Purified Thyroid Peroxidase Research Articles

Cellular thyroid peroxidase (TPO), unlike purified TPO and adjuvant, induces antibodies in mice that resemble autoantibodies in human autoimmune thyroid disease.

Autoantibodies to several protein antigens in human autoimmunity interact with a restricted range of epitopes, whereas diverse epitopes are recognized by antibodies induced in animals using antigen and adjuvant. To examine the basis for this difference, we compared the qualitative nature of antibodies developing in AKR/N mice injected with purified thyroid peroxidase (TPO) and adjuvant or with TPO expressed on major histocompatibility complex (MHC) class II+ fibroblasts. Mice injected with purified TPO had higher TPO antibody levels than TPO+/class II+ fibroblast-treated mice. Despite lower titers, recipients of TPO+/class II+ cells developed very high affinity antibodies (Kd = approximately 10(-10) M), comparable with those of human TPO autoantibodies and about 10-fold higher than those in purified TPO plus adjuvant-immunized mice. Moreover, more than 90% of TPO antibodies in TPO+/class II+ fibroblast-injected mice, compared with only approximately 50% in TPO plus adjuvant-immunized mice, were to the immunodominant region recognized by patients' autoantibodies. Consistent with this epitopic restriction, TPO+/class II+ fibroblast-injected mice had TPO antibody epitopic fingerprints similar to those of human autoantibodies. In conclusion, mice injected with TPO+/class II+ fibroblasts, but not those injected with purified TPO and adjuvant, develop antibodies closely resembling autoantibodies in human disease. These observations indicate that some animal models based on conventional immunization may not be representative of human diseases with a major humoral component.

Thyroid peroxidase: kinetics, pH optima and substrate dependency.

The oxidation of iodide, guaiacol and 2,2'-azino-di[3-ethyl-benzthiazoline-(6)-sulphonic acid] and the iodination of tyrosyl residues in bovine serum albumin, catalysed by partly purified thyroid peroxidase, were studied. The enzyme showed pH optima with all electron donors. With the exception of guaiacol, the position of the pH optima depended upon both the electron donor and hydrogen peroxide concentrations. With increased hydrogen peroxide concentrations the optima shifted to lower pH, and with increased iodide concentration to higher pH. For monoiodotyrosine (MIT) formation in bovine serum albumin the position of the pH optimum was also dependent on the hydrogen peroxide concentrations. The position of the pH optimum of the oxidation of guaiacol was pH 9 and independent of substrate and hydrogen peroxide concentrations. It is obvious from these findings that iodination reactions must be studied under well-defined conditions.

Thyroid peroxidase/microsomal antibodies are not identical with thyroid cytotoxic antibodies in autoimmune thyroiditis

Cytotoxic activity in sera of patients with Hashimoto's thyroiditis was measured with an antibody-dependent cell-mediated cytotoxicity assay. Cytotoxicity was determined in a 51chromium release assay using human thyroid cell targets incubated with heat-inactivated serum or IgG from patients with Hashimoto's thyroiditis. Effector cells were obtained from peripheral mononuclear cells of normal subjects. Cytotoxicity was significantly increased in patients with Hashimoto's thyroiditis (median specific lysis 20.2%, range 2.1-58.8) compared with normals (median specific lysis 8.1%, range 0-19.5; p less than 0.00001). The amount of percent specific lysis did not correlate with the titres of microsomal antibodies determined by different methods: passive hemagglutination technique (r = 0.2), enzyme immunoassay with microsomal antigen (r = 0.16), and radioimmunoassay for thyroid peroxidase antibody (r = 0.02). The cytotoxic activity was located in the IgG fraction, both in microsomal antibody positive and negative sera. After pre-incubation of microsomal antibody/thyroid peroxidase antibody positive or negative sera with purified thyroid peroxidase followed by analysis in the antibody-dependent cell-mediated cytotoxicity assay, cytotoxicity decreased in only 2 cases but was unchanged in the remaining sera. Western blot experiments with solubilized thyroid membranes and immunoblotting with cytotoxic-positive/microsomal antibody negative sera showed no binding to thyroid peroxidase. Our data suggest that cytotoxicity in sera from patients with Hashimoto's thyroiditis is not mediated by antibodies against thyroid peroxidase, but by antibodies not yet identified.

Studies with Purified Human Thyroid Peroxidase and Thyroid Microsomal Autoantibodies

We have isolated highly purified thyroid peroxidase (TPO) from human thyroid tissue to study further the relationship between TPO and the thyroid microsomal antigen that elicits the production of microsomal autoantibodies in patients with autoimmune thyroid disease. Serum samples were obtained from 24 patients with suspected autoimmune thyroid disease, and from 7 normal subjects. Microsomal autoantibodies in the patient sera, as determined by the microsomal hemagglutination assay (MCHA), varied between 1:100 and 1:102,400. Antithyroglobulin antibodies, however, were very low (less than 1:100). Binding of serum autoantibodies to purified human TPO, as determined by enzyme-linked immunosorbent assay, correlated fairly well with MCHA titers (r = 0.72; P less than 0.001). An immunoblot procedure was developed to study the binding of serum antibodies to the major active fragment of TPO (93 kDa), after sodium dodecyl sulfate-polyacrylamide gel electrophoresis under both reducing and nonreducing conditions. Binding under both conditions correlated very well with MCHA titers (r = 0.80-0.84; P less than 0.001). Studies were performed to determine the inhibitory effect of patient serum on the enzymatic activity of purified human TPO. A marked inhibitory effect on guaiacol activity was observed when TPO was preincubated with as little as 10 microL high titer serum. There was a significant correlation (r = 0.47; P less than 0.01) between MCHA titer and inhibitory effect. The addition of 2 micrograms purified human TPO completely or almost completely inhibited the binding of serum antibodies to thyroid microsomes (enzyme-linked immunosorbent assay) in 10 of 11 patient sera with high MCHA titers (1:25,600 or greater).

Metabolism of35S- and14C-Labeled Propylthiouracil in a Model in Vitro System Containing Thyroid Peroxidase*

In previous communications we described an in vitro model system containing highly purified thyroid peroxidase (TPO) for studying the mechanism of inhibition of thyroid hormone biosynthesis by the antithyroid drugs, 6-propylthiouracil (PTU) and 1-methyl-2-mercaptoimidazole (MMI). We showed that inhibition of iodination of thyroglobulin in this system may be reversible or irreversible depending on the relative concentrations of iodide and drug and the TPO concentration. Metabolism of the drugs occurred under both conditions, but was more limited under irreversible conditions of inhibition. It was of interest to examine the nature of the drug metabolites associated with reversible and irreversible conditions of inhibition. For this purpose we have employed the 35S- and 14C-labeled drugs and a recently developed reverse phase HPLC procedure. Results of a similar study with MMI were reported in an earlier communication. In the present study we report our findings with PTU. Under conditions of reversible inhibition, PTU was readily metabolized and by 15 min was reduced to a few percent of the starting value. The earliest detectable metabolite with both [35S]- and [14C]PTU was the disulfide, which reached a peak in about 15 min and then slowly declined. Coincident with the decline in the disulfide was the appearance of more polar metabolites. In the case of [35S]PTU, these corresponded to sulfate/sulfite, PTU sulfonate, and a product tentatively identified as PTU sulfinate. The latter two were also observed as 14C-labeled metabolites produced from [14C]PTU. Two nonpolar desulfurated 14C-labeled metabolites were also observed. Surprisingly, these did not correspond to either propyluracil or propyldeoxyuracil, the anticipated most likely products of PTU desulfuration. The identity of these desulfurated metabolites of PTU in the TPO model system remains to be determined. Under conditions of irreversible inhibition of iodination, a relatively small fraction of PTU was metabolized. PTU disulfide was, again, the earliest detectable metabolite, and it declined with time. However, only small amounts of other metabolites were observed, in contrast to the results obtained under conditions of reversible inhibition of iodination. As in the case of MMI, the difference in metabolic pattern between reversible and irreversible conditions is primarily related to the rapid inactivation of TPO that occurs under irreversible conditions. In general, the metabolism of PTU by the TPO model system resembled that previously observed with MMI. With both drugs, the disulfide was the earliest detectable metabolite, and under conditions of reversible inhibition of iodination, an appreciable fraction of the sulfur was oxidized as far as sulfate/sulfite.(ABSTRACT TRUNCATED AT 400 WORDS)

Interaction of highly purified thyroid peroxidase with anti-microsomal antibodies in autoimmune thyroid diseases.

Among the several antigens involved in autoimmune thyroid diseases, the microsomal antigen has been recently identified as the human thyroid peroxidase (TPO). The availability of highly purified TPO and anti-TPO monoclonal antibody (mAb) allowed us to study in more details anti-TPO autoimmune antibodies (aAb) and their relationship with anti-microsomal aAb. Only sera with anti-microsomal aAb, as assayed by passive hemagglutination, highly immunoprecipitated purified TPO; anti-thyroglobulin aAb did not contribute to this effect. IgG binding to TPO and inhibition of mAb binding to TPO were also observed using anti-microsomal positive and anti-thyroglobulin negative sera. The correlation between anti-TPO and anti-microsomal aAb titers was found significant (p less than 0.02). These data suggest that anti-microsomal and anti-TPO immunoreactivity are presented by the same aAb. It could not be ruled out, however, that some of the anti-microsomal aAb detected by passive hemagglutination could be directed against antigens different from TPO. The use of highly purified TPO and anti-TPO mAb would allow large scale studies of anti-TPO aAb, necessary for investigating their pathological significance in thyroid disorders.

Detection of a catalytic intermediate of peroxidase in hog thyroid microsomes

A catalytic intermediate, Compound II of peroxidase was detected spectrophotometrically in thyroid microsomes. From comparison with the spectral data on purified thyroid peroxidase, the content of the peroxidase was estimated to be 0.019 nmol per mg of the microsomal protein, being about one-eighths of the amount of cytochrome b5. It was concluded that thyroid peroxidase exhibits the same peroxidase activity for guaiacol or ascorbate in the free and the microsome-bound forms.

Mechanism of action of thioureylene antithyroid drugs: factors affecting intrathyroidal metabolism of propylthiouracil and methimazole in rats.

Experiments were performed with rats to test the physiological significance of a previously proposed mechanism of action of thioureylene antithyroid drugs, which had been derived from results obtained with a model system containing purified thyroid peroxidase. Two features of the previously proposed scheme were tested:1) the effects of drug dosage and 2) the effects of iodine deficiency. In the dosage experiments, rats were injected with graded doses of [35S]PTU (0.18-59 mumol or [35S]mmi (0.16-18 mumol). Thyroid glands were removed 1 and 6--8 h later and 35S distribution in the homogenates was determined by paper chromatography. Serum samples were also analyzed by the same procedure. From the measured 35S activity in the various components and from the known specific activity of the injected drugs, it was possible to calculate thyroidal concentrations of unchanged drug and drug metabolites. At low doses, thyroidal concentrations of unchanged 6-propyl-2-thiouracil (PTU) and 1-methyl-2-mercaptoimidazole (MM) significantly exceeded their concentrations in serum, as reported by previous investigators. A major new finding in the present investigation was the observation that intrathyroidal metabolism of PTU and MMI is greatly affected by dosage. Marked inhibition of intrathyroidal drug metabolism was observed at 6-8 when the dosage was increased from 5.9 to 18 mumol for [35S]PTU and from 0.88 to 2.2 mumol for [35S]MMI (per 200 g rat). These findings demonstrate that with increasing dosage, PTU and MMI inhibit their own intrathyroidal metabolism. These dosage effects are similar to results previously reported for in vitro oxidation of PTU and MMI by the thyroid peroxidase system, and they offer support for the physiological significance of the previously proposed scheme. Further evidence for the physiological validity of this scheme was obtained in the experiments with iodine-deficient rats. As predicted from the in vitro findings, intrathyroidal metabolism of [35S]PTU and [35S]MMI was markedly reduced in rats on low iodine diet.

Dissociation between degree of iodination and iodoamino acid distribution in thyroglobulin.

The iodoamino acid distribution in thyroglobulin is known to be largely determined by the degree of iodination of the protein. Data presented in this communication indicate that the degree of iodination is not the sole determinant of iodoamino acid distribution. This was shown in two different incubation systems employing purified thyroid peroxidase, which is known to catalyze both iodination and coupling in thyroglobulin. In one system, iodination and coupling were measured simultaneously. In the other, coupling alone was measured in the absence of iodination. Formation of both T3 and T4 was markedly stimulated in both systems by the addition of very low concentrations of free diiodotyrosine (DIT; 0.1–1.0 /IM), without a significant change in the degree of iodination. The increase in T3 and T4 was demonstrated both with 131I-labeling and with RIA techniques. Formation of T3 was preferentially increased, especially at lower levels of iodination, resulting in an increase in the T3:T4 ratio. Experiments with [14C]DIT indicated that the added DIT was not itself converted to iodothyronines. Its mode of action, therefore, must be through stimulation of intramolecular coupling, involving iodotyrosyl residues within the thyroglobulin polypeptide chains. Although the physiological significance of the in vitro observations with DIT has not been established, the present findings demonstrate that factors other than the degree of iodination of thyroglobulin can markedly affect its hormonal content. It seems likely, therefore, that similar factors may exist in vivo which can modulate the thyroid hormone content of thyroglobulin at a given level of iodination.

Interaction of thyroid peroxidase with concanavalin A covalently coupled to agarose.

We have investigated the interaction between concanavalin A-agarose (Con A-agarose) and thyroid peroxidase, an integral membrane protein found in the 105,000 X g, 1-h particulate fraction of thyroid tissue. An intact form of porcine thyroid peroxidase was obtained by solubilization with the nonionic detergent Triton X-100 and two fragmented, hydrophilic forms of the enzyme were prepared by trypsin treatment of the membrane. The three types of thyroid peroxidase bind to Con A-agarose and can be eluted with alpha-methyl-D-mannoside. The alpha-methyl-D-mannoside eluate of the most purified thyroid peroxidase preparation has been analyzed by polyacrylamide gel electrophoresis. Peroxidase activity corresponds with a glycoprotein band. The binding of thyroid peroxidase to Con A-agarose can be inhibited by sugars in the following order: alpha-methyl-D-mannoside greater than D-mannose greater than alpha-methyl-D-glucoside greater than D-glucose greater than D-galactose. This order of specificity is typical of Con A-sugar interactions. Furthermore, inactivation of the carbohydrate binding site of Con A by demetallization greatly reduces the extent of thyroid peroxidase binding. Reactivation of the carbohydrate binding site by the addition of Ca2+ and Mn2+ to demetallized Con A-agarose restores thyroid peroxidase binding. These and other experiments suggest that htyroid peroxidase is, like several other peroxidases, a glycoprotein. In addition, the interaction between thyroid peroxidase and Con A-agarose may provide a new purification tool for thyroid peroxidase.

The mechanism of action of the thioureylene antithyroid drugs.

A model incubation system containing purified thyroid peroxidase (TPO) was used to study the mechanism of action of the thioureylene anti-thyroid drugs--propylthiouracil (PTU), methylmercapto imidazole (MMI) and carbimazole. Two general types of experiments were performed: a) measurement of the inhibitory effects of the drugs on TPO-catalyzed iodination and on TPO-catalyzed oxidation of guaiacol, and b) studies of the metabolism of PTU and MMI by the TPO model system. The major observations can be summarized as follows: 1) The thioureylene drugs are potent inhibitors of TPO-catalyzed iodination of protein and tyrosine. Their potency increases greatly as the concentration of I- decreases. 2) The thioureylene drugs are also potent inhibitors of TPO-catalyzed oxidation of guaiacol, a reaction that does not involve iodide. 3) MMI and PTU are readily oxidized in the model incubation system when iodide is present but not in the absence of iodide. The rate of oxidation increased as the iodide concentration was increased from 10 to 100 muM. 4) Oxidation of PTU and MMI by the model incubation system is inhibited by relatively slight increases in the concentration of PTU and MMI. These drugs are capable of inhibiting their own and each other's metabolism. 5) Inhibition of iodination is competitively antagonized by iodide at low drug concentrations, but not at higher drug concentrations. 6) Inhibition of iodination by MMI and PTU may be either reversible (low ratio of drug to iodide), or irreversible (higher ratio of drug to iodide). In reversible inhibition the iodination is inhibited for a period which may be as brief as 2 min or as long as 20 min, but thereafter, iodination begins, and there is escape from inhibition. During the lag-period there is extensive metabolism of the drug. In the case of irreversible inhibition of iodination is inhibited completely or almost completely for 60 min, and drug oxidation during this period is relatively low. 7) Irreversible inhibition may be transformed into reversible inhibition by increasing the concentration of TPO or the concentration of iodide. However, increasing the concentration of H2O2 or of tyrosine does not overcome irreversible inhibition. On the basis of these findings and of current views concerning the mechanism of enzymatic iodination, a scheme is proposed for the mechanism of inhibition by thioureylene drugs of TPO-catalyzed iodination of protein and tyrosine.

Evidence for a catalytic role for thyroid peroxidase in the conversion of diiodotyrosine to thyroxine.

When thyroglobulin and other proteins are incubated with iodide in the presence of purified thyroid peroxidase and glucose plus glucose oxidase, the iodotyrosines MIT and DIT are formed by iodination of tyrosyl residues, and significant amounts of T4 are also produced. Formation of T4 is greatest when goiter thyroglobulin is the iodine acceptor. The present study was designed to determine whether thyroid peroxidase plays a catalytic role in the coupling reaction to form T4, or whether it serves only to form the precursor of T4, diiodotyrosine, which might then couple non-enzymatically to form T4. Two lines of evidence are presented in support of the view that thyroid peroxidase plays a role in T4 formation beyond that of simply providing the DIT precursor for the coupling reaction. In one group of experiments, thyroglobulin, casein, and fibrinogen were iodinated chemically with molecular iodine and enzymatically with thyroid peroxidase. At any given level of iodination the number of DIT and MIT residues p...

Purification and Iodinating Activity of Hog Thyroid Peroxidase

Abstract 1. A peroxidase was purified from the particulate fraction of hog thyroid tissue by solubilization with trypsin, chromatography on diethylaminoethyl cellulose, and filtration through Biogel P-100. The final product, although not homogeneous, represented about a 400-fold purification over the starting particulate fraction. 2. The molecular weight of thyroid peroxidase was estimated by gel filtration studies. Elution volumes of proteins of known molecular weight were compared with the elution volume of the peroxidase, the latter being localized by its enzyme activity. The average molecular weight in three determinations was 64,000. 3. An absorption maximum was observed at 410 mµ, suggestive of a hemoprotein. The progressive increase in A410:A280 during the purification procedure suggested that the enzyme activity was associated with a hemoprotein. Inhibitor studies provided further support for this view. 4. When supplemented with H2O2 or, more effectively, with the H2O2-generating system, glucose-glucose oxidase, purified thyroid peroxidase readily catalyzed the iodination of protein, free tyrosine, or free monoiodotyrosine. Iodination was catalyzed over a wide range of iodide concentrations, from l10-7 m to g5 x 10-3 m. In an incubation system containing 1.2 µg of peroxidase, 0.5 mg of purified thyroglobulin, 1.0 mg of glucose, 0.83 µg of glucose oxidase, and I- varying from 50 µm to 4 mm, in 1.0 ml of 0.05 m phosphate buffer, pH 7.0, Vmax was 1.2 mµmoles of I- per min per µg of enzyme protein and Km was 2.5 x 10-4 m I-. 5. No consistent effect of pH on iodination was observed in the range, pH 5 to 7 (the only range tested). Iodination occurred about as readily at pH 5 as at pH 7. 6. During the peroxidase-catalyzed iodination of thyroglobulin or other protein, iodide was rapidly bound as 3-monoiodotyrosine, 3,5-diiodotyrosine, and thyroxine. The best yields of thyroxine were obtained with thyroglobulin as acceptor. The formation of 3',3,5-triiodothyronine was not observed. 7. The iodination of protein by thyroid peroxidase was markedly inhibited by low concentrations of antithyroid drugs. This was true for compounds of the aromatic inhibitor group—resorcinol, p-aminobenzoic acid, sulfathiazole, and others—as well as those of the thioamide group—thiourea, thiouracil, and 1-methyl-2-mercaptoimidazole. Glutathione and thiocyanate were also effective inhibitors, but perchlorate was not. Inhibition by 1-methyl-2-mercaptoimidazole and by thiocyanate was competitive with iodide under certain conditions. 8. Cyanides, and especially azide, were potent inhibitors of thyroid peroxidase, supporting the view that enzyme activity was associated with a hemoprotein. 9. At pH 5.0, iodination of thyroglobulin or of bovine serum albumin with thyroid peroxidase was inhibited at concentrations of I- greater than 1 x 10-4 m. This suggests a possible mechanism for the antithyroid action of excess iodide. However, for reasons which are not yet clear, this inhibition was not observed at pH 7.0. 10. Results obtained with purified thyroid peroxidase support the view that it is unnecessary to postulate the existence of a separate tyrosine iodinase in the thyroid gland. Moreover, the findings suggest that a peroxidase may be involved in the coupling reaction as well as in iodination of protein in the thyroid. 11. No evidence was obtained that 3,5-diiodo-4-hydroxyphenylpyruvic acid is an intermediate in thyroxine formation during the iodination of thyroglobulin with thyroid peroxidase. The results of this study favor the view that the coupling of diiodotyrosine to form thyroxine occurs within the matrix of the protein molecule.