasma membrane.

Specificite des hormones thyroidiennes et de l'hormone de croissance sur les proprietes et la proteinogenese des particules subcellulaires. II. Souris genetiquement naines 'dwarf'.

Summary Experiments were undertaken in order to compare the action of GH and thyroid hormones on a genetically dwarf mouse which is a mutant whose adenohypophysis is almost entirely devoid of growth hormone (GH) and thyroid stimulating hormone (TSH). The Mg++-activated DNA-dependent RNA polymerase reaction of liver nuclei was about 50 p. cent less in dwarf than in normal (same strain) mice. Single or multiple administrations of GH stimulated the RNA polymerase ; effect of 3,5,3′-triiodo-L-thyronine (T3) was less pronounced. Probably the two hormones affected two different sites of RNA polymerase, their effects being additive and complementary. L-thyroxine induced swelling of liver mitochondria isolated from normal and dwarf mice. The respiratory control index and the P/O ratios were similar. Hypothyroidism due to the lack of TSH secretion seemed responsible for a decreased turnover rate, determined by a double isotope procedure, of liver subcellular fractions especially in mitochondria. Differences were much less significant with kidney proteins. Growth hormone had small effects on protein catabolism. Chronic treatement of dwarf mice with GH has little incidence on turnover rate whereas T3 increased it markedly, especially with fast turnover rate fractions. GH stimulated early incorporation of L-leucine when the radioactive amino acid was supplemented with a large amount of carrier. In conclusion it appears that GH stimulates protein synthesis at the molecular level by a direct or indirect action on RNA polymerase and probably by an increase of the intracellular aminoacid pool. Thyroid hormones seem to affect especially the biological degradation of subcellular components mainly those of mitochondria.

Influence du diiodo-3,5-hydroxy-4-benzoate de n-butyle sur les fonctions des mitochondries hepatiques et corticosurrenaliennes de rat

Abstract The effect of n -butyl-3,5-diiodo-4-hydroxybenzoate (DIBB), considered to be an antithyroxine drug, on the oxidative phosphorylation of mitochondria isolated from rat liver and adrenal cortex was studied. In the presence of liver mitochondria the iodinated compound inhibited state 3 oxidation, stimulated state 4 oxidation and lowered P O ratio when β-hydroxybutyrate, succinate, glutamate-malate and malate-isocitrate were used as substrates. The Lineweaver-Burk plot of DIBB inhibition of the rate of O 2 consumption with variable concentrations of substrates, in the presence of ADP, produced straight lines. The inhibition was non-competitive, with succinate and competitive, with β-hydroxybutyrate. The swelling of liver mitochondria induced by DIBB was faster and more pronounced than with thyroxine at similar concentrations. Oxygen consumption by adrenal cortex mitochondria was activated by DIBB at low concentrations in respiratory states 3 and 4. When DOC replaced ADP the 11β-hydroxylation rate was decreased by the iodinated product. It is possible that there are at least two distinct sites at which DIBB can inhibit respiration; one site being probably located in the respiratory chain close to the entry of dehydrogenases and a second site in relation with the coupling of high-energy intermediates. The effects of DIBB on oxidative phosphorylation of rat mitochondria which are similar in some aspects to those observed with thyroid hormones could explain its antithyroxine activity.