Biomedical subjects
Publications and source records attributed to D Deme.
In this study, it is shown that NADPH iodination occurs in a thyroid peroxidase-H2O2 system in presence of thyroglobulin, the normal iodination substrate. Previous data suggested that thyroid H2O2 generation is a NADPH-dependent system. Present results support the concept of a compartmentalization of the sites of NADPH oxidation and peroxidasic iodination.
The action of Ca2+ on H2O2 formation catalyzed by an NADPH-dependent H2O2-generating system was studied in a thyroid particulate fraction. It was shown that H2O2 formation was inhibited by EGTA and restored in the presence of micromolar concentrations of free Ca2+, and that Mg2+ was unable to restore the activity of the EGTA-inhibited particulate fraction. Consequently, NADPH-dependent H2O2 formation requires Ca2+, and the Ca2+ stimulation of this process is specific and reversible.
We have examined the respective roles played by guaiacol and scopoletin in NADPH oxidation catalyzed by the peroxidase/H2O2 system. It was shown that NADPH was not oxidized by either the horseradish or lactoperoxidase/H2O2 systems alone; oxidation occurred immediately after the addition of guaiacol or scopoletin. In both cases, the oxidation product was enzymatically active NADP+. Differences were observed in the NADPH oxidation mechanism depending on whether guaiacol or scopoletin was the mediator molecule. In guaiacol-mediated NADPH oxidation, the stoichiometry between H2O2 and oxidized NADPH was about 1; superoxide dismutase did not affect the oxidation rate. In scopoletin-mediated oxidation, the stoichiometry was much higher (1:14 in the present experiments); superoxide dismutase considerably increased the oxidation rate. It is concluded that catalysis of NADPH oxidation by the horse radish peroxidase/H2O2 system requires the presence of a mediator molecule. The NADPH oxidation mechanism depends on the intermediary oxidation state of this molecule.
Oxidation of NADPH catalyzed by the peroxidase/H2O2 system is known to require the presence of mediating molecules. Using either lactoperoxidase or horseradish peroxidase, we demonstrated that in the peroxidase/H2O2 system, NADPH oxidation was mediated by iodide. The oxidation product was the iodinated NADP. This product was shown to possess spectral characteristics different from those of NADP+ and NADPH, since for iodinated NADP, increased absorbance was observed in the 280-nm region and was directly proportional to the rate of iodination. It is suggested that oxidation and iodination of NADPH proceed via a single reaction between the intermediary iodide oxidation species and NADPH. Experiments with different molecules of NADPH analogues indicated that iodination occurred in the nicotinamide part of the NADPH molecule.
Cytochrome c peroxidase (CcP) and horseradish peroxidase (HRP), when combined with a stoichiometric amount of H2O2, form stable compounds I which are known as FeIV Ro and FeIV o pi + structures, respectively. These compounds were assayed in the catalysis of thyroid hormone synthesis and the iodination reaction. As previously shown for the lactoperoxidase FeIV Ro compound, the CcP FeIV Ro compound was involved in the coupling and not in the iodination reactions. In contrast, the HRP FeIV o pi + compound catalyzed both iodination and hormone formation. The possible role of the different peroxidase-H2O2 compounds in the two sequential reactions, thyroglobulin iodination and thyroid hormone formation, is discussed.
In thyroid gland, iodination takes place on the apical plasma membrane and requires the presence of the thyroid peroxidase and H2O2 generating system. H2O2 generation and NBT (nitro blue tetrazolium) reductase activity (both of which are NADPH-dependent) as well as peroxidase activity were compared for their respective orientations in membrane vesicles. The possible role of NADPH-NBT reductase activity in H2O2 generation was also examined. Results favor the conclusion that thyroid peroxidase is oriented towards the luminal side of the vesicles, whereas the NADPH site of NADPH oxidase-dependent H2O2 generation is located on the external side of the same or of different vesicles. Furthermore, it is shown that different NADPH-NBT reductase activities are present on both the outer and inner surfaces of the membrane vesicles, and that none of these activities is able to produce either H2O2 or O-2. The idea that a multi-component complex is involved in H2O2 generation is discussed, and a model is proposed which takes into account the possible spatial separation of the thyroid peroxidase site from the NADPH site of this H2O2 generation system on the apical membrane of the thyrocyte.
The titration by ferrocyanide and the localization of the oxidizing equivalents of lactoperoxidase "compound II" were studied as a function of pH. It was demonstrated that 1) whatever the pH, the structure of lactoperoxidase "compound II" was compatible with a Fe IV R degree state, 2) at acidic pH, ferrocyanide preferentially reduced the oxidizing equivalent localized on the heme iron to give an Fe III R degree compound, 3) at pH 4.2 only the Fe III R degree form was obtained after reduction of lactoperoxidase "compound II" with one mole of ferrocyanide and whereas at pH greater than 4.2, a mixture of both Fe III R degree and Fe IV R forms was present, 4) lowering the pH from 7.2 to 4.0 induced a transition of Fe IV R state to Fe III R degree state, but increasing the pH from 4.0 to 7.2 did not permit the formation of Fe IV R compound from Fe III R degree compound.
A NADPH-dependent H2O2 generating system associated with a thyroid particular fraction is described. H2O2 is measured by two different methods: iodination of NADPH itself when the system is supplemented with lactoperoxidase and [125I]iodide, and by the scopoletin method. It is shown that: H2O2 generation is inhibited by catalase and is dependent on NADPH or particulate protein concentration; radical scavengers of OH and of singlet oxygen have no effect while superoxide dismutase has only a marginal effect; disruption of the particular fraction by phospholipase A2 or digitonin treatment completely abolished H2O2 generation activity while thyroid peroxidase activity appears, suggesting different sites for the two activities in the membrane vesicles.
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The effect of a pseudohalide, SCN-, an anion with the same molecular size as iodide, was studied on two reactions: thyroglobulin iodination and thyroid hormone synthesis (coupling reaction) catalyzed by peroxidases. The coupling reaction was studied separately from the iodination reaction by using labelled thyroglobulin samples previously iodinated but containing little or no hormones. 1. SCN- inhibits iodide oxidation (I- leads to I2) whatever the enzyme, thyroid, lactoperoxidase or horseradish peroxidase. The amount of SCN- required to completely inhibit this reaction varies depending on the enzyme. Similarly tyrosine iodination is inhibited by SCN- with large variations, depending on the peroxidase, in the concentration of this anion required for inhibition. 2. In contrast SCN- stimulates the coupling reaction: (a) this affect is seen with the thyroid and lactoperoxidases but not with horseradish peroxidase; (b) the concentration of SCN- required for half-maximal stimulation of the coupling reaction is much lower (0.5-1 microM) than that required for the inhibition of iodide oxidation (60-80 microM); (c) ClO4(-), an anion with the same molecular size as SCN- and I-, has no effect on the coupling reaction; (d) this stimulatory effect of SCN- does not depend on a modification of the thyroglobulin molecule since it is not seen with horseradish peroxidase or in purely chemical coupling conditions. 3. The stimulatory effect of SCN- is therefore seen as resulting from the binding of this anion to a limited number of high-affinity sites present at the surface of both thyroid and lactoperoxidases. The inhibitory effect depends, in contrast, on the binding of SCN- to the substrate site with lower affinities. Since iodide also behaves both as a substrate for the iodination reaction and as a stimulatory ligand for the coupling reaction, these data provide further support in favour of the existence of an enzyme-iodide (or SCN-) complex with catalytic properties different from those of the native peroxidase.
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3,5,3'-triiodotyronine formation was studied in vitro after iodination of goiter thyroglobulin catalyzed by purified thyroid peroxidase. A fractionnal number of T3 is always obtained per mole of thyroglobulin in both in vitro and in vivo. This result cannot be explained by a heterogeneity in thyroglobulin iodination or in a partial conversion of T3 to T4. It is suggested that thyroglobulin is heterogenous either in its primary sequence or in its teritiary configuration. Thyroglobulin contains tyrosine residues which are specific for the T3 formation and moreover T3 is not the precursor of T4. The efficiency of T3 formation was studied: the maximal number of T3 molecules is obtained with 30 iodine atoms per mole of thyroglobulin. In addition the results suggest that the tyrosines which are coupled with a high efficiency are iodinated sequentially.
Almost non-iodinated human goiter thyroglobulin has been iodinated in vitro by thyroid peroxidase to levels as high as 75 iodine atoms per mol of protein. The following results were obtained. 1. The iodine distribution obtained in vitro with human thyroglobulin strongly ressembles that obtained in vivo for rat thyroglobulin. Thus the distribution of iodine seems to depend essentially on the structure of thyroglobulin and on the reactivity of the different tyrosine residues. 2. Although the number of hormone residues increased with iodination the highest efficiency of hormone synthesis was obtained in a very narrow range of iodination: in vitro (40%) between 25 and 30 iodine atoms, and in vivo (48%) between 10 and 20 atoms. This result suggests that the tyrosines which are coupled with a high efficiency are iodinated sequentially. 3. Maximal thyroxine content was found to be lower than approximately 3 mol/mol of thyroglobulin. This result might mean that the two 12-S subunits of thyroglobulin are not identical and that one of them is able to produce 2 mol of hormone while the second only 1 mol.
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