Cell biology of H2O2 generation in the thyroid: investigation of the control of dual oxidases (DUOX) activity in intact ex vivo thyroid tissue and cell lines.

H2O2 generation by dual oxidase (DUOX) at the apex of thyroid cells is the limiting factor in the oxidation of iodide and the synthesis of thyroid hormones. Its characteristics have been investigated using different in vitro models, from the most physiological thyroid slices to the particulate fraction isolated from transfected DUOX expressing CHO cells. Comparison of the models shows that some positive controls are thyroid specific (TSH) or require the substructure of the in vivo cells (MßCD). Other controls apply to all intact cell models such as the stimulation of the PIP(2) phospholipase C pathway by ATP acting on purinergic receptors, the activation of the Gq protein downstream (NaF), or surrogates of the intracellular signals generated by this cascade (phorbol esters for protein kinase C, Ca(++) ionophore for Ca(++)). Still, other controls, exerted by intracellular Ca(++) or its substitute Mn(++), the intracellular pH, or arachidonate bear directly on the enzyme. Iodide acts at the apical membrane of the cell through an oxidized form, presumably iodohexadecanal. Cooling of the cells to 22°C blocks the activation of the PIP(2) phospholipase C cascade. All these effects are reversible. Their kinetics and concentration-effect characteristics have been defined in the four models. A general scheme of the thyroid signaling pathways regulating this metabolism is proposed. The probes characterized could be applied to other H2O2 producing cells and to pathological material.

TGFbeta1 effects on functional activity of porcine thyroid cells cultured in suspension.

Thyrotropin (TSH) and transforming growth factor beta 1 (TGFbeta1) have major roles in the regulation of folliculogenesis and differentiation in thyroid cells. Isolated porcine thyroid cells cultured in the presence of TSH on a plastic surface recover a follicular architecture and exhibit normal functional properties. The addition of TGFbeta1 to the culture medium induces important morphological changes and extracellular matrix remodelling. Similarly, thyroid cells lose their ability to organify iodine and their responsiveness to adenylate cyclase. The aim of this study was to analyse the influence of TGFbeta1 on the functional activity of thyrocytes in suspension culture, independent of follicle disruption. In this system, we demonstrate that TGFbeta1 inhibits expression of thyroperoxidase, NADPH oxidase activity, iodine uptake and, consequently, iodine organification. Moreover, TGFbeta1 decreases basal and TSH-stimulated cAMP production and TSH receptor expression. Taken together, these data converge to demonstrate an essential role of TGFbeta1 in the regulation of the thyroid cell function.

Expression of reduced nicotinamide adenine dinucleotide phosphate oxidase (ThoX, LNOX, Duox) genes and proteins in human thyroid tissues.

The large homolog of NADPH oxidase flavoprotein LNOX2, and probably LNOX1, are flavoproteins involved in the thyroid H(2)O(2) generator. Western blot analysis of membrane proteins from normal human thyroid, using antipeptide antibodies, indicated that LNOX1,2 are 164-kDa glycoproteins and that N-glycosylated motifs account for at least 10-20 kDa of their total apparent molecular mass. Northern blot analysis of 23 different human tissues demonstrated that LNOX2 messenger RNA (mRNA) is strongly expressed only in the thyroid gland, although blast analysis of expressed sequence tags databases indicated that LNOX genes are also expressed in some nonthyroid cells. We investigated LNOX1,2 gene and protein expressions in normal and pathological human thyroid tissues using real-time kinetic quantitative PCR and antipeptide antibodies, respectively. In normal tissue, LNOX1,2 are localized at the apical pole of thyrocytes. Immunostaining for LNOX1,2 was heterogeneous, inside a given follicle, with 40-60% of positive follicular cells. Among normal and pathological tissues, variations of LNOX1 and LNOX2 mRNA levels were parallel, suggesting a similar regulation of both gene expressions. Whereas LNOX mRNAs seemed slightly affected in benign disease, the expression of protein was highly variable. In multinodular goiters, 40-60% of cells were stained. In hypofunctioning adenomas, LNOX immunostaining was highly variable among follicles, whereas sodium/iodide (Na+/I-) symporter immunostaining was decreased. In hyperfunctioning thyroid tissues, only few cells (0-10%) were weakly stained, whereas sodium/iodide symporter staining was found in the majority of follicular cells. In conclusion, LNOX proteins are new apical glycoproteins with a regulation of expression that differs from other thyroid markers.

Expression of nicotinamide adenine dinucleotide phosphate oxidase flavoprotein DUOX genes and proteins in human papillary and follicular thyroid carcinomas.

Duox2, and probably Duox1 are glycoflavoproteins involved in the thyroid H2O2 generator functionally associated to thyroperoxidase (TPO). We investigated both DUOX1 and DUOX2 gene expressions using quantitative reverse transcription-polymerase chain reaction (RT-PCR) in 47 thyroid carcinomas, including 10 paired normal/tumoral tissues. In carcinomas, variations of DUOX1 and DUOX2 mRNA levels were parallel, indicating that control mechanisms of both gene expressions operate in tumors as well as in normal thyroid tissues; DUOX1 expression was in the normal range in 20, was decreased up to 50-fold in 8, and increased up to 7-fold in 19 samples. DUOX2 expression was in the normal range in 15, was decreased up to 200-fold in 10, and increased up to 5-fold in 22 samples. In the 10 paired samples, variations of DUOX and TPO gene expressions were not correlated. We analyzed Duoxl/2 protein expression in 86 tumor samples using an antipeptide antiserum reacting with both Duox proteins. In normal tissue, Duox proteins are localized at the apical pole of thyrocytes, with 40% to 60% of thyrocytes being stained. In the 86 cancer tissues, immunostaining was absent in 19 samples, was low in 32, and normal or even slightly increased in the other 35 samples. The expression of Duox proteins was related to tumor differentiation, being more frequently found in neoplastic tissues that were able to pick up radioiodine, and in those with a detectable expression of sodium iodide symporter (NIS), pendrin and TPO.

Thyroid oxidase (THOX2) gene expression in the rat thyroid cell line FRTL-5.

A cDNA encoding an NADPH oxidase flavoprotein was isolated from the rat thyroid gland. The predicted 1517-residue polypeptide was 82.5% identical to the human THOX2/DUOX2 and 74% similar to THOX1/DUOX1. Rat THOX2 lacks a stretch of 30 residues, corresponding to one exon in the human gene sequence. THOX2 mRNA was found to be expressed in cultured FRTL-5 cells. The level of THOX2 mRNA was increased by cAMP in these cells and it was decreased in the thyroids of rats treated with the antithyroid drug methimazole, unlike the TPO and NIS mRNAs. Since it was found in the intestine, duodenum, and colon, in addition to thyroid, we suggest that it be called LNOX, the new family of long homologs of NOX flavoproteins rather than THOX and/or DUOX.

Purification of a novel flavoprotein involved in the thyroid NADPH oxidase. Cloning of the porcine and human cdnas.

Hydrogen peroxide is the final electron acceptor for the biosynthesis of thyroid hormone catalyzed by thyroperoxidase at the apical surface of thyrocytes. Pig and human thyroid plasma membrane contain a Ca(2+)-dependent NAD(P)H oxidase that generates H(2)O(2) by transferring electrons from NAD(P)H to molecular oxygen. We purified from pig thyroid plasma membrane a flavoprotein which constitutes the main, if not the sole, component of the thyroid NAD(P)H oxidase. Microsequences permitted the cloning of porcine and human full-length cDNAs encoding, respectively, 1207- and 1210-amino acid proteins with a predicted molecular mass of 138 kDa (p138(Tox)). Human and porcine p138(Tox) have 86.7% identity. The strongest similarity was to a predicted polypeptide encoded by a Caenorhabditis cDNA and with rbohA, a protein involved in the Arabidopsis NADPH oxidase. p138(Tox) shows also similarity to the p65(Mox) and to the gp91(Phox) in their C-terminal region and have consensus sequences for FAD- and NADPH-binding sites. Compared with gp91(Phox), p138(Tox) shows an extended N-terminal containing two EF-hand motifs that may account for its calcium-dependent activity, whereas three of four sequences implicated in the interaction of gp91(Phox) with the p47(Phox) cytosolic factor are absent in p138(Tox). The expression of porcine p138(Tox) mRNA analyzed by Northern blot is specific of thyroid tissue and induced by cyclic AMP showing that p138(Tox) is a differentiation marker of thyrocytes. The gene of human p138(Tox) has been localized on chromosome 15q15.

Biochemical characterization of a Ca2+/NAD(P)H-dependent H2O2 generator in human thyroid tissue.

An NAD(P)H-dependent H2O2 forming activity has been evidenced in thyroid tissue from patients with Grave's disease. Its biochemical properties were compared to those of the NADPH oxidase previously described in pig thyroid gland. Both were Ca2+-dependent and activated by inorganic phosphate anions in the same range of concentrations. Both are flavoproteins using FAD as cofactor, but the human enzyme was also able to utilize FMN. The apparent Km for NADPH of the human enzyme (100 microM) was 5-10 times higher than that of porcine enzyme. Vm was 3 to 10 times higher in pig (150 nmol x h(-1) x mg(-1)) than in man (14 to 45). Total content in human tissue was 7 to 9% of that in porcine tissue. An unidentified inhibitor has been detected in the 3000 g particulate fraction from most patients, which could account for this apparently low enzyme content. An NADH-dependent H2O2 production has also been observed in porcine and human thyroid tissues. This activity was only partly Ca2+-dependent (man, 50-70%; pig, 80-90%) and presented similar apparent Km values for NADH (man, 100 microM; pig, 200 microM). In pig thyrocytes, the expression of the Ca2+-dependent part of the NADH-oxidase activity was induced by TSH and down-regulated by TGFbeta, as was the NADPH oxidase activity. Furthermore, NADPH and NADH-dependent activities were not additive. We conclude that a single, inducible, NAD(P)H-oxidase can use NADPH or NADH as substrate to catalyse H2O2 formation, and that human and porcine NAD(P)H-oxidases are highly similar. Differences observed could be attributed to minor differences in enzyme structure and/or in membrane microenvironment. The NADH-dependent Ca2+-independent activity observed in human and porcine thyroid fractions could be attributed to a distinct and constitutive enzyme.

Regulation of the thyroid NADPH-dependent H2O2 generator by Ca2+: studies with phenylarsine oxide in thyroid plasma membrane.

Pig thyroid plasma membranes contain a Ca(2+)-dependent NADPH:O2 oxidoreductase, the thyroid NADPH-dependent H2O2 generator. This provided the H2O2 for the peroxidase-catalysed synthesis of thyroid hormones. The effect of the tervalent arsenical, phenylarsine oxide (PAO), on the NADPH oxidase was studied. PAO caused two directly related dose-dependent effects with similar half-effect concentrations of PAO (3 nmol of PAO/mg of protein): (i) partial inactivation of H2O2 formation by the Ca(2+)-stimulated enzyme, and (ii) desensitization of the enzyme activity to Ca2+. PAO had no effect on membranes that had been Ca(2+)-desensitized by alpha-chymotrypsin treatment. The NADPH oxidase in membranes treated with excess PAO had the same Vmax with and without Ca2+. This value was half the Vmax of the native enzyme. However, the K(m) for NADPH determined with Ca2+ (18 microM, identical with that of the native enzyme) was approx, one-third of the K(m) measured without Ca2+, showing the direct action of Ca2+ on the PAO-enzyme complex. PAO had the same effects, partial inactivation and Ca2+ desensitization, on the NADPH: ferricyanide oxidoreductase activity of the NADPH oxidase, suggesting that PAO acts on the flavodehydrogenase entity of the enzyme. Both partial inactivation and Ca2+ desensitization were completely and specifically reversed by 2.3-dimercaptopropanol, partly reversed by dithiothreitol and not reversed by 2-mercaptoethanol, indicating that PAO binds to vicinal thiol groups. These results suggest that thiol groups are involved in the control of thyroid NADPH oxidase by Ca2+; PAO bound to vicinal thiols might alter the structure of the enzyme so that electron transfer occurs without Ca2+ but more slowly.

Solubilization and characterization of a thyroid Ca(2+)-dependent and NADPH-dependent K3Fe(CN)6 reductase. Relationship with the NADPH-dependent H2O2-generating system.

The thyroid plasma membrane contains a Ca(2+)-regulated NADPH-dependent H2O2-generating system which provides H2O2 for the thyroid-peroxidase-catalyzed biosynthesis of thyroid hormones. The molecular nature of the membrane-associated electron transport chain that generates H2O2 in the thyroid is unknown, but recent observations indicate that a flavoprotein containing a FAD prosthetic group is involved. Solubilization was reinvestigated using 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (Chaps), Triton X-100, and high salt concentrations. Chaps eliminated about 30% of the proteins, which included a ferricyanide reductase, without affecting the H2O2-generating system. Similarly, Triton X-100 alone did not extract the NADPH oxidase. An NADPH-oxidase activity, which was measured in the presence of the artificial electron acceptor potassium ferricyanide, was solubilized by increasing the ionic strength to 2 M KCl. This NADPH-ferricyanide reductase activity was shown to belong to the H2O2-generating system, although it did not produce H2O2. It was still Ca2+ dependent and H2O2 production was restored by decreasing the ionic strength by overnight dialysis. No H2O2 production activity was detected after sucrose density gradient centrifugation of the dialyzed solubilized enzyme, but a well-defined peak of NADPH oxidation activity with a sedimentation coefficient of 3.71 S was found in the presence of K3Fe(CN)6. These results suggest that some unknown component(s) (phospholipid or protein) is removed during sucrose density gradient centrifugation. Finally, thyrotropin, which induces NADPH oxidase and regulates H2O2 production in porcine thyrocytes in primary culture, also induced the NADPH-K3Fe(CN)6 reductase activity associated with the H2O2-generating system. Thus, this enzyme seems to be another marker of thyroid differentiation.

The Ca2+- and reduced nicotinamide adenine dinucleotide phosphate-dependent hydrogen peroxide generating system is induced by thyrotropin in porcine thyroid cells.

Hydrogen peroxide (H2O2) is an essential electron acceptor for thyroid peroxidase-catalyzed iodination and coupling reactions. In the presence of iodide, its production is a limiting step in thyroid hormone biosynthesis. Several studies have demonstrated that the thyroid particulate fraction contains a Ca2+- and NADPH- dependent H@O@ generator (NADPH-O2:oxidoreductase), the so- called thyroid NADPH-oxidase. It has recently been demonstrated that cellular H2O2 release is under the tonic control of TSH in primary cultures of dog thyrocytes. The present study evaluates the effect of TSH on the thyroid NADPH-oxidase and cytochrome c reductase activities, two enzymes believed to be involved on H2O2 generation in the thyroid gland. There was almost no detectable NADPH-dependent H2O2 generator in the membranes of cells grown for 18 h without TSH. But cells grown in the presence of TSH (0.1 mU/ml) had a CA2+- and NADPH-dependent H2O2-generating activity that increased up to the third day in culture, as did the cell iodide organification capacity. This increase was also partially blocked by 12-O-tetradecanoylphorbol 13-acetate and cycloheximide. Forskolin and 8-bromo-cAMP both reproduced the action of TSH on the Ca2+- and NADPH-dependent H2O2 generator. In contrast, the thyroid NADPH-cytochrome c reductase activity in particles from control cells was similar to that of TSH-treated cells and was unaffected by forskolin or 12-O-tetradecanoylphorbol 13-acetate. These results suggest that NADPH-cytochrome c reductase activity is not regulated by TSH and, thus, reinforce the idea that this enzyme is not involved in thyroid H2O2 generation. On the other hand, the Ca2+- and NADPH-dependent H2O2 generator, so-called thyroid NADPH- oxidase, is induced by TSH through the cAMP cascade. Thus, it seems to be another marker of thyroid differentiation, in addition to thyroperoxidase and thyroglobulin, and could play a key role in thyroid hormone production.

The Ca2+/NADPH-dependent H2O2 generator in thyroid plasma membrane: inhibition by diphenyleneiodonium.

The thyroid plasma membrane contains a Ca(2+)-regulated NADPH-dependent H2O2-generating system which provides H2O2 for the peroxidase-catalysed biosynthesis of thyroid hormones. The electron transfer from NADPH to O2 catalysed by this system was studied by using diphenyleneiodonium (DPI), an inhibitor of flavo- and haemo-proteins. The prosthetic group of the H2O2 generator was removed by incubation with 5 mM CHAPS at 40 degrees C, and an active holoenzyme was reconstituted with FAD, but not with FMN. The H2O2-generating system also had an intrinsic Ca(2+)-dependent NADPH:ferricyanide reductase activity which is probably linked to its flavodehydrogenase component (or domain). Both activities, H2O2 production and ferricyanide reductase activity, were inhibited by DPI, with similar K1/2 (2.5 nmol/mg of protein). DPI only inhibited a system reduced with NADPH in the presence of Ca2+. NADPH could not be replaced by NADP+, NADH or sodium dithionite, suggesting the need for specific mild reduction of a redox centre in a particular conformation. Ferricyanide protected both activities against inhibition by DPI; the NADPH:ferricyanide reductase activity was completely protected at a ferricyanide concentration 20 times lower than that needed to protect the H2O2 formation, implying at least two target sites for DPI. One might be the flavodehydrogenase component; the other was beyond, on the entity which transfers the electrons to O2. This second site has not been identified.

Inhibition of thyroid NADPH-oxidase by 2-iodohexadecanal in a cell-free system.

The major nonpolar iodolipid formed in horse thyroid cells has recently been identified as 2-iodohexadecanal (2-IHDA). We have investigated in vitro the effect of 2-IHDA on the NADPH-oxidase, NADPH-cytochrome c reductase, and thyroid peroxidase (TPO) activities of a porcine thyroid plasma membrane preparation. 2-IHDA inhibited NADPH-oxidase activity, with half-inhibition at 3-5 microM, but it had no effect on NADPH-cytochrome c reductase. It inhibited the TPO-catalyzed iodination of protein, but not iodide oxidation. Hexadecanal also inhibited NADPH-oxidase. Inhibition by the non-iodinated lipid aldehydes depended on the length of their aliphatic chain: dodecanal and tridecanal gave maximal inhibition. Free iodide, 2-iodohexadecanol and palmitic acid all had no inhibitory effect. Washing treated membranes showed that the inhibition of NADPH-oxidase by hexadecanal was fully reversible, whereas that of 2-IHDA and other iodinated or brominated alkanals was irreversible. Thus the interaction between some residues of the thyroid NADPH-oxidase and the lipid aldehyde groups was favored or stabilized by the iodine atom. Modification of primary amine and thiol groups of NADPH-oxidase inhibited its activity. These groups could also be the target of lipid aldehydes. We suggest that 2-IHDA, because it inhibits TPO and more profoundly the H2O2-generating system in thyroid plasma membrane, modulates iodide metabolism in the thyrocyte and may mediate the Wolff-Chaikoff effect.

Activation of the NADPH-dependent H2O2-generating system in pig thyroid particulate fraction by limited proteolysis and Zn2+ treatment.

The NADPH-dependent H2O2-generating system in a pig thyroid particulate fraction requires micromolar concentrations of Ca2+ for activity. The H2O2 generator could be Ca(2+)-desensitized (i.e. made fully active in the absence of Ca2+) by limited proteolysis with alpha-chymotrypsin or by treatment with ZnCl2. The Zn2+ effect was temperature- and dose-dependent with an apparent half-maximum concentration of 0.15 mM at 40 degrees C. Ca2+ desensitization was not reversed by adding the Zn2+ chelators, 1,10-phenanthroline and EGTA, but about one-third of the Ca(2+)-sensitivity was recovered after addition of 10 mM-dithiothreitol. The proteolysed enzyme and the Zn(2+)-treated enzyme had different Km values for NADPH. The Zn2+ effect did not seem to involve proteolysis or membrane fusion. These results indicate that Ca2+ regulation occurs via an autoinhibitory domain or inhibitory protein component of the H2O2-generator system. Its inhibitory effect may be removed by proteolysis or conformational changes, making the catalytic site accessible to the substrate NADPH and/or enabling electrons to be transferred from NADPH to O2.

Mechanism of NADPH oxidation catalyzed by horse-radish peroxidase and 2,4-diacetyl-[2H]heme-substituted horse-radish peroxidase.

The mechanism of NADPH oxidation catalyzed by horse-radish peroxidase (HRP) and 2,4-diacetyl-[2H]heme-substituted horse-radish peroxidase (DHRP) was studied. The roles of the different H2O2/peroxidase compounds were examined by spectral studies. The oxidized NADPH species were identified using the superoxide dismutase effect and by measuring the stoichiometry between NADPH oxidized and H2O2 used. In the presence of a mediating molecule, like scopoletin, both enzymes acted via a similar mechanism, producing only NADP degrees, which in turn reacted with O2 producing O2-. Consequently H2O2 was completely regenerated in the presence of superoxide dismutase and partially regenerated in its absence. In the absence of a mediating molecule, the H2O2 complex of both enzymes (compound I) catalysed NADPH oxidation by single-electron transfer, producing NADP degrees; compound II of these enzymes catalyzed NADPH oxidation more slowly by a direct two-electron transfer, producing NADPH+. There were difference between HRP and DHRP. HRP compound II was produced by the oxidation of 1 mol NADPH/mole compound I, while DHRP compound II was formed by the spontaneous conversion of compound I to compound II. The NADPH oxidation catalyzed by DHRP compound I did not lead to the formation of compound II. When H2O2 was produced slowly by the glucose/glucose-oxidase system, compound II was never formed and a pure O2- adduct of DHRP (compound III) accumulated.

Mechanism of hydrogen peroxide formation catalyzed by NADPH oxidase in thyroid plasma membrane.

The thyroid plasma membrane contains a Ca2(+)-regulated NADPH-dependent H2O2 generating system which provides H2O2 for the thyroid peroxidase-catalyzed biosynthesis of thyroid hormones. The plasma membrane fraction contains a Ca2(+)-independent cytochrome c reductase activity which is not inhibited by superoxide dismutase. But it is not known whether H2O2 is produced directly from molecular oxygen (O2) or formed via dismutation of super-oxide anion (O2-). Indirect evidence from electron scavenger studies indicate that the H2O2 generating system does not liberate O2-, but studies using the modified peroxidase, diacetyldeuteroheme horseradish peroxidase, to detect O2- indicate that H2O2 is provided via the dismutation of O2-. The present results provide indirect evidence that the cytochrome c reductase activity is not a component of the NADPH-dependent H2O2 generator, since it was removed by washing the plasma membranes with 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid without affecting H2O2 generation. Spectral studies with diacetyldeuteroheme-substituted horseradish peroxidase showed that the thyroid NADPH-dependent H2O2 generator does not catalyze superoxide anion formation. The O2- adduct compound (compound III) was formed but was completely inhibited by catalase, indicating that the initial product was H2O2. The rate of NADPH oxidation also increased in the presence of diacetylheme peroxidase. This increase was blocked by catalase and was greatly enhanced by superoxide dismutase. The O2- adduct compound (compound III) was produced in the presence of NADPH when glucose-glucose oxidase (which does not produce O2-) was used as the H2O2 generator. NADPH oxidation occurred simultaneously and was enhanced by superoxide dismutase. We conclude that O2- formation occurs in the presence of an H2O2 generator, diacetylheme peroxidase and NADPH, but that it is not the primary product of the H2O2 generator. We suggest that O2- formation results from oxidation of NADPH, catalyzed by the diacetylheme peroxidase compound I, producing NADP degree, which in turn reacts with O2 to give O2-.

Nonenzymatic NADPH-dependent reduction of 2,6-dichlorophenol-indophenol.

The reduction of 2,6-dichloroindophenol (DCIP) by direct interaction with NADPH was studied. The results indicate that reduction proceeds via a direct electron transfer from NADPH to DCIP, with no oxygen consumption, and a rate constant of k = 4.69 M-1.s-1. The reduced DCIP can rapidly transfer its electrons to potassium ferricyanide (K3Fe(CN)6) or ferricytochrome c, but not to nitro blue tetrazolium. Superoxide dismutase inhibits DCIP reduction in an oxygen-dependent manner by favoring the reoxidation of the reduced DCIP. We therefore conclude DCIP is not suitable for detecting O2- when the nucleotides NADH or NADPH are present.

NADPH-dependent H2O2 generation catalyzed by thyroid plasma membranes. Studies with electron scavengers.

Hog thyroid plasma membrane preparations containing a Ca2+-regulated NADPH-dependent H2O2-generating system were studied. The Ca2+-dependent reductase activities of ferricytochrome c, 2,6-dichloroindophenol, nitroblue tetrazolium, and potassium ferricyanide were tested and the effect of these scavengers on H2O2 formation, NADPH oxidation and O2 consumption were measured, with the following results. 1. Thyroid plasma membrane Ca2+-independent cytochrome c reduction was not catalyzed by the NADPH-dependent H2O2-generating system. This activity was superoxide-dismutase-insensitive. 2. Of the three other electron scavengers tested, only K3Fe(CN)6 was clearly, but partially reduced in a Ca2+-dependent manner. 3. Though the NADPH-dependent reduction of nitroblue tetrazolium was very low and superoxide-dismutase-insensitive, nitroblue tetrazolium inhibited O2 consumption, H2O2 formation and NADPH oxidation, indicating that nitroblue tetrazolium inhibits the H2O2-generating system. We conclude that the thyroid plasma membrane H2O2-generating system does not or liberate O2- and that Ca2+ controls the first step (NADPH oxidation) of the H2O2-generating system.

Ca2+ regulation of thyroid NADPH-dependent H2O2 generation.

A thyroid particulate fraction contains an NADPH-dependent H2O2-generating enzyme which requires Ca2+ for activity. A Chaps solubilized extract of the thyroid particulate fraction partially purified by DEAE chromatography did not show a dependence on Ca2+ for activity. Preincubation of the particulate fraction with Ca2+ yielded a preparation insensitive to Ca2+. The non-particulate fraction obtained after incubation of the particles in the presence of Ca2+ was able to inhibit, in the presence of EGTA, the Ca2+-desensitized particulate fraction and the enzyme isolated on DEAE. It is concluded that the reversible Ca2+ activation of the NADPH-dependent H2O2 generation was modulated in porcine thyroid tissue by (a) calcium-releasable inhibitor protein(s).

Requirement of a compartmentalization for NADPH oxidizing site and peroxidase-H2O2 in the thyroid iodinating system.

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.

Solubilization and characteristics of the thyroid NADPH-dependent H2O2 generating system.

Solubilization of the thyroid particulate-associated NADPH-dependent H2O2 generating system has been tested with different detergents; (3-(3-cholamidopropyl)-dimethylammonio)1-propane sulfonate (CHAPS) was found to be the best of the six detergents tested. The ratio of H2O2 generation to NADPH oxidation was similar for CHAPS extract and native particulate material. CHAPS was also the only detergent able to preserve the Ca++-sensitivity of the NADPH oxidase. Solubilization of this enzyme allowed the determination of some of its characteristics: specificity for divalent cations, apparent Km for NADPH, optimum pH and sensitivity to SH- reagents.