Bovine Thyroid Membranes Research Articles

Comparison of Two In Vitro Methods for the Measurement of Recombinant Human TSH Bioactivity

Thyroid stimulating hormone (TSH), a pituitary glycoprotein hormone, is a potent inducer of intracellular cAMP production. Two methods for measuring TSH bioactivity were evaluated and compared. One assay is based on using a radioimmunoassay (RIA) to measure the recombinant human TSH-induced increase in cAMP using a bovine thyroid membrane isolate. The other is based on a Chinese hamster ovary (CHO) cell line that has been transfected with the TSH receptor and a cAMP-responsive luciferase reporter. The within-assay coefficient of variation for the membrane-based assay was determined to be approximately 35% compared with approximately 25% for the cell-based assay. Twenty-one preparations of recombinant human TSH (rhTSH) were tested using both methods. No significant difference was detected between the data sets and no assay bias was present. Both assay systems provide a suitable means for measuring the activity of rhTSH. The advantage of the membrane-based assay is the relatively small quantity of TSH needed for analysis. However, the average time required to analyse a sample using the membrane-based method was more than twice as long as that needed to test a sample in the cell-based assay. Other advantages of the cell-based method include the use of a 96-well format, which facilitates the analysis of several concentrations of rhTSH within one assay plate, and the use of a non-radioactive endpoint. Copyright 2002 The International Association for Biologicals. Published by Elsevier Science Ltd. All rights reserved.

Serum unmasks the binding of thyroid-stimulating hormone to endogenous and transfected receptors: evidence for a soluble form of the receptor in human thyroid

A specific homologous radioligand receptor assay for thyroid-stimulating hormone (TSH) using bovine thyroid membranes was adapted for use with human thyroid. Specific 125I-labelled TSH binding was detected in the 3000 g membrane pellet from bovine thyroid but predominantly in the 3000 g supernatant of the human thyroid homogenate. Both assays required incubation in the presence of 10% serum, whilst the assay using human thyroid could only be precipitated using polyethylene glycol (PEG). The serum requirement transcended a possible role as carrier protein and unmasked specific TSH binding. Molecular sieving determined that the active fraction of the serum had an apparent size of 30,000-100,000. The requirement for PEG-assisted precipitation of the TSH receptor assay was a consequence of the TSH-binding entity from Graves' thyroid behaving like a soluble 'receptor': it did not sediment with the membranes, passed a 0.2 microns filter and, upon molecular sieving, had an apparent size of 300,000-1,000,000. A full-length TSH receptor cDNA was cloned from a human Graves' thyroid library and stably transfected cell lines expressing the TSH-receptor protein were constructed using human HeLa and murine 3T3 cells. Specific TSH binding was unmasked by serum in the human cell lines, as observed for the human thyroid TSH receptor, whereas serum hindered TSH binding in the murine cell lines. A soluble form of the receptor was not released from the cells and was not produced in conditions which demonstrated a soluble receptor-like binding component in human thyroid tissue.

The role of the oligosaccharide chains of thyrotropin alpha- and beta-subunits in hormone action.

TSH, a dimeric glycoprotein hormone, has two N-linked complex-type oligosaccharide chains on the alpha-subunit and one on the beta-subunit. The oligosaccharide chains of TSH are important for the expression of hormonal activity, but the contribution of those on each of the subunits to the activity is not clear. In the current study we have determined the relative importance of the oligosaccharide chains of TSH subunits using a recently reported method of enzymatic deglycosylation. The alpha- and beta-subunits of bovine TSH were deglycosylated with endoglycosidase-F and recombined to obtain differentially deglycosylated TSH. The derivatives showed no differences in their receptor-binding activities. The in vitro bioactivity of these derivatives was assessed by measuring adenylyl cyclase activity in bovine thyroid membranes and stimulation of cAMP production and growth in FRTL-5 cells. In the adenylyl cyclase assay, deglycosylation of the alpha-subunit alone had a more profound effect on the activity [maximal stimulatory activity (Vmax), 13% that of alpha.beta) than when the beta-subunit alone was deglycosylated (Vmax, 50% that of alpha.beta). In the FRTL-5 assays, removal of carbohydrate from TSH alpha, but not the beta-subunit, caused a 2- to 3-fold increase in the concentration required for half-maximal stimulation, with minimal change in the apparent Vmax. The adenylyl cyclase assay in bovine membranes was more sensitive to carbohydrate removal than the assays of rat FRTL-5 cells, in which the derivatives with lower activity were able to stimulate cAMP and growth to near-maximal levels, albeit at 3-fold higher concentrations. These results indicate that the carbohydrate chains in both subunits of TSH, particularly those in the alpha-subunit, are important in hormone action. In contrast to previous reports, our study shows that the beta-subunit plays a role in signal transduction.

Biological Activity and Metabolic Clearance of a Recombinant Human Thyrotropin Produced in Chinese Hamster Ovary Cells

The presence and specific structures of the oligosaccharides on TSH have been shown to be important for its production and bioactivity. Since the carbohydrate structure of a protein reflects the glycosylation apparatus of the host cells in which the protein is expressed, we examined the biological activity and metabolic clearance of a preparation of purified recombinant human (rh) TSH derived from a stable transfectant of Chinese hamster ovary cells. Carbohydrate compositional analysis of this rTSH showed it to be more highly sialylated than a nonrecombinant, cadaver-derived pituitary hTSH. In addition, no N-acetyl galactosamine was detectable in rhTSH, which implies the absence of terminal sulfate moieties, both of which are present in pituitary-derived TSH. The immunologic activity and porcine TSH receptor-binding activity of the preparation of rhTSH were 3- to 4-fold lower than those of a standard pituitary hTSH. The rhTSH showed a maximum stimulatory activity similar to that of pituitary hTSH in two different in vitro bioassays. However, rhTSH elicited about 3-fold and 5-fold less cAMP than pituitary TSH after stimulation of adenylyl cyclase in bovine thyroid membranes and the rat FRTL-5 cell line, respectively. Removal of sialic acid did not alter the immunologic activity of rhTSH. However, the potencies of rhTSH in receptor-binding, adenylyl cyclase, and FRTL-5 assays were increased 2.4-, 2.6- and 26.7-fold, respectively after sialic acid removal. These data suggest that the in vitro biological activity of rhTSH is influenced by its highly sialylated oligosaccharide chains. The rhTSH had a 2-fold lower metabolic clearance rate than pituitary TSH, resulting in a greater than 10-fold higher serum concentration of rhTSH at 3 h as compared to pituitary hTSH. After sialic acid removal, the rhTSH was cleared faster (7.5-fold) than pituitary hTSH, showing that its longer plasma half-life was due to its higher sialylation. Biologically active rhTSH should be of clinical value in the diagnosis and treatment of patients with thyroid cancer and as a pure hTSH reference preparation.

The role of carbohydrate in thyrotropin action assessed by a novel approach using enzymatic deglycosylation.

Deglycosylation of thyrotropin (TSH) and gonadotropins by chemical methods virtually abolishes their biological activity without impairing receptor binding activity. Recent reports have suggested that enzymatic deglycosylation, using endoglycosidases caused a much smaller decrease, if any, in the potency of the glycoprotein hormones without altering the Vmax. However, in these studies complete removal of the carbohydrate chains from the hormones was not unequivocally documented. We have prepared completely deglycosylated bovine TSH by endoglycosidase F digestion of its subunits, which were more readily deglycosylated than the intact hormone. The deglycosylated subunits were separated from any incompletely digested subunits by concanavalin A affinity chromatography. Carbohydrate compositional analysis, using a highly sensitive pulsed amperometric detection method coupled to ion-exchange high performance liquid chromatography, was performed to ascertain the complete removal of the glycan moieties from the subunits. The deglycosylated subunits thus prepared were recombined to obtain deglycosylated TSH dimer. Receptor binding activity of bTSH was minimally affected by the carbohydrate removal. In an in vitro bioassay using stimulation of cyclic AMP production in FRTL-5 cells, deglycosylated bTSH showed reduced activity with a potency 5-10-fold lower than that of control, although the Vmax remained unaltered. In contrast, the deglycosylated bTSH showed a reduction in Vmax, when assayed for its adenylyl cyclase stimulating activity in bovine thyroid membranes. Previous reports using chemical methods have apparently overestimated the effects of deglycosylation, probably because of altered protein conformation, while those using endoglycosidases have apparently underestimated these effects, probably because of incomplete deglycosylation.

Monoclonal Antibodies to the Thyrotropin Receptor Raised by an Autoantiidiotypic Protocol and Their Relationship to Monoclonal Autoantibodies from Graves' Patients*

Monoclonal antibodies that bind to the TSH receptor were obtained by an autoantiidiotypic approach in which immunization of BALB/c mice was performed with mixtures of bovine (b) and human (h) TSH. Two of 28 positive wells were selected for cloning and characterization: D2 and 4G11. Their antiidiotypic character was evidenced by TSH-inhibitable binding to affinity-purified polyclonal anti-TSH. The specificity of D2 and 4G11 for the hormone-binding region of the TSH receptor was demonstrated by several findings: 1) they inhibited the binding of [125I]iodo-bTSH to receptor in a dose-dependent manner; 2) their binding to partially purified thyroid plasma membranes could be completely inhibited by bTSH and hTSH; and 3) they inhibited the TSH-dependent growth and adenylate cyclase stimulation in FRTL-5 cells in a dose-dependent manner. By Western blot analysis of bovine thyroid membranes, D2 bound to a polypeptide of 188,000-195,000 mol wt under nonreducing conditions and 54,000-59,000 mol wt after treatment of membranes with beta-mercaptoethanol; the 4G11 epitope was undetectable. Scatchard analysis of the binding of 125I-labeled antibodies to receptor showed that 4G11 bound to a single site with a Kd of 5.7 X 10(-9) M, whereas D2 showed complex binding characterized by high affinity (Kd = 1.74 X 10(-11) M) and low affinity (Kd = 1.3 X 10(-8) M) sites. Binding studies in which D2 and 4G11 competed with each other for the TSH receptor showed mutual but unequal inhibition. The data suggest that portions of the D2 and 4G11 epitopes overlap, but that there is a high affinity binding site(s) for D2 for which 4G11 competes less effectively. The binding of D2 and 4G11 to TSH receptor was inhibited by monoclonal antibodies secreted by Graves' heterohybridomas, showing that D2 and 4G11 share characteristics with autoantibodies of Graves' disease and lending support to the hypothesis that idiotypic network interactions may play a role in the pathogenesis of Graves' disease.

Multiple isoforms of ADP-ribosylated G-like proteins from mammalian thyroid membranes

Bovine, canine, and porcine thyroid membrane proteins which were [32P] ADP-ribosylated by cholera and pertussis toxin in vitro were analyzed by one and two-dimensional polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. These three mammalian species have similar cholera toxin substrates (Mr 42,000 and 48,000) and pertussis toxin substrates (Mr 40,000). Resolution by two dimensional gel electrophoresis of these ribosylated proteins revealed that they each consist of at least 6 distinct polypeptides with similar isoelectric points ranging from approximately 5.5-7.0.

Characterization of a Bioeffective Monoclonal Antibody against Thyrotropin β-Subunit*

To describe a region of the TSH molecule participating in binding to receptor, a monoclonal antibody specific for a TSH epitope shared by beta-subunits of bovine (b), ovine (o), and human (h) TSH was obtained by immunization with mixtures of purified bTSH and hTSH. RIAs showed that the antibody also bound the beta-subunits of bLH, oLH, hLH, and hCG, but not the beta-subunits of porcine LH and TSH. Preincubation of [125I]iodo-bTSH with the antibody completely inhibited binding of the hormone to the TSH receptor of bovine thyroid membrane preparations at pH 7.4 in 50 mM NaCl (ED50 = 10 nM). The antibody also inhibited TSH-induced mitogenesis of FRTL-5 cells (ED50 = 50 nM). We conclude that the antibody binds to a site on the bTSH molecule that participates in high affinity binding of hormone to physiological TSH receptor. The target epitope includes a conserved structural determinant in beta-subunits of the glycoprotein hormones as well as a feature that allows discrimination of porcine hormones from those of bovine, ovine, and human origin.

Identification and characterization of a specific dolichylmonophosphate phosphatase activity in bovine thyroid microsomes

1. 1. Bovine thyroid membranes are able to dephosphorylate exogenous [1- 3H]DMP as well as endogenous prelabeled [ 32P]DMP. 2. 2. The kinetics, properties and specificity of the dolichylmonophosphatase activity have been studied by monitoring respectively the release of [1- 3H]dolichol from [1- 3H]DMP and the residual amount of [ 32P]DMP. 3. 3. The DMP-phosphatase activity is not linear with respect to time and exhibits a neutral pH optimum. There is only linearity in a narrow range of protein concentration when 0.25% Triton X-100 is included in the incubation mixture. 4. 4. Studying the enzymatic activity in function of protein concentration, the detergent requirement shows to be very critical. 5. 5. Triton X-100 is necessary for enzymatic activity with [1- 3H]DMP (only 10% of enzymatic activity in the absence of detergent) although the detergent inhibits the hydrolysis of endogenous prelabeled [ 32P]DMP. 6. 6. Divalent cations are not essential for enzymatic activity, Ca 2+-ions being even inhibitory. In accordance, EDTA (EGTA) is slightly stimulatory. 7. 7. The DMP-Pase activity is not influenced by the ionic strength of the incubation system and sulphydryl groups are not involved. NaF, VOS and VO 4 3− are strongly inhibitory. 8. 8. The inhibition by dolichol and PO 4 3− can be explained as the result of product inhibition. An apparent K m -value of 2.5·10 −5 M is computed for [1- 3H]DMP. 9. 9. Bacitracin inhibits DMP-phosphatase in contrast with other reports. 10. 10. Propylthiouracyl, cAMP, TSH and several other bio-effectors are without effect on the in vitro system. 11. 11. The specificity of the DMP-Pase activity is discussed, showing that the phosphatase is distinctly different from other phosphatases especially phosphatidic acid phosphohydrolase.

Topography, purification and characterization of thyroidal NAD+ glycohydrolase.

Subcellular fractionation of bovine thyroid tissue by differential pelleting and isopycnic gradient centrifugation in a zonal rotor indicated that NAD(+) glycohydrolase is predominantly located and rather uniformly distributed in the plasma membrane. Comparison of NAD(+) glycohydrolase activities of intact thyroid tissue slices, functional rat thyroid cells in culture (FRT(l)) and their respective homogenates indicated that most if not all of the enzyme (catalytic site) is accessible to extracellular NAD(+). The reaction product nicotinamide was predominantly recovered from the extracellular medium. The diazonium salt of sulphanilic acid, not penetrating into intact cells, was able to decrease the activity of intact thyroid tissue slices to the same extent as in the homogenate. Under the same conditions this reagent almost completely abolished NAD(+) glycohydrolase activity associated with intact thyroid cells in culture. The triazine dye Cibacron Blue F3GA and its high-M(r) derivative Blue Dextran respectively completely eliminated or caused a severe depression in the NAD(+) glycohydrolase activity of FRT(l) cells. The enzyme could be readily solubilized from bovine thyroid membranes by detergent extraction, and was further purified by gel filtration and affinity chromatography on Blue Sepharose CL-6B. The overall procedure resulted in a 1940-fold purification (specific activity 77.6mumol of nicotinamide released/h per mg). The purified enzyme displays a K(m) of 0.40mm for beta-NAD(+), a broad pH optimum around pH7.2 (0.1 m-potassium phosphate buffer) and an apparent M(r) of 120000. Nicotinamide is an inhibitor (K(i) 1.9mm) of the non-competitive type. The second reaction product ADP-ribose acts as a competitive inhibitor (K(i) 2.7mm). The purified enzyme splits beta-NAD(+), beta-NADP(+), beta-NADH and alpha-NAD(+) at rates in the relative proportions 1:0.75:<0.02:<0.02 and exhibits transglycosidase (pyridine-base exchange) activity. Anionic phospholipids such as phosphatidylinositol and phosphatidylserine inhibit the partially purified enzyme. A stimulating effect was observed upon the addition of histones.

Monoclonal antibodies to the thyrotropin receptor: stimulating and blocking antibodies derived from the lymphocytes of patients with Graves disease.

Human monoclonal antibodies have been generated from heterohybridomas obtained by fusing mouse myeloma cells with peripheral lymphocytes from patients with active Graves disease. This report characterizes four antibodies as presumptive thyrotropin receptor antibodies because they specifically inhibit thyrotropin binding and competitively inhibit thyrotropin-induced cAMP levels in human thyroid cells. Two of these antibodies, 208F7 and 206H3, are representative of autoimmune stimulators in Graves disease sera because they stimulate thyroid function in all assays, including the mouse bioassay; their ability to inhibit thyrotropin-induced cAMP increases in thyroid cells competitively is complemented by more than additive agonism at low (10 pM) thyrotropin concentrations. These stimulating antibodies interact more potently with human thyroid ganglioside preparations than with bovine thyroid or brain gangliosides; in contrast, they are poor inhibitors of 125I-labeled thyrotropin binding to liposomes containing the glycoprotein component of the human thyrotropin receptor. Antibodies 129H8 and 122G3 appear to be representative of inhibiting or "blocking" antibodies in Graves disease sera. Thus they have no intrinsic stimulatory action in assays of thyroid function but rather inhibit thyrotropin activity in the assays tested. These two antibodies do not react with human thyroid gangliosides but are strong inhibitors of thyrotropin binding to liposomes containing the high-affinity glycoprotein component from human, bovine, and rat thyroid membranes. The data unequivocally establish the pluritopic nature of the immunoglobulins in Graves disease and relate individual components or determinants of the thyrotropin receptor structure with specific autoimmune immunoglobulins.

Monoclonal antibodies to the thyrotropin receptor: the identification of blocking and stimulating antibodies

Monoclonal antibodies to the thyrotropin (TSH) receptor have been obtained from fusions of mouse myeloma cells with spleen cells immunized with solubilized thyroid membrane preparations. Two monoclonal antibodies which inhibit 125I-TSH binding and are reactive with the glycoprotein component of the bovine TSH receptor (11E8 and 13D11), are shown to inhibit basal and TSH stimulated adenylate cyclase activity in bovine thyroid membranes and human thyroid cells. Both antibodies also inhibit 125I-TSH binding in vitro, whether binding is measured at pH 6.0 in low salts and at 0-4 C or at pH 7.4 in 50 mM NaCl and at 37 C. The glycoprotein component is thus a portion of the physiologic TSH receptor in vivo and 125I-TSH binding studies apparently measure the high affinity glycoprotein component under nonphysiologic conditions and conditions more representative of the physiologic milieu. A third monoclonal antibody whose interaction with thyroid membranes is prevented by TSH is shown to stimulate adenylate cyclase activity in bovine thyroid membranes and human thyroid cells. This stimulating antibody only weakly inhibits 125I-TSH binding to thyroid membranes or to the glycoprotein component of the TSH receptor. The 22A6 antibody does, however, immunoprecipitate mixed brain gangliosides, in distinct contrast to the monoclonal antibodies to the glycoprotein receptor component, i.e., 11E8 and 13D11. The results support the speculation that autoimmune antibodies which inhibit TSH binding to thyroid membranes are not necessarily identical to antibodies which stimulate function; that antibodies directed at the high affinity initial site of TSH interaction with a cell can behave as blocking rather than stimulating antibodies and that a possible relationship exists between stimulating antibodies and the low affinity TSH binding sites (gangliosides) on thyroid membranes.

Thyrotropin stimulation of the ADP-ribosyltransferase activity of bovine thyroid membranes.

Thyrotropin increases the ADP-ribosylation activity of bovine thyroid membranes. Rapid ADP-ribosylation of membrane components is followed by increasing ADP-ribosylation of components in the supernatant of the reaction mixture. One of the major membrane proteins ADP-ribosylated in the thyrotropin-stimulated reaction has an approximate molecular weight of 40,000; this same protein is also a major ADP-ribosylated product of the A promoter of cholera toxin and appears to be related to the G regulatory subunit of the adenylate cyclase complex. The ADP-ribosylated products appearing in the supernatant solution comigrate with thyrotropin and preparations of 125I-labeled alpha subunit of thyrotropin; the alpha subunit, but not the beta subunit, of thyrotropin can be ADP-ribosylated by the membrane ADP-ribosyltransferase activity. NAD can be shown to enhance the ability of thyrotropin to stimulate the adenylate cyclase activity of bovine thyroid membrane preparations and of membrane preparations of a rat thyroid tumor whose adenylate cyclase activity is otherwise unresponsive to thyrotropin. The beta subunit of thyrotropin inhibits thyrotropin stimulation of both the ADP-ribosylation and adenylate cyclase activities of the thyroid membrane.

Effect of methyldopa on thyroid stimulating immunoglobulins producted by peripheral lymphocytes from patients with juvenile Graves'disease

In an attempt to test effects of methyldopa (inhibitor for suppressor T-cell) on the production of thyroid stimulating immunoglobulins (TSI), peripheral lymphocytes were preincubated with or without the drug followed by cultures with human thyroid plasma membrane for 7 days. Thyroid stimulating activities (TSA) in the supernatants of cultures were measured as cAMP generated during incubation of ATP with bovine thyroid membrane, and then were expressed as a percentage of control level. When the cells were preincubated in the presence of methyldopa, IgG synthesis from both patients and normal subjects generally increased. The pretreatment of methyldopa enhanced mean TSA from a level of 120±48% to 192±57% in the patients (P 0.01), while the effect was not observed in normal subjects. All these activities producted by lymphocytes in vitro were well correlated with those of IgG purified from the sera through DEAE-cellulose column chromatography. These results demonstrated that methyldopa is of use for in vitro production of TSI. The findings suggest that the inhibitory effect of methyldopa on T-cell proliferation may enhance the TSI production.

Thyroid membrane ADP ribosyltransferase activity. Stimulation by thyrotropin and activity in functioning and nonfunctioning rat thyroid cells in culture.

Bovine thyroid membranes possess both ADP ribosyltransferase and NAD glycohydrolase activities with the same Km values for NAD and the same pH optima. In intact membranes, the ADP ribosyltransferase is limited in its extent by the amount of available membrane acceptor which can be ADP-ribosylated; in membranes solubilized with lithium diiodosalicylate, an artificial acceptor, L-arginine methyl ester, can be substituted to eliminate this limitation. The product of the ADP ribosyltransferase is a mono-ADP-ribosylated acceptor whether the intact or solubilized membrane provides the enzyme activity and whether membrane or exogenous acceptor, L-arginine methyl ester, is utilized. The intact membranes and the solubilized preparation also have an enzyme activity which can release AMP from the mono-ADP-ribosylated acceptor whether formed by the action of the membrane ADP ribosyltransferase or the A promoter of cholera toxin. The NAD glycohydrolase activity appears to represent the half-reaction of the ADP ribosyltransferase, i.e. an activity measurable substituting water for a membrane acceptor or L-arginine methyl ester. Membranes from functional rat thyroid cells in culture, i.e. cells chronically stimulated by thyrotropin and unresponsive to further additions of thyrotropin, have low ADP-ribosylation but high NAD glycohydrolase activities. In contrast, membranes from nonfunctional rat thyroid cells, i.e. cells unresponsive to thyrotropin, have high ADP-ribosylation and low NAD glycohydrolase activities. NAD hydrolysis by the NAD glycohydrolase activity cannot account for the low ADP-ribosylation activity in membranes from the functioning cells, and its low level of ADP-ribosylation can be eliminated by solubilizing the membranes and substituting an artificial acceptor, L-arginine methyl ester. The ADP ribosyltransferase activity of rat thyroid cell membrane preparations can be enhanced by thyrotropin in a dose-dependent manner but not by insulin, glucagon, hydrocortisone, adrenocorticotropin, or its glycoprotein hormone analog, human chorionic gonadotropin. It is thus suggested (i) that, in analogy to cholera toxin, thyrotropin-stimulated ADP-ribosylation may be important in the regulation of the adenylate cyclase response and (ii) that the level of membrane acceptor available for ADP-ribosylation may relate both to a stable "'activated" state of the adenylate cyclase system in cells chronically stimulated with thyrotropin and/or to a desensitized state with regard to a failure of more thyrotropin to elicit additional functional responses.

The effect of desialylation on the in vitro interaction of human chorionic gonadotropin with human thyroid plasma membranes.

Previous studies have indicated that crude preparations of hCG (hCG-c) are much more active than purified preparations (hCG-p) in displacing 125I-labeled bovine TSH ([l25I]iodo-bTSH) from receptors in human and bovine thyroid membranes when the two are compared on the basis of their gonadotropic activity. In an attempt to elucidate the nature of the TSH-displacing factor in hCG-c and in view of the past reports indicating that such preparations contain a spectrum of desialylated hCG molecules, we have desialylated both hCG-c and hCG-p by treatment with neuraminidase and have studied their interactions with human thyroid membranes. Exposure of hCG-c to neuraminidase for increasing lengths of time yielded preparations with progressively greater ability to inhibit the binding of [125I]iodo-bTSH to the membranes, and the increase in inhibitory potency was directly related to the quantity of sialic acid released into the medium. On an equal weight basis, desialylated hCG-c was approximately 5 times more active...

Binding of thyroglobulin to bovine thyroid membranes. Role of specific amino acids in receptor recognition.

Bovine thyroglobulin was treated with increasing ratios of succinic anhydride, trinitrobenzene sulfonic acid, tetranitromethane, and N-acetylimidazole in an attempt to assess the role of lysine or tyrosine residues in binding to thyroid membrane receptors. Extensive succinylation results in dissociation to 12 S thyroglobulin with retention of a considerable portion of the three-dimensional structure. Only 25% of the lysine residues can be modified by trinitrophenylation without affecting inter-subunit interactions. Succinylation as well as trinitrophenylation increases the affinity of thyroglobulin for the membrane receptor by a factor of 2. The binding of thyroglobulin to the membrane was reduced after nitration of 30% of the tyrosyl residues with tetranitromethane. O-Acetylation of 40-70% of the tyrosyl residues by N-acetylimidazole nearly abolished the ability of thyroglobulin to bind to the membrane. Removal of the O-acetyl group with hydroxylamine restored the binding properties. The results indicate that tyrosyl residues play an important role in thyroglobulin interactions with thyroid membranes.

Thyroglobulin interactions with thyroid membranes. Relationship between receptor recognition of N-acetylglucosamine residues and the iodine content of thyroglobulin preparations.

Bovine thyroglobulin has been subjected to sequential glycohydrolase treatment in order to define further the components of the carbohydrate chain which are important in binding of the glycoprotein to bovine thyroid membranes. Preparations of asialoagalactothyroglobulin exhibit the best binding, suggesting that exposed N-acetylglucosamine residues on the B carbohydrate chain of thyroglobulin play an important role in the interaction of thyroglobulin with the thyroid membranes. Enhanced binding of asialoagalactothyroglobulin to microsomal, lysosomal, and Golgi membranes, as well as to thyroid cells in culture, was also observed. Isopycnic rubidium chloride gradient centrifugation, a procedure used in the isolation of thyroglobulin molecules with a low iodine content, also isolates thyroglobulin molecules with a low sialic acid content and with an increased ability to interact with wheat germ agglutinin, a lectin which recognizes exposed N-acetylglucosamine residues. The studies further indicate that there is a correlation between iodine content, exposed N-acetylglucosamine residues, and the binding of thyroglobulin to thyroid membranes.

Monoclonal antibodies to the thyrotropin receptor: implications for receptor structure and the action of autoantibodies in Graves disease.

Hybridoma cells have been obtained by fusing P3-NS1/1-Ag4-1 mouse myeloma cells with spleen cells from mice immunized with solubilized preparations of the thyrotropin receptor. Five clones were produced that secrete a monoclonal antibody whose binding to thyroid membranes is specifically inhibited by unlabeled thyrotropin. The antibody interacts with functioning thyroid cells in culture but not with nonfunctioning cells; this interaction is prevented by thyrotropin. The antibodies are capable of competitively blocking thyrotropin binding to bovine thyroid membrane preparations; they prevent 125I-labeled thyrotropin binding to a solubilized preparation of the glycoprotein component of the bovine thyrotropin receptor but are unable to inhibit 125I-labeled thyrotropin binding to liposomes containing gangliosides at comparable concentrations. They prevent 125I-labeled thyrotropin binding to rat, bovine, or human (Graves disease) thyroid membrane preparations. They do not stimulate adenylate cyclase activity in thyroid membrane preparations but can inhibit thyrotropin-stimulated iodide uptake by functioning thyroid cells in culture.

GUANYLATE CYCLASE ACTIVITY OF BOVINE THYROID MEMBRANE

In this comunication we described about a method for measuring guanylate cyclase activity of bovine thyroid membrane using competitive protein assay for c-GMP and cold GTP as substrate for the enzyme. The guanylate cyclase was found in cytosol and plasma membrane of bovine thyroid. The enzyme was relatively stable compared with adenylate cyclase of same thyroid membrane. TSH, LATS failed stimulation of guanylate cyclase of bovine thyroid membrane.