Molecular cloning of a cDNA for the human phospholysine phosphohistidine inorganic pyrophosphate phosphatase

We previously reported the isolation from bovine liver of a novel 56-kDa inorganic pyrophosphatase named phospholysine phosphohistidine inorganic pyrophosphate phosphatase (LHPPase). It is a unique enzyme that hydrolyzes not only oxygen-phosphorus bonds in inorganic pyrophosphate but also nitrogen-phosphorus bonds in phospholysine, phosphohistidine and imidodiphosphate in vitro. In this study, we determined the partial amino acid sequence of the purified bovine LHPPase. To investigate whether humans have the same enzyme, we isolated a cDNA clone from a HeLa cell cDNA library that encodes for the human homologue of LHPPase. Although its sequence does not include the consensus sequence of a typical inorganic pyrophosphatase, it does contain a similar sequence of the active site in other phosphatases such as protein-tyrosine phosphatase, dual-specific phosphatase and low molecular weight acid phosphatase. Human LHPPase was highly expressed in the liver and kidney, and moderately in the brain. The recombinant protein was produced in E. coli. Its ability to hydrolyze oxygen-phosphorus bonds and nitrogen-phosphorus bonds was confirmed. The enzymatic characteristics of this human protein were similar to those of purified bovine LHPPase. Thus, we concluded that the cDNA encoded the human counterpart of bovine LHPPase.     

Suppression of thyroid cell growth by serum IgG in Graves' disease.

To determine whether serum immunoglobulin in addition to epidermal growth factor (EGF) augment growth in human thyroid cells, effects of these factors on thyrocytes were tested using IgG derived from 34 patients with Graves' disease and 12 normal subjects. The cell growth was estimated by [3H]-thymidine uptake, cell cycle determined by FACS analysis and the expression of c-fos mRNA in monolayer thyrocytes enzymatically prepared from Graves' thyroid. The addition of IgG taken from patients with Graves' disease inhibited the [3H]-thymidine uptake compared to that taken from control subjects. IgG taken from Graves' disease suppressed EGF-induced increase of S + G2/M phase in cell cycle and the expression of c-fos mRNA, while those taken from normal subjects did not affect at all. [3H]-thymidine uptake was more suppressed by IgG from patients with a smaller-sized goiter than by those with a larger-sized one. There was a negative correlation between the suppression of [3H]-thymidine uptake and levels of TBII (p less than 0.05). There was no correlation between the degree of suppression and the levels of T3, T4, TSAb, TSBAb or MCHA. Thus, in conclusion, IgG derived from sera of Graves' may inhibit the growth of Graves' thyrocytes, leading to the determination of the goiter size.     

Dimethylsulfoxide maintains human thyroid cells in suspension culture, facilitating synthesis and release of thyroid hormone

Usually, human thyrocytes in primary culture rapidly lose their thyroid function and fail to synthesize or release thyroid hormone after 3-5 days of culture. By culturing thyroid follicles obtained from patients with Graves' disease in medium supplemented with TSH and a low concentration of fetal calf serum (1%), thyrocytes can maintain thyroid function for several days. We have found that the addition of dimethylsulfoxide to culture medium (1.7%) furthermore enhanced and maintained thyroid function (de novo synthesis and release of [125I] thyroxine) for more than 13 days, probably by inhibiting dedifferentiation of thyrocytes. The present bioassay will be also useful for detecting thyroid stimulating immunoglobulin in patients with Graves' disease.     

Stimulation of cellular prion protein expression by TSH in human thyrocytes

The cellular isoform of prion protein (PrP(C)) is a cell-surface glycosyl-phosphatidylinositol-anchored protein which is ubiquitously expressed on the cell membrane. It may function as a cell receptor or as a cell adhesion molecule. Thyroid follicles, obtained from patients with Graves' disease at thyroidectomy, were cultured in F-12/RPMI-1640 medium supplemented with 0.5% fetal bovine serum and bovine thyroid stimulating hormone (bTSH). Northern blot analyses revealed that bTSH increased the steady-state expression levels of PrP mRNA in a time- and dose-dependent manner. This increase was reproduced by dibutyryl-cAMP and 12-decanoylphorbol-13-acetate. The mRNA expression was greater in thyroid follicles in suspension culture than in thyrocytes cultured in a monolayer. These findings suggest that TSH stimulates PrP mRNA expression in thyrocytes through the protein kinase A and C pathways. The greater mRNA expression in thyroid follicles than in monolayer cells suggests that PrP(C) may be involved in structure formation or maintenance of thyroid follicles.     

Identification of new protein complexes of Escherichia coli inorganic pyrophosphatase using pull-down assay

Inorganic pyrophosphatase (PPase) is a conserved and essential enzyme catalyzing the hydrolysis of pyrophosphate PP_i. Its activity is required to promote a lot of thermodynamically unfavorable reactions including biosynthesis of activated precursors of sugars and amino acids. Several protein partners of PPase were found so far in Escherichia coli by large-scale approaches. Functional role of these interactions was not studied. In this paper we report the identification of three protein partners of E. coli PPase not found earlier. Pull-down assay on the Ni²⁺-chelating column using 6His-tagged PPase as bait was used to isolate PPase complexes from stationary-phase cells. Of several isolated protein components, five were identified by MALDI-TOF mass-spectrometry: two chaperones (DnaK and GroEL) and three enzymes of carbohydrate and amino acid metabolism (FbaB, fructose-1,6-bisphosphate aldolase, class I; GadA, l-glutamate decarboxylase; and KduI, 5-keto-4-deoxyuronate isomerase). These three proteins were cloned, expressed and purified in 6His-tagged and/or tag-free forms. Their binary interactions with PPase were verified by independent approaches. Initial characterization of the complexes indicates that PPase may stabilize its protein partners against unfolding or degradation. Comparative analysis of the PPase protein partners allowed an insight into its possible involvement in the cell metabolic regulation.     

Evidence that catalysis by yeast inorganic pyrophosphatase proceeds by direct phosphoryl transfer to water and not via a phosphoryl enzyme intermediate

In this work, we show that adenosine 5'-O-(3-thiotriphosphate) (ATP gamma S) is a substrate for yeast inorganic pyrophosphatase (PPase) (EC 3.6.1.1) and further, using chirally labeled [gamma-17O,18O]ATP gamma S, that enzyme-catalyzed hydrolysis to produce chiral inorganic thio[17O,18O]phosphate proceeds with inversion of configuration. Both the synthesis of chiral ATP gamma S and the determination of inorganic thiophosphate configuration were carried out as described by Webb [Webb, M. R. (1982) Methods Enzymol. 87, 301-316]. We also show in a single turnover experiment performed in H2(18)O that 1 mol each of 18O16O3P and 16O4P is produced per mol of inorganic pyrophosphate hydrolyzed, a strong indication that oxygen uptake to form inorganic phosphate on PPase catalysis of inorganic pyrophosphate hydrolysis comes directly from H2O. These two results provide strong evidence for the conclusion that PPase catalyzes inorganic pyrophosphate hydrolysis via a single-step direct phosphoryl transfer to water and does not involve formation of a phosphorylated enzyme intermediate.     

Facilitation of polymerase chain reaction with thermostable inorganic pyrophosphatase from hyperthermophilic archaeon Pyrococcus horikoshii

An inorganic pyrophosphatase (PPases) was cloned from the hyperthermophilic archaeon Pyrococcus horikoshii and was expressed in and purified from Escherichia coli. The recombinant inorganic pyrophosphatase (PhPPase) exhibited robust catalytic activity of the hydrolysis of pyrophosphate into two orthophosphates at high temperatures (70°C to 95°C). Thermostable pyrophosphatase activity was applied into polymerase chain reaction (PCR) due to its ability to push chemical equilibrium toward the synthesis of DNA by removing pyrophosphate from the reaction. A colorimetric method using molybdate and reducing agents was used to measure PCR progress by detecting and quantifying inorganic phosphate in the PhPPase-coupled PCR mixture. Compared to PCR mixtures without PhPPase, the thermostable PhPPase enhanced the amount of PCR product in the same number of cycles. Thus, thermostable PPase may overcome the limitations of thermodynamically unfavorable DNA polymerization in PCR by yielding more products.     

Inorganic pyrophosphatase activity in cell-free extracts of Ureaplasma urealyticum

Cell-free extracts of Ureaplasma urealyticum strains Pi and T960 (CX8) (serovars 6 and 8, respectively) metabolized inorganic pyrophosphate (PPi). The inorganic pyrophosphatase (PPase) activity was greatest with Mg2+ as cofactor, but Mn2+ acted as a poor substitute. The PPases of the two serovars differed electrophoretically. Although the highest PPase activity was obtained using PPi as substrate, the enzyme could also utilize to a lesser degree both tripolyphosphate and trimetaphosphate. No activity was observed against beta-glycerophosphate, naphthyl phosphates, glucose 6-phosphate, fructose 6-phosphate, fructose 1,6-bisphosphate, thiamin pyrophosphate, phosphoribosylpyrophosphate, ADP or ATP. Acid- and alkaline-phosphatase activities were observed with naphthyl phosphates as substrates, but they did not have the same electrophoretic mobility on gels as the PPase activity. U. urealyticum PPase was inhibited by oxidized glutathione, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, phenylglyoxal, p-chloromercuribenzoic acid, Mn2+, Zn2+ and Ca2+. Neither reduced glutathione, L-cysteine nor Co2+ enhanced activity. PPi can act as a substrate or regulator of certain metabolic reactions, and PPi metabolism can function in bacterial bioenergetics; its role in ureaplasmas is presently unclear.     

Expression of TPO and ThOXs in human thyrocytes is downregulated by IL-1alpha/IFN-gamma, an effect partially mediated by nitric oxide

Morphological and functional alterations in Hashimoto's thyroiditis (HT) are predominantly mediated by Th1 cytokines through apoptotic cell death. This ultimate step could be preceded by functional injuries in thyroid hormone synthesis. The action of two Th1 cytokines (IL-1alpha/IFN-gamma) on thyroperoxidase (TPO) and thyroid oxidase (ThOXs) expression was tested in human thyrocytes isolated from normal tissues, Graves' disease (GD) tissues, and autonomous toxic nodules. There was no evidence of cell death. Nitric oxide (NO) release was induced by cytokines but was absent when NG-nitro-L-arginine methyl ester (L-NAME) was coincubated. When thyrotropin (TSH)-incubated normal and GD thyrocytes were treated with IL-1alpha/IFN-gamma, TPO and ThOXs protein and mRNA expression dropped, a decrease partially prevented by L-NAME, suggesting that NO acts as a mediator of Th1 effects. In thyrocytes from autonomous toxic nodules, the high level of TPO and ThOXs protein expression was not influenced by TSH or by cytokines, a finding partially reproduced when normal thyrocytes were treated with increasing concentrations of TSH. In conclusion, incubation of normal or GD thyrocytes with Th1 cytokines induces a significant reduction in TSH-increased expression of both TPO and ThOXs, an effect partially mediated by NO. The thyroid cell function can therefore be severely affected in HT, even when cells remain viable. In autonomous toxic nodules, cells become partially insensitive to exogenous Th1 cytokines.     

PYP-1, inorganic pyrophosphatase, is required for larval development and intestinal function in C. elegans

Inorganic pyrophosphatase (PPase) catalyzes the hydrolysis of inorganic pyrophosphate (PPi) into phosphate (Pi), which provides a thermodynamic driving force for important biosynthetic reactions. The nematode Caenorhabditis elegans gene C47E12.4 encodes a PPase (PYP-1) which shows 54% amino acid identity with human PPase. PYP-1 exhibits specific enzyme activity and is mainly expressed in the intestinal and nervous system. A null mutant of pyp-1 reveals a developmental arrest at early larval stages and exhibits gross defects in intestinal morphology and function. The larval arrest phenotype was successfully rescued by reintroduction of the pyp-1 gene, suggesting that PYP-1 is required for larval development and intestinal function in C. elegans.     

Method for real-time detection of inorganic pyrophosphatase activity

A sensitive and simple method for real-time detection of inorganic pyrophosphatase (PPase) (EC 3.6.1.1) activity has been developed. The method is based on PPase-induced activation of the firefly luciferase activity in the presence of inorganic pyrophosphate (PPi). PPi inhibits the luciferase activity, but in the presence of PPase the luciferase activity is restored and the luminescence output increases. The assay yields linear responses between 8 and 500 mU. The detection limit was found to be 8 mU PPase. The method was used to detect the hydrolytic activity of PPases from Saccharomyces cerevisiae, Escherichia coli, and Bacillus stearothermophilus. As substrate for the luciferase, adenosine 5'-phosphosulfate can replace ATP, which is an advantage for detection of PPase activity in crude extracts containing ATP-hydrolyzing activities. The method can be used for kinetic and inhibition studies as well as for detection of PPase activity during different purification procedures.     

Characterization of the Family I inorganic pyrophosphatase from Pyrococcus horikoshii OT3

A gene encoding for a putative Family I inorganic pyrophosphatase (PPase, EC 3.6.1.1) from the hyperthermophilic archaeon Pyrococcus horikoshii OT3 was cloned and the biochemical characteristics of the resulting recombinant protein were examined. The gene (Accession No. 1907) from P. horikoshii showed some identity with other Family I inorganic pyrophosphatases from archaea. The recombinant PPase from P. horikoshii (PhPPase) has a molecular mass of 24.5 kDa, determined by SDS-PAGE. This enzyme specifically catalyzed the hydrolysis of pyrophosphate and was sensitive to NaF. The optimum temperature and pH for PPase activity were 70 degrees C and 7.5, respectively. The half-life of heat inactivation was about 50 min at 105 degrees C. The heat stability of PhPPase was enhanced in the presence of Mg2+. A divalent cation was absolutely required for enzyme activity, Mg2+ being most effective; Zn2+, Co2+ and Mn2+ efficiently supported hydrolytic activity in a narrow range of concentrations (0.05-0.5 mM). The K(m) for pyrophosphate and Mg2+ were 113 and 303 microM, respectively; and maximum velocity, V(max), was estimated at 930 U mg(-1).     

Analysis of Fas, FasL and Caspase-8 expression in thyroid gland in young patients with immune and non-immune thyroid diseases

INTRODUCTION: Apoptosis, programmed cell death is a regulating mechanism enabling the removal of superabundantly produced and unnecessary at the certain moment cells. Disturbances of the apoptosis regulation contribute to the pathogenesis of many diseases, including autoimmune thyroid disorders. The aim of this study was to estimate expression of proapoptotic Fas/FasL and caspase-8 in thyroid tissues in patients with Graves' disease (GD), non-toxic nodular goiter (NTNG) and Hashimoto's thyroiditis (HT). MATERIAL AND METHODS: Inclusion criteria of Graves' patients were: large goiter, ophthalmopathy, TRAb > 5 U/L, positive titre of anti-TPO and anti-TG antibodies and concentration of TSH < 0.45 microIU/mL for more the 2-3 months from an onset of the disease. Isolated thyrocytes were identified by indirect method: in the first stage mouse monoclonal antibodies (mAbs) anti-TPO were bound to rabbit anti-mouse antibodies IgG (Fab')2 labeled FITC. To obtained cellular suspension mAbs directed against apoptotic Fas/FasL molecules labeled with PE (Phycoerythrin) was added. All investigations were performed on Coulter EPICS XL flow cytometer. Detection of apoptotic proteins was confirmed by Western Blot and immunohistochemistry methods using mAbs in DAB chromogene visuality and marked by Mayer's haematoxylin. Evaluation of caspase-8 expression in thyroid follicular cells was performed by Western Blot test. RESULTS: The analysis of Fas and FasL expression on surface of thyroid follicular cells was higher in patients with Hashimoto's thyroiditis (38%, 26%) in comparison with patients with Graves' disease (18%, 14%). In case of patients with Hashimoto's thyroiditis significantly lower percentage of thyroid tissue infiltrating immune Fas+ (13%) and FasL+ (22%) T cells in comparison with Graves' patients (33%, 43% respectively) was observed . Identification of proapoptotic Fas and FasL molecules in the thyroid follicular cells revealed higher expression of both proteins in patients with GD (++,++) and HT (+++; +++, respectively) in comparison with NTNG patients (+/0; +/0). Caspase-8 expression was detected in band 55 kDa using Western Blot test in patients with thyroid autoimmune diseases. CONCLUSIONS: We conclude that alteration in the expression of proapoptotic proteins in thyroid follicular cells may play a role in pathogenesis of thyroid autoimmune disorders. In addition, suppression of apoptosis in Graves' disease led to increased proliferation of thyroid follicular cells which is responsible for goiter formation.     

Expression and distribution of S-100 protein, CD83 and apoptosis-related proteins (Fas, FasL and Bcl-2) in thyroid tissues of autoimmune thyroid diseases

Previous studies have shown that dendritic cells (DCs) and apoptosis-related proteins play a critical role in the pathogenesis of autoimmune thyroid diseases (ATD).This study was designed to investigate the expression and distribution of S-100 protein, CD83 and apoptosis-related proteins (Fas, FasL and Bcl-2) in the thyroid tissues of ATD and their role in ATD pathogenesis as determined by immunochemical staing techniques and other methods. Pathological tissues of 30 patients with Hashimoto's thyroiditis (HT), 30 patients with Graves' disease (GD) and 30 cases of thyroid follicular adenoma (TFA, as control) were used for this study. A higher expression of S-100 in HT (4.2+/-3.1%) and GD (3.9+/-2.8%) vs TFA (0.95+/-0.64%) (p<0.001). was observed as well as a higher expression of CD83 in HT (22.58+/-13.96% and GD (29.92+/-14.43%) vs TFA (5.19+/-8.08%) (p<0.001). HT thyrocytes adjacent to thyroid infiltrating lymphocytes (TILs) showed greater increases in the levels of Fas and FasL than did the GD thyrocytes while HT TILs exhibited lower expression of Fas and FasL than did the GD TILs. GD thyrocytes expressed increased levels of the antiapoptotic protein Bcl-2 as compared to the low levels detected in HT thyrocytes. An opposite pattern was observed in the TILs in GD (low expression of Bcl-2) and HT (high expression of Bcl-2). The findings suggest that the high expression of DC markers is related to the pathogenesis of HT and GD. Up-regulation of both the number and matured functions of DCs may lead to the presentation of more antigens to lymphocytes which are related to the development of autoimmune thyroid diseases. The regulation of Fas/FasL/Bcl-2 in GD favors apoptosis of infiltrating lymphocytes and thyrocyte survival. The regulation of Fas/FasL/Bcl-2 in HT may promote thyrocyte apoptosis leading to hypothyroidism.     

Proton pumping inorganic pyrophosphatase of endoplasmic reticulum-enriched vesicles from etiolated mung bean seedlings

Endoplasmic reticulum (ER)-enriched vesicles from etiolated hypocotyls of mung bean seedlings (Vigna radiata) were successfully isolated using Ficoll gradient and two-phase (polyethylene glycol-dextran) partition. The ER-enriched vesicles contained inorganic pyrophosphate (PPi) hydrolysis and its associated proton translocating activities. Antiserum prepared against vacuolar H+-pyrophosphatase (V-PPase, EC 3.6.1.1) did not inhibit this novel pyrophosphatase-dependent proton translocation, excluding the possible contamination of tonoplast vesicles in the ER-enriched membrane preparation. The optimal ratios of Mg2+/PPi (inorganic pyrophosphate) for enzymatic activity and PPi-dependent proton translocation of ER-enriched vesicles were higher than those of vacuolar membranes. The PPi-dependent proton translocation of ER-enriched vesicles absolutely required the presence of monovalent cations with preference for K+, but could be inhibited by a common PPase inhibitor, F-. Furthermore, ER H+-pyrophosphatase exhibited some similarities and differences to vacuolar H+-PPases in cofactor/substrate ratios, pH profile, and concentration dependence of F-, imidodiphosphate (a PPi analogue), and various chemical modifiers. These results suggest that ER-enriched vesicles contain a novel type of proton-translocating PPase distinct from that of tonoplast from higher plants.     

The role of lipids in the regulation of ATP and PPi synthesis in mitochondria

It was demonstrated previously that mitochondria of higher and lower eukaryotes can synthesize, in the course of oxidative phosphorylation, not only ATP but also inorganic pyrophosphate (PPi). Two PPases were isolated from bovine heart mitochondria (soluble--PPase I and membrane--PPase II). Coupling PPase II, in contrast to PPase I, contains phosphatidyl choline, but PPase I is lipidized readily in the presence of different phospholipids. Reconstitution experiments of the PPi synthesis system have shown that after lipidization PPase I is able to incorporate into submitochondrial particles (SMP) and becomes a coupling factor for oxidation and PPi synthesis. It seems that phospholipid is indispensible for incorporation into the membrane and the manifestation of the coupling activity of the enzyme. The effect of lipids on the activity of soluble and membrane-bound pyrophosphatase was studied. It is shown that PPase II phospholipid is involved in the regulation of the hydrolase activity of the isolated enzyme. However, hydrolysis of PPi by SMP and its synthesis by mitochondria are affected by cooperative rearrangements of the entire lipid component of the membrane rather than by changes in the phase state of phosphatidyl choline contained in PPase II. An opposite response of ATP and PPi synthesis to changes in viscosity makes it likely that the viscosity of the mitochondrial inner membrane may control the levelling of these two processes in mitochondria.     

Expression of integrins of the beta1 family in thyroid cells from patients with Graves' disease in vivo and in vitro.

The expression of the beta1 family of integrins was determined in thyroid follicular cells from patients with Graves' disease (GD). Integrin expression was quantitated by flow fluorocytometry of single cell suspensions with antibodies against the common beta1 chain and the alpha1-alpha6 subunits. Results indicated that also in thyroid glands of GD, as previously observed in nodular goiters, two follicular cell populations with different patterns of beta1 integrin expression coexist (VLAalpha3beta1 and VLAalpha1,3,5,6beta1). The VLAalpha1,3,5,6beta1 thyrocyte population in GD was more abundant than in nodular goiters, ranging from 40 to 70% of the total follicular cells and the overall expression of the beta1 integrins was a two-fold higher. In thyrocytes from patients with GD cultured in vitro, alpha3 and alpha2 expression was regulated by cell-to-cell contact as previously described in normal thyroid cells, while the expression of alpha1, alpha5 and alpha6 was quickly lost during the culture. Our data suggest that the integrin profile of the VLAalpha1,3,5,6beta1 thyrocyte population in GD is induced by micro-environmental conditions rather than being the expression of a constitutive phenotype.     

Small-angle X-ray scattering studies on yeast inorganic pyrophosphatase and its interactions with divalent metal ions, inorganic phosphate, and hydroxymethane bisphosphonate

Small-angle x-ray scattering studies have been carried out on the enzyme yeast inorganic pyrophosphatase (PPase), and its overall conformational changes on interaction with divalent metal ions (Mg²⁺ and Mn²⁺) and with phosphoryl ligands [inorganic phosphate (P_i) and hydroxymethane bisphosphonate (PCHOHP), a nonhydrolyzable inorganic pyrophosphate analog] were assessed. The enzyme undergoes an apparent reduction in size on simultaneous addition of Mg²⁺ and high P_i concentration, although neithough neither Mg²⁺ nor P_i added separately induced any measurable conformational changes. By contrast, simultaneous addition of Mn²⁺ and P_i to PPase does not result in an observable conformational change. However, the overall structure of the enzyme appears to enlarge in the simultaneous presence of Mn²⁺ ions and PCHOHP. The significance of the structural changes seen in PPase under various conditions is discussed.     

Heterogeneous responses of recombinant human thyrotropin receptor to immunoglobulins from patients with Graves' disease.

Non-thyroid mammalian cells, CHO-K1 cells, stably expressing human thyrotropin receptor (CHO-TSH-R cells) were used for the assay of thyroid stimulating antibody (TSAb) activities of IgGs from 24 patients with Graves' disease and we compared them with the values obtained in porcine thyroid cells. A significant positive correlation was observed between the results given by CHO-TSH-R cells (hTSAb) and porcine thyrocytes (pTSAb) (r = 0.94, p less than 0.001). However, we found that hTSAb values of IgGs from 5 patients were extremely different from their hTSAb values. Four out of these 5 IgGs showed strong pTSAb activity but exhibited a weak or negative hTSAb activity. Conversely, one out of 5 autoantibodies was very strong for hTSAb but its pTSAb was low. These heterogeneous responses of recombinant hTSH-R to Graves' IgGs suggest that there exist different types of TSAb and also that the epitope(s) for TSAb may be different from case to case.     

The thyroid "microsomal" antigen is an epitope on the thyrotropin receptor

Antimicrosomal antibodies are present in the sera of most patients with autoimmune thyroiditis, and Graves' disease. It has, in general, been difficult to separate antimicrosomal activity from that directed against the thyrotropin (TSH) receptor in Graves' IgG preparations. The "microsomal" antigen has been localized to the endoplasmic reticulum and microfollicular aspect of thyrocytes; its structure is however unknown. In an attempt to identify the thyroid microsomal antigen, we studied the interaction of Hashimoto's IgG with high microsomal antibody titre and negative for thyroglobulin with purified thyroid plasma and light microsomal membranes. We allowed Hashimoto's, Graves', and control IgGs to bind to protein blots of thyroid plasma membranes resolved on SDS-PAGE under non-reducing conditions. All seven Hashimoto's IgG at a concentration of 2 mg/ml interacted with an M approximately 197,000 polypeptide corresponding to the TSH holoreceptor. By contrast to Graves' IgG (which were positive at 1 mg/ml), however, this binding was not blocked by pretreatment of the protein blots with TSH. Normal IgGs showed no binding at concentrations of up to 2 mg/ml. Both Hashimoto's and Graves' IgG interacted with TSH-affinity column-purified receptor preparations. Two of the Hashimoto's IgGs induced adenylate cyclase activation in thyroid plasma membranes, three inhibited TSH-stimulated enzyme activation, and two were without effect. Two classes of autoantibodies, other than TSH receptor directed, were encountered; one class raised to antigens common to all seven patients and another class unique to individual patients, eg, Mr 210,000 and Mr 20,000 polypeptides. We propose that the TSH receptor has multiple epitopes (functional domains), and the one to which antimicrosomal antibody bind is likely to be spatially separated from that with which Graves' IgG and TSH interact. Differences in affinity or number of sites allows for the demonstration of Graves' IgG against a background of antimicrosomal antibody.