Inflammatory disease in HLA-B27 transgenic rats.

UNLABELLED: A spontaneous inflammatory disease in rats transgenic for HLA-B27 resembles the B27-associated human spondyloarthropathies. Colitis and arthritis, the two most important features, require T cells, gut bacteria, and high expression of B27 in bone marrow-derived cells. Control rats with HLA-B7 remain healthy. Most rats with HLA-Cw6 (associated with psoriasis vulgaris) remain healthy; a minority develop mild and transient disease. Rats with a mutant B27 with a Cys67-->Ser substitution resemble wild-type B27 transgenics, but with a lower prevalence of arthritis. A similar phenotype is seen in B27 rats co-expressing a viral peptide that binds B27. Disease-prone LEW but not F344 B27 rats develop high serum IgA levels concurrent with disease progression. Colitis is associated with high interferon-gamma, arthritis with high interleukin-6. Disease is similar in B27 LEW, F344, and PVG rats, but the DA background is protective. CONCLUSIONS: The spondyloarthropathy-like disease in rats is specific for HLA-B27 but does not require Cys67. Arthritis but not colitis is particularly sensitive to B27 peptide-binding specificity. Genetic background exerts a strong influence, but some phenotypic differences exist between permissive strains that do not influence disease susceptibility. The data favor a role for B27 peptide presentation in arthritis, but other mechanisms to explain the role of B27 have not been excluded.

The specificity of peptides bound to human histocompatibility leukocyte antigen (HLA)-B27 influences the prevalence of arthritis in HLA-B27 transgenic rats.

Human histocompatibility leukocyte antigen B27 is highly associated with the rheumatic diseases termed spondyloarthropathies, but the mechanism is not known. B27 transgenic rats develop a spontaneous disease resembling the human spondyloarthropathies that includes arthritis and colitis. To investigate whether this disease requires the binding of specific peptides to B27, we made a minigene construct in which a peptide from influenza nucleoprotein, NP383-391 (SRYWAIRTR), which binds B27 with high affinity, is targeted directly to the ER by the signal peptide of the adenovirus E3/gp19 protein. Rats transgenic for this minigene, NP1, were made and bred with B27 rats. The production of the NP383-391 peptide in B27(+)NP1(+) rats was confirmed immunologically and by mass spectrometry. The NP1 product displaced approximately 90% of the 3H-Arg-labeled endogenous peptide fraction in B27(+)NP1(+) spleen cells. Male B27(+)NP1(+) rats had a significantly reduced prevalence of arthritis, compared with B27(+)NP- males or B27(+) males with a control construct, NP2, whereas colitis was not significantly affected by the NP1 transgene. These findings support the hypothesis that B27-related arthritis requires binding of a specific peptide or set of peptides to B27, and they demonstrate a method for efficient transgenic targeting of peptides to the ER.

Selectivity in tyrosyl iodination sites in human thyroglobulin.

Previous studies indicate that when low iodine thyroglobulin (Tg) is iodinated enzymatically with thyroid peroxidase (TPO), the tyrosyl residues that are used for the formation of thyroid hormone (hormonogenic sites) are selected for early iodination. The aim of the present study was to assess the relative importance of the substrate (Tg) and the enzyme (TPO) in the selection of the early tyrosyl sites that undergo iodination. For this purpose, low iodine human Tg (2.0 atoms I per 660,000 dimer) was iodinated chemically with (125)I-(3) and enzymatically with TPO + 125I- to a matched low level of iodination (approximately 8 added I atoms per molecule). After reduction and alkylation, the two Tg preparations were digested with trypsin, and the tryptic digests were separated by reverse-phase HPLC into 10 125I-containing pools. Each pool was further fractionated by HPLC to provide purified 125I-peptides suitable for sequence analysis. From the sequence information and the known amino acid sequence of Tg, it was possible to define the location of the iodinated tyrosyl residues. Surprisingly, almost identical results were obtained with chemically and enzymatically iodinated Tg. Not only were the 125I-peptide maps very similar, but all of the recovered 125I in the purified peptides from both samples was located in only three different tyrosyl sites, 5, 2553, and 2520. Tyr 5 and Tyr 2553 are well-established sites of thyroxine formation, while Tyr 2520 has previously been proposed by us to be a donor site. Our observation that the same hormonogenic tyrosyl sites are iodinated by chemical as well as enzymatic iodination indicates that preferential iodination of hormonogenic sites is dependent primarily on the native structure of Tg. TPO plays a minor role, if any, in the selection of early tyrosyl iodination sites in Tg. Consistent with this conclusion was our finding that chemical iodination, as well as enzymatic iodination, led to formation of uniformly iodinated Tg, as determined by isopycnic centrifugation in rubidium chloride. However, we observed a slightly higher diiodotyrosine (DIT) content and a correspondingly lower monoiodotyrosine content in enzymatically iodinated Tg, compared to matched chemically iodinated Tg. This was not observed with two other proteins, bovine serum albumin and trypsinogen, or with free tyrosine, as substrates for iodination. The same preferential formation of DIT in Tg was, however, observed when lactoperoxidase was substituted for TPO. Preferential formation of DIT, therefore, appears to involve interaction between Tg and the peroxidase.

Mechanism of simultaneous iodination and coupling catalyzed by thyroid peroxidase.

Thyroid peroxidase (TPO) simultaneously catalyzes two very different types of reaction in the thyroid gland- iodination and coupling. The present study addresses the mechanism of this simultaneous dual activity. Compound I, the two-electron oxidation product of TPO, exists in two different forms--an oxoferryl porphyrin pi-cation radical and an oxoferryl protein radical. It has been proposed that iodination is mediated by the porphyrin pi-cation radical form of TPO compound I, while coupling is mediated by the protein radical form. However, results obtained in the present study favor the view that both iodination and coupling are mediated by the porphyrin pi-cation radical form of compound I. In the first part of the study, we compared coupling and iodination activities of two peroxidases with very similar crystal structures--cytochrome c peroxidase (CcP) and lignin peroxidase (LiP). Although these two peroxidases have very similar three-dimensional structures, CcP forms a compound I only of the protein radical type, whereas compound I of LiP exists only as a porphyrin pi-cation radical. Comparison of the catalytic activities of the two enzymes showed that diiodotyrosine (DIT)-stimulated coupling activity of LiP was significantly greater than that of CcP. Moreover, lignin peroxidase displayed very significant iodinating activity at acid pHs, whereas iodination with CcP was negligible at all pHs tested. Our findings with these two structurally similar peroxidases suggested that TPO-catalyzed iodination and coupling could both be mediated by the porphyrin pi-cation radical form of compound I. More direct evidence in support of this view was obtained in the second part of this study, employing TPO and lactoperoxidase (LPO) model systems in which iodination and coupling occurred simultaneously. Heme spectral analysis was used to correlate formation of the protein radical form of compound I with the kinetics of the iodination and coupling reactions. Formation of the compound I protein radical was not observed until the iodination and coupling reactions had almost been completed. In separate experiments it was shown that the spontaneous conversion of the porphyrin pi-cation radical form of TPO or LPO compound I to the protein radical form was markedly inhibited by a low concentration of iodide, especially in the presence of an iodide acceptor. These studies provide compelling evidence that both iodination and coupling are mediated by the porphyrin pi-cation radical form of compound I. This was further substantiated by the finding that coupling was inhibited in the presence of excess iodide, an observation readily explained by competition between iodide and DIT residues in thyroglobulin for oxidation by the porphyrin pi-cation radical.

Minocycline and the thyroid: antithyroid effects of the drug, and the role of thyroid peroxidase in minocycline-induced black pigmentation of the gland.

Minocycline (MN), a member of the tetracycline family of antibiotics, is known to induce a black discoloration of the thyroid in several species, including humans. Antithyroid effects of MN have also been reported. The aim of the present study was two-fold: (1) to determine whether thyroid peroxidase (TPO) is involved in the MN-induced black thyroid, and (2) to obtain information on the effect of MN on TPO-catalyzed iodination and coupling in model systems containing highly purified TPO. Treatment of MN with TPO in the presence of the H2O2 generating system, glucose-glucose oxidase, resulted in the formation of a black product (or products). In phosphate buffer, pH 7.0, the color intensity reached its peak in about 90 min. Control samples without TPO showed little or no color change during this interval. Formation of the black product(s) did not require the presence of iodide. Other members of the tetracycline family were not oxidized to dark products by the TPO system. These results provide definitive evidence that TPO is involved in the MN-induced black thyroid. MN is an inhibitor of TPO-catalyzed iodination in model systems, with a potency comparable to that of MMI and PTU. At low drug concentrations (approximately 25 microM), MN appeared to act as a competitive inhibitor, as previously shown for lower concentrations of MMI and PTU. However, when the drug concentration was increased, MN and the thioureylene drugs inhibited iodination by different mechanisms. With PTU and MMI, iodination was irreversibly inhibited through inactivation of TPO. However, inhibition of iodination by MN (100 microM) was not associated with inactivation of TPO and was at least partially reversible. The most potent inhibitory effect of MN was on TPO-catalyzed coupling. This was demonstrated both in a coupling test system, designed to measure coupling in the absence of iodination, and in an iodination system, in which iodination and coupling occurred simultaneously. In both systems, MN was several times more potent than PTU and MMI, or other tetracycline drugs. Based on the potent antithyroid effects of MN observed in our in vitro studies, it seems advisable to monitor thyroid function in patients receiving long-term MN therapy.

The selenium analog of 6-propylthiouracil. Measurement of its inhibitory effect on type I iodothyronine deiodinase and of its antithyroid activity.

6-Propylthiouracil (PTU), a widely used antithyroid drug for the treatment of Graves' disease, is also a potent inhibitor of Type I iodothyronine deiodinase (ID-1). Inhibition of ID-1 was attributed initially to the formation of a mixed disulfide between PTU and a putative cysteine residue at the active site. It has been demonstrated recently that ID-1 is a selenium-containing enzyme, with selenocysteine, rather than cysteine, at the active site. It seemed possible, therefore, that the selenium analog of PTU (PSeU) might be a more potent inhibitor of ID-1 than PTU. To test this possibility, we developed a procedure for the synthesis of PSeU, and we compared PSeU and PTU as inhibitors of ID-1 in a test system containing 125I-rT3, rat liver microsomes, and dithiothreitol. Deiodinase activity was measured by the increase in 125I-iodide. PTU and PSeU were tested at 0.1, 0.3, 1 and 3 microM. Based on results of four separate experiments, the drugs were essentially equipotent as inhibitors of ID-1, although statistical analysis suggested that PSeU may be slightly more potent than PTU. PTU and PSeU were also compared for antithyroid activity in vivo and in vitro. As inhibitors of the catalytic activity of thyroid peroxidase (TPO), the two drugs were essentially equipotent in iodination and guaiacol assays involving measurements made shortly after the addition of H2O2. However, in in vivo experiments with rats, PSeU showed no appreciable inhibition of organic iodine formation in the thyroid, whereas PTU, as expected, was a potent inhibitor. The lack of inhibition of organic iodine formation in vivo by PSeU suggests that, unlike PTU, it is not concentrated by the thyroid gland. In an iodination system in which H2O2 was generated by glucose-glucose oxidase, both PTU and PSeU, when present at 10 microM, acted as reversible inhibitors of iodination. However, when the drug concentration was raised to 50 microM, TPO was inactivated and iodination was irreversibly inhibited. These results suggest that PTU and PSeU inhibit TPO-catalyzed iodination by similar mechanisms. Under the same conditions, the selenium analog of methimazole (another widely used antithyroid drug) does not inactivate TPO. It acts primarily as a reversible inhibitor of TPO-catalyzed iodination.

Evidence for a radical mechanism in peroxidase-catalyzed coupling. II. Single turnover experiments with horseradish peroxidase.

Single turnover experiments were performed with horseradish peroxidase (HRP) to study the mechanism of peroxidase-catalyzed coupling and its stimulation by low concentrations of free diiodotyrosine (DIT). HRP was used because, unlike thyroid peroxidase (TPO) and lactoperoxidase (LPO), the spectral properties of compounds I and II are readily distinguishable. This made it possible to correlate the kinetics and stoichiometry of T4 + T3 formation with spectral data. Incubation of 2 microM preformed HRP-I with 2 microM [125I]Tg (thyroglobulin of low hormone content, high iodotyrosine content) in the presence of 1 microM free DIT yielded about 0.8 residue T4 and 0.2 residue T3 per molecule of Tg. This represents the theoretical maximum for iodothyronine formation, indicating remarkably efficient use of the oxidizing equivalents in HRP-I for coupling. The time course for formation of T4 + T3 was biphasic. During a rapid initial phase (about 1 min), HRP-I was completely converted to HRP-II, coincident with the formation of about 0.65 residues of T4 + T3. During the second slower phase, lasting 10-15 min, HRP-II was completely reduced to the native enzyme, with formation of the remaining T4 + T3. In the absence of DIT, the coupling yield was reduced to 0.5-0.6 residue T4 + T3 per molecule Tg, and the reaction, although considerably slower, was still biphasic. The rapid phase again corresponded to the conversion of HRP-I to HRP-II, and the slower phase to the conversion of HRP-II to native enzyme. To gain insight into the mechanism of the stimulatory effect of free DIT on coupling, we studied the reaction of DIT with HRP-I and HRP-II. Free DIT reacted with both HRP-I and HRP-II in one-electron transfer reactions, and the time course for these reductions resembled those observed with DIT + Tg. These observations suggest that in DIT-stimulated coupling, free DIT radicals act as a shuttle for transferring oxidizing equivalents from the peroxidase intermediates to the DIT residues in Tg. The remarkable efficiency of the HRP-I-mediated coupling reaction implies that (i) only hormonogenic residues in Tg are oxidized and (ii) oxidation of two hormonogenic residues occurs within the same molecule of Tg. A scheme which attempts to explain both kinetic and stoichiometric features of the coupling reaction observed in this study is proposed. This scheme is based on a radical mechanism, consistent with the conclusions reached in the companion paper.

The selenium analog of methimazole. Measurement of its inhibitory effect on type I 5'-deiodinase and of its antithyroid activity.

Methimazole (MMI), unlike propylthiouracil (PTU) is a poor inhibitor of type I iodothyronine deiodinase (ID-1). Inhibition of the enzyme by PTU was attributed initially to formation of a mixed disulfide between PTU and a cysteine residue at the active site. Presumably, MMI was unable to form a stable mixed disulfide and thus did not inhibit the enzyme. However, it has been demonstrated recently that ID-1 is a selenium-containing enzyme, with selenocysteine, rather than cysteine, at the active site. This observation raised the possibility that the selenium analog of MMI, methyl selenoimidazole (MSeI), might be a better inhibitor of ID-1 than MMI itself, as formation of the Se-Se bond with the enzyme would be expected to occur more readily than formation of the S-SE bond. To test this possibility, we developed a procedure for the synthesis of MSeI and compared MSeI with MMI and PTU for inhibition of ID-1 and for antithyroid activity. For inhibition of ID-1, MMI and MSeI were tested at concentrations of 10-300 microM. No significant inhibition was observed with MMI. MSeI showed slight but significant inhibition only in the 100-300 microM range. PTU, on the other hand, showed marked inhibition at 1 microM. Thus, replacement of the sulfur in MMI with selenium only marginally increases its inhibitory effect on ID-1. As an inhibitor of ID-1, MSeI is much less than 1% as potent as PTU. MMI and MSeI were also compared for antithyroid activity, both in vivo and in vitro. As an inhibitor of the catalytic activity of thyroid peroxidase, MMI was 4-5 times more potent than MSeI in a guaiacol assay, but only twice as potent in an iodination assay. In in vivo experiments with rats, MMI was at least 50 times more potent than MSeI in inhibiting thyroidal organic iodine formation. The relatively low potency of MSeI in vivo suggests that it is much less well concentrated by the thyroid than in MMI.

An unexpected side reaction in the guaiacol assay for peroxidase.

In routine guaiacol assays for thyroid peroxidase and lactoperoxidase employing a newly purchased bottle of guaiacol from Aldrich Chemical Co., we were surprised to find the formation of a blue color instead of the expected amber color classically associated with this assay. This was observed also with horseradish, myelo-, and cytochrome c peroxidase. The blue color (Amax approximately 650 nm) was not formed with guaiacol reagents obtained from two other chemical companies, nor was it seen with a bottle of old Aldrich guaiacol that had been in use in the laboratory for more than 10 years. In the present investigation we provide evidence that formation of the blue color is closely associated with the presence of a low concentration of catechol (approximately 0.5 mol%) in the new Aldrich guaiacol reagent. Catechol itself, even in much higher concentration, is a very weak donor for peroxidase, forming a light pink color. The blue color in Aldrich new guaiacol is not formed to the exclusion of 470-nm-absorbing product(s). Formation of the latter is, however, inhibited, and use of Aldrich new guaiacol for assay leads to low values for peroxidase activity. Other dihydroxyphenols (resorcinol and hydroquinone) do not mimic the action of catechol in formation of the blue color. Resorcinol is a very potent inhibitor of peroxidation of guaiacol. Possible schemes are proposed for formation of the products that may be associated with the amber and blue colors.

Myeloperoxidase-catalyzed iodination and coupling.

Myeloperoxidase (MPO), which displays considerable amino acid sequence homology with thyroid peroxidase (TPO) and lactoperoxidase (LPO), was tested for its ability to catalyze iodination of thyroglobulin and coupling of two diiodotyrosyl residues within thyroglobulin to form thyroxine. After 1 min of incubation in a system containing goiter thyroglobulin, I-, and H2O2, the pH optimum of MPO-catalyzed iodination was markedly acidic (approximately 4.0), compared to LPO (approximately 5.4) and TPO (approximately 6.6). The presence of 0.1 N Cl- or Br- shifted the pH optimum for MPO to about 5.4 but had little or no effect on TPO- or LPO-catalyzed iodination. At pH 5.4, 0.1 N Cl- and 0.1 N Br- had a marked stimulatory effect on MPO-catalyzed iodination. At pH 4.0, however, iodinating activity of MPO was almost completely inhibited by 0.1 N Cl- or Br-. Inhibition of chlorinating activity of MPO by Cl- at pH 4.0 has been previously described. When iodination of goiter thyroglobulin was performed with MPO plus the H2O2 generating system, glucose-glucose oxidase, at pH 7.0, the iodinating activity was markedly increased by 0.1 N Cl-. Under these conditions iodination and thyroxine formation were comparable to values observed with TPO. MPO and TPO were also compared for coupling activity in a system that measures coupling of diiodotyrosyl residues in thyroglobulin in the absence of iodination. MPO displayed very significant coupling activity, and, like TPO, this activity was stimulated by a low concentration of free diiodotyrosine (1 microM). The thioureylene drugs, propylthiouracil and methimazole, inhibited MPO-catalyzed iodination both reversibly and irreversibly, in a manner similar to that previously described for TPO-catalyzed iodination.

Peroxidase-catalyzed bromination of tyrosine, thyroglobulin, and bovine serum albumin: comparison of thyroid peroxidase and lactoperoxidase.

A recent paper (Buchberger, W., 1988, J. Chromatogr. 432, 57) on lactoperoxidase-catalyzed bromination of tyrosine and thyroglobulin stated, without evidence, that thyroid peroxidase (TPO) is able to use bromide as a substrate. This was in disagreement with unpublished experiments previously performed in this laboratory, and we undertook, therefore, to examine this subject further. Highly purified porcine TPO was compared with lactoperoxidase (LPO) and chloroperoxidase (CPO) for ability to catalyze bromination of tyrosine, thyroglobulin, and bovine serum albumin (BSA). The incubation mixture contained 50-100 nM peroxidase, 10-500 microM 82Br-, tyrosine (150 microM), thyroglobulin (0.3 or 1 microM), or BSA (7.5 microM), and a source of H2O2. The latter was either generated by glucose (1 mg/ml)-glucose oxidase (0.5 or 1 micrograms/ml), or added initially as a bolus (100 microM). With TPO, formation of organically bound 82Br was undetectable under all conditions in the pH range 5.4-7.0. Lactoperoxidase and CPO, on the other hand, displayed considerable brominating activity. Lactoperoxidase was much more active at pH 5.4 than at pH 7.0 and was more active with BSA as acceptor than with tyrosine or thyroglobulin. The distribution of 82Br among the various amino acids in LPO-brominated thyroglobulin and BSA was determined by HPLC. As expected, monobromotyrosine and dibromotyrosine together comprised the greatest part of the bound 82Br. However, a surprisingly high percentage (20-25%) was present as monobromohistidine. Evidence was also obtained for the presence of a small percentage of the bound 82Br as tetrabromothyronine. Peroxidase-catalyzed bromination probably depends on the oxidation of Br- to Br+ by the Compound I form of the enzyme. Since oxidation of Br- to Br+ requires a stronger oxidant than oxidation of I- to I+, our results suggest that Compound I of LPO and of CPO has a higher oxidation potential than Compound I of TPO. In vivo experiments with rats on a low iodine diet injected with 82Br- showed that even under conditions of high stimulation by thyrotropic hormone, there is negligible formation of organic bromine in the thyroid. Measurements of thyroid:serum concentration ratios for 82Br- in similar rats provided no evidence that Br- is a substrate for the iodide transport system of the thyroid.

Effect of iodine deficiency and cold exposure on thyroxine 5'-deiodinase activity in various rat tissues.

We measured thyroxine 5'-deiodinase I (T(4)5'D-I) activity in thyroid, liver, and kidney and thyroxine 5'-deiodinase II (T(4)5'D-II) activity in brown adipose tissue (BAT) in rats on a low-iodine diet (LID) to test the possibility that increased deiodinase activity in these tissues might contribute to the maintenance of ther serum 3,5,3'-triiodothyronine (T3) level. Control rats received LID plus KI. Experiments were also performed with LID and LID plus KI rats exposed to cold. T(4)5'D-I activity was greatly increased in the thyroids of LID rats but not in liver or kidney. We consider it likely that increased thyroxine (T4)-to-T3 conversion in the greatly enlarged thyroids of LID rats contributed to the maintenance of serum T3. T(4)5'D-II activity in BAT was markedly increased in LID rats and was further greatly increased on cold exposure. However, we were unable to demonstrate an increase in uncoupling protein mRNA levels in BAT in response to cold in LID rats. We attribute this to the very low serum T4 level, which limits substrate availability. This factor also makes it unlikely that BAT contributes to maintenance of serum T3 in LID rats.

Enzymatic deglycosylation of porcine thyroid peroxidase: effects on catalytic activity and immunoreactivity.

Thyroid peroxidase is a heme-containing, membrane-bound, glycoprotein enzyme that catalyzes iodination and coupling in the thyroid gland. It is also the antigen for microsomal autoantibodies that are commonly found in the serum of patients with autoimmune thyroid disease. We examined the effect of deglycosylation on the catalytic functions and the immunoreactivity of this enzyme. A highly purified, solubilized, large tryptic fragment of porcine thyroid peroxidase, retaining all of the N-linked glycosylation sites of the native enzyme and displaying full catalytic activity was used. It was deglycosylated by treatment with N-glycanase under nondenaturing conditions. The loss in relative molecular mass after treatment, determined by gel electrophoresis, was about 75% of the estimated molecular weight of the glycan portion of porcine thyroid peroxidase. Lectin blots performed with horseradish peroxidase-conjugated concanavalin A showed a similar loss in relative molecular mass but some residual carbohydrate. The intensity of the carbohydrate stain was consistent with the loss of about 75% of the glycans. Despite this loss, three different assays for catalytic activity of porcine thyroid peroxidase were not significantly decreased. Immunoreactivity measured by immunoblotting and by enzyme-linked immunosorbent assay was also unimpaired. These findings suggest that N-glycanase-sensitive glycans in porcine thyroid peroxidase do not act as antigenic determinants and play a minor role, if any, in catalytic activity and, presumably therefore, in the maintenance of protein conformation.

Lack of effect of propylthiouracil and methylmercaptoimidazole on thyroglobulin biosynthesis.

Experiments were performed both in vivo and in vitro to test a previous proposal that part of the antithyroid action of the thioureylene drugs, propylthiouracil (PTU) and methylmercaptoimidazole, can be attributed to inhibition of thyroglobulin (Tg) biosynthesis. Rat thyroid lobes were incubated in leucine-free Eagle's medium containing bovine thyroid-stimulating hormone and 0, 0.1-0.2, or 1 mM drug. After a 30-min preincubation, 5 mu Ci of [14C]leucine were added and the incubation was continued for 4 hr. The soluble fraction was analyzed by sucrose density gradient centrifugation, and the fractions corresponding to the 19S Tg peak were pooled and assayed for 14C. No inhibition of 14C incorporation into 19S Tg was observed, even in thyroid lobes incubated in the presence of 1 mM methylmercaptoimidazole or 2 mM PTU. At the same time, 14C incorporation into 19S Tg was completely inhibited when lobes were incubated in the presence of 0.1 mM puromycin. In vivo, rats received an injection of PTU (1 mumol/100 g body wt), followed 60 min later by an injection of 25 mu Ci of [14C]leucine. Blood samples and thyroids were taken 5 hr after the [14C]leucine injection. Serum thyroid-stimulating hormone was not significantly affected by the PTU injection. The thyroid-soluble fraction was analyzed by sucrose density gradient centrifugation. No significant differences between saline and PTU-injected groups were observed in [14C]leucine incorporation into 19S Tg. We conclude from both our in vitro and our in vivo studies that PTU and methylmercaptoimidazole have no inhibitory effect on thyroglobulin synthesis in rat thyroids and that such inhibition does not play a significant role in the antithyroid action of these drugs.

Purification and characterization of a large, tryptic fragment of human thyroid peroxidase with high catalytic activity.

Thyroid peroxidase (TPO) was purified from human thyroid tissue, obtained at surgery from patients with Graves' disease, by a procedure similar to one that we had previously used for the purification of porcine TPO. The membrane-bound enzyme was solubilized by treatment of the thyroid particulate fraction with trypsin plus detergent. After precipitation with ammonium sulfate, the enzyme was purified by a series of column treatments, including ion-exchange chromatography on DEAE-cellulose, gel filtration through Bio-Gel P-100, and hydroxylapatite chromatography. Although a high degree of purification was achieved, the finally isolated product was considerably more heterogeneous than the TPO obtained from porcine thyroids. Several pools of active enzyme differing in values for A412/A280 and in specific activity were collected. Gel electrophoresis was performed under native, denaturing [sodium dodecyl sulfate (SDS)] and denaturing plus reducing conditions. Native gel electrophoresis indicated that the active enzyme (93 kDa) was heavily contaminated with an inactive 60-kDa fragment, which we were unable to remove by HPLC. The inactive fragment was highly antigenic when tested on immunoblots with an antibody to TPO. The presence of the inactive fragment greatly reduced values for A412/A280 in the finally purified human TPO. Two of the pools, with A412/A280 values of 0.159 and 0.273, were used for further testing. Catalytic activity was very similar in these two pools when measured on the basis of heme content by several different assays. Moreover, the specific activities of both, based on heme content, were very similar to those observed with a porcine TPO preparation with A412/A280 = 0.48. These findings indicate that the inactive 60-kDa fragment most likely did not contain heme. On SDS-polyacrylamide gel electrophoresis under reducing conditions, the 60-kDa fragment completely disappeared and was replaced by a 36- and a 24-kDa component. Amino terminal sequence information obtained on these components indicated that the 24-kDa component represents the amino terminal portion of the active 93-kDa fragment, whereas the 36-kDa fragment represents the carboxyl terminal portion. A model is proposed suggesting that the 60-kDa fragment was generated by trypsin cleavage of native TPO at two internal sites within a disulfide loop (res approximately 300 and res 564) and at one further internal site (res 280). In addition, trypsin cleavage is proposed at sites near the amino and carboxyl ends common to both the active 93-kDa and the inactive 60-kDa fragments.(ABSTRACT TRUNCATED AT 400 WORDS)

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 of 35S- and 14C-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)

A reexamination of the proposed inactivation of thyroid peroxidase in the rat thyroid by propylthiouracil.

The antithyroid drug 6-propylthiouracil (PTU) was previously shown in our laboratory to have an unexpectedly prolonged inhibitory effect on iodination in the thyroid glands of rats. Eighteen hours after injection of a relatively small dose, iodination in the thyroid remained inhibited by more than 90%. We previously suggested that the prolonged inhibitory effect might be due to inactivation of thyroid peroxidase (TPO), a reaction previously shown to occur under certain conditions in an in vitro iodinating system containing highly purified TPO. However, the analytical procedure used in our earlier study did not exclude the possibility that sufficient PTU remained in the thyroid even after 18 h to inhibit TPO-catalyzed iodination by a reversible mechanism. Development of an improved analytical procedure, based on HPLC, led us to reexamine the mechanism of the prolonged inhibitory effect of PTU on iodination in rat thyroid glands. Rats were injected with [35S]PTU (1 mumol/100 g BW), and ultrafiltrates prepared from their homogenized thyroid glands were analyzed by HPLC. The major 35S-labeled metabolites were identified as sulfate/sulfite, PTU sulfinate, and PTU sulfonate. However, even after 18 h, a significant amount of unchanged [35S]PTU was also present. The calculated mean concentration of residual PTU was 20 microM, a sufficiently high level to explain the observed inhibition of iodination on the basis of a reversible mechanism. Experiments were also performed to examine the intrathyroidal distribution of 35S at intervals after the injection of [35S]PTU. All of the oxidation products of PTU showed marked increases between 2 and 16 h after injection. Based on our view that TPO is the major mediator of intrathyroidal metabolism of PTU, this observation is inconsistent with our previous proposal that TPO is inactivated after PTU injection. The results of the present study, therefore, lead us to withdraw our previous suggestion that TPO is inactivated after injection of PTU into rats. It is more likely that inhibition of iodination by PTU in the rat thyroid involves competition between PTU and tyrosyl residues of thyroglobulin for oxidized iodine, comparable to the reversible mechanism of inhibition observed in the TPO model system.

Metabolism of 35S- and 14C-labeled 1-methyl-2-mercaptoimidazole in vitro and in vivo.

We previously described an in vitro incubation system for studying the mechanism of inhibition of thyroid peroxidase (TPO)-catalyzed iodination by the antithyroid drug 1-methyl-2-mercaptoimidazole (MMI). Inhibition of iodination in this system may be reversible or irreversible, depending on the relative concentrations of iodide and MMI and on the TPO concentration. Metabolism of the drug occurs under both conditions, and in the present investigation we used 35S- and 14C-labeled MMI together with reverse phase HPLC to examine the metabolic products associated with reversible and irreversible inhibition of iodination by MMI. Under conditions of reversible inhibition, MMI was rapidly metabolized and disappeared completely from the incubation mixture. With [35S]MMI, the earliest detectable 35S-labeled product was MMI disulfide, which reached a peak after a few minutes and then declined to undetectable levels. Coincident with the decrease in disulfide was the appearance of two 35S peaks, the major one corresponding to sulfate/sulfite, and the other to a component eluting at 7.5 min. Similar results were obtained for the disulfide and for the 7.5 min metabolite with [14C]MMI. The major 14C-labeled metabolite containing no S appeared to be 1-methylimidazole. Under conditions of irreversible inhibition, MMI disulfide was also the earliest detectable 35S-labeled metabolite. However, MMI decreased more slowly, and after reaching a nadir at about 6 min returned gradually to a level about halfway between the initial and the minimum value. The reformation of MMI appeared to involve the nonenzymatic disproportionation of MMI disulfide. Formation of the 7.5 min peak was also observed, but there was no formation of sulfate/sulfite. The difference in metabolic pattern between the reversible and irreversible conditions is primarily related to the rapid inactivation of TPO that occurs under irreversible conditions. The metabolism of [35S]MMI in thyroids of rats injected with the labeled drug resembles more closely conditions of reversible inhibition, since sulfate/sulfite is the only 35S-labeled metabolite. Neither [35S]MMI disulfide nor the 7.5 min component was detected in rat thyroids in vivo. However, it was demonstrated that these components do not survive homogenization with thyroid tissue, and failure to detect them in vivo does not exclude them as likely intermediates in intrathyroidal MMI metabolism. Based on the observations reported in this study, we present a revised scheme for the mechanism of inhibition of TPO-catalyzed iodination by MMI.

Propylthiouracil and methimazole display contrasting pathways of peripheral metabolism in both rat and human.

We have developed HPLC procedures for analyzing the metabolites of [35S]methylmercaptoimidazole [( 35S] MMI) and [35S]propylthiouracil [( 35S]PTU) in bile, urine, serum, and liver of rats. We also studied urinary metabolites of [35S] MMI and [35S]PTU in one human subject. In bile collected from [35S]MMI-injected rats, two major metabolites accounted for 80-90% of the total 35S. Incubation of these metabolites either with or without beta-glucuronidase led to the appearance of a 35S-labeled compound less polar than MMI. In contrast, the major [35S]PTU metabolite in bile (greater than 50% of total 35S) was completely converted to [35S]PTU on incubation with beta-glucuronidase and showed no conversion in a control incubation. From these results we conclude that the major biliary metabolites of MMI in rats are not glucuronides. They appear to be labile conjugates of a metabolite of MMI. After [35S]MMI injection into rats, two major and at least four minor metabolites were observed in urine. In one human who received [35S]MMI orally, the HPLC profile of 35S in urine was similar to that of the rat. Incubation of human urine or of its isolated major component with beta-glucuronidase had no significant effect on the HPLC profile. On the other hand, the major urinary metabolite of [35S]PTU in human and rat urine was completely converted to [35S]PTU on incubation of whole urine with beta-glucuronidase. These results indicate that glucuronides comprise at most only a minor fraction of MMI metabolites in urine of rats or humans. Based on similarities in elution time, the metabolites of [35S]PTU in urine closely resembled those in bile of rats. In contrast, the metabolites of [35S]MMI in urine were strikingly different from those in bile. PTU displays noncovalent binding to serum protein to a much greater extent than does MMI. However, after injection of [35S]MMI into rats, a significant fraction of the 35S was firmly bound to protein in both serum and liver. This binding appeared to be covalent and involved metabolism of [35S]MMI. This type of binding was much less detectable after the injection of [35S]PTU into rats.