Monoclonal antibodies to human beta-interferon produced by adoptive transfer in irradiated mice.

Three murine anti-human beta-interferon (IFN-beta) monoclonal antibodies have been isolated following adoptive transfer of immune spleen cells. Adoptive transfer was used to increase the specific efficiency of the fusion. These antibodies have been used to define two epitopes on IFN-beta; the antiviral, antiproliferative, and immunomodulatory effects of IFN-beta are associated with one of these epitopes.

Pattern of medium proteins radiolabelled after culture of Day 13 to 16 pig conceptus tissue with [3H]leucine and absence of chorionic gonadotrophin-like activity.

Day 13-16 pig conceptus tissue was cultured for 24 h in medium containing [3H]leucine. The patterns of radioactively labelled proteins that were released into the medium during culture were analysed by SDS-polyacrylamide gel electrophoresis and fluorography. Day-13 conceptuses released two major radiolabelled proteins of Mr 23 000 and 26 000 and Day 14-16 conceptuses released these as well as proteins of Mr 14 000, 19 000, 44 000, 50 000 and 88 000. Various immunological and biological tests for a human chorionic gonadotrophin-like activity were performed on tissue extracts and on culture medium, but there was no evidence for its presence in the pig conceptus at Day 13-16 of gestation.

Resolution of radioiodinated thyrotropin into receptor active and inactive components by column chromatography.

Following radioiodination by the lactoperoxidase method and subsequent purification on Sephadex G100, it was found that [125I]TSH exhibited varying degrees of binding activities to the thyrotropin receptor. In order to further purify the radiolabeled hormone, the [125I]TSH preparation was chromatographed on Sepharose 6B. Two peaks of radioactive material (Peaks I and II) were recovered, containing approx. 60% of the applied radioactivity. Upon elution with Mg2+, the remainder of the radiolabeled material was recovered as a single peak (Peak III). Characterization of these 3 peaks by radioimmunoassay demonstrated that all 3 were immunocompetent, although Peaks I and III were 3-4-fold more immunoreactive than Peak II. Analysis by radioreceptor assay indicated that Peak III showed an increase in receptor-binding capacity (in comparison with the [125I]TSH preparation purified by Sephadex G100 alone), while both Peaks I and II exhibited significantly reduced binding activity. In contrast, human TSH (NIH) chromatographed mainly as a receptor inactive peak, although it was fully immunocompetent. Scatchard analysis of receptor binding to bovine [125I]TSH from Peak III yielded a curvilinear plot with affinities similar to those we have previously reported for [125I]TSH purified by Sephadex G100 chromatography. The total number of binding sites, however, increased proportionally with the active fraction of the [125I]TSH preparation. Since the mass of bound hormone is calculated from the percent bound of total radioactivity and only a fraction of the measured total participates in the binding, it is therefore necessary to correct for the inactive fraction when calculating the total receptor number.

Regulation of thyroid adenylate cyclase: guanyl nucleotide modulation of thyrotropin receptor-adenylate cyclase function.

The role of guanyl nucleotides in regulating the hormonal responsiveness of adenylate cyclase was studied in thyroid plasma membranes. Guanyl-5'-yl-imidodiphosphate [Gpp(NH)p] alone stimulated the enzyme. At a low ATP concentration (0.2 mM), TSH alone had little or no effect, but when added with Gpp(NH)p, it resulted in a dose-dependent increase of enzyme activity. Kinetic studies revealed that Gpp(NH)p alone stimulated adenylate cyclase activity only after a 10-min lag. TSH abolished the lag, resulting in an apparent increase in activity and a lowering of the activation constant for Gpp(NH)p. GTP caused an initial increase in activity at 2 min, followed by a gradual decline below basal levels. This inhibition was not prevented by TSH. Further examination revealed that GDP caused inhibition of Gpp(NH)p-stimulated activity in a competitive manner, suggesting that conversion of GTP to GDP may be responsible for the time-dependent decay seen with GTP. To study the effects of guanyl nucleotides on coupling of the TSH receptor to adenylate cyclase, plasma membranes were preactivated with saturating amounts of Gpp(NH)p and washed extensively to remove unbound Gpp(NH)p. Incubation of preactivated membranes with either Gpp(NH)p or TSH gave no further stimulation of adenylate cyclase. However, Gpp(NH)p plus TSH produced 30% more stimulation. In contrast, the addition of TSH plus GDP to preactivated membranes led to a dose-dependent decrease in activity. Furthermore, promotion of further stimulation by TSH plus Gpp(NH)p was competitively inhibited by GDP, in the same manner as untreated membranes. This suggested that the dual action of TSH, i.e, stimulation and inhibition, is governed and mediated by specific guanyl nucleotides. Analysis of guanyl nucleotides effects on the binding of [125I[iodo-TSH to membranes revealed that although some inhibition of binding exists, this effect is 1) unrelated to the concentration effect of nucleotides on adenylate cyclase, and 2) not specific for guanosine phosphates. Scatchard analysis of TSH binding in the presence of guanyl nucleotides demonstrated that even at high concentrations, GTP had no effect on the high affinity, low capacity receptor for TSH. These results suggest that GDP or Gpp(NH)p plays no role in modulating TSH-receptor interaction. Thus, guanyl nucleotide regulation of TSH action appears to be limited to adenylate cyclase.

Dissociation kinetics of the thyrotropin-receptor complex. Characterization of a slowly dissociable component.

The kinetics of the dissociation of thyrotropin (TSH) from human thyroid plasma membranes were studied in an attempt to further understand the molecular dynamics of the TSH--receptor interaction. Dissociation of bound [125I]TSH from thyroid plasma membranes was a biphasic process consisting of rapidly and slowly dissociable components, RDC and SDC, respectively. The dilution induced dissociation of bound [125I]TSH was enhanced by the addition of excess TSH (DEC). DEC was proportional to the dose of unlabeled TSH and its magnitude increased linearly with temperature. These results are in contrast to those reported for the kinetics of [125I]insulin dissociation. The functional significance of DEC remains largely unexplained. It was found that the fraction of SDC was dependent upon time of association in a temperature-dependent and apparently saturable process. It could not be attributed to alterations in the electrophoretic, immunologic or binding properties of [125I]TSH. Furthermore, no correlation was observed between generation of SDC and change in the Scatchard profile of TSH binding, in contrast to studies on growth hormone. These data suggest that, like some other polypeptide hormones, binding of TSH to its receptor does not proceed according to laws describing simple, rapidly reversible, bimolecular reactions. Furthermore, bound TSH undergoes a receptor-mediated conversion from a rapidly to a slowly dissociable state with time of incubation.

Thyrotropin receptors in normal human thyroid. Nonclassical binding kinetics not explained by the negative cooperativity model.

Saturation analysis of equilibrium binding of iodinated thyrotropin (125I-TSH) to normal human thyroid preparations yielded linear Scatchard plots under non-physiological conditions of pH 6.0 or 20 mM Tris/acetate buffer, pH 7.4. The apparent equilibrium dissociation constant of this binding was approximately 10(-8) M. By contrast, nonlinear plots were obtained under standard conditions of pH 7.4 and 40 mM Tris/acetate buffer. Resolution of the components of these curves by computer analysis revealed the presence of at least two classes of binding sites, one of which is of a low capacity and high affinity (approximately 10(-10) M) consistent with receptor binding. The other component is of a high capacity and lower affinity. Binding to non-target tissues of muscle, parathyroid, mammary carcinoma, and placenta was only demonstrable at pH 6.0 or in 20 mM Tris/acetate buffer, pH 7.4, yielding linear Scatchard plots with similar binding affinity (approximately 10(-8)M) to normal thyroid but much reduced capacity. Preincubation of thyroid tissue at 50 degrees C resulted in an apparent selective loss of the high affinity component of binding measured under standard conditions. Kinetic experiments on the dissociation of bound 125I-TSH were undertaken to determine whether the non-linearity of Scatchard plots was due to two or more classes of binding sites or negative cooperativity. It was found that the experimental determinant that is presently ascribed to a negative cooperativity phenomenon regulating receptor affinity (i.e. an enhanced dilution-induced dissociation rate in the presence of excess native hormone), although apparently hormone-specific, was demonstrated under nonphysiological binding conditions and in non-target tissue. Significantly, the phenomenon was found under conditions of pH 6.0 or 20 mM Tris where a linear Scatchard plot was obtained. The evidence thus suggests that 125I-TSH binds to heterogeneous binding sites (of which the high affinity is probably the receptor for TSH) and that the enhanced dilution-induced dissociation of bound hormone by native hormone for this system, is only a characteristic of the low affinity binding site (maybe gangliosides).

Contribution of negative cooperativity to the thyrotropin-receptor interaction in normal human thyroid: kinetic evaluation.

The kinetics of 125I-labeled thyrotropin (125I-TSH) binding to human thyroid receptors are presented. At pH 6.0, binding was maximal (30--35%) and there was one class of binding sites [Kd = 6.8 X 10(-9) M; binding capacity (Ro) = 57 pmol/mg of protein]. At pH 7.4, Scatchard plots of binding were nonlinear, indicating either a single class of negatively cooperative sites (Kd = 3.7 X 10(-9) M; Ro = 26 pmol/mg of protein) or, alternatively, independent high- (Kd = 5.0 X 10(-10) M; Ro = 3 pmol/mg of protein) and low-affinity (Kd = 1.7 X 10(-8) M; Ro = 26 pmol/mg of protein) binding sites. The role of negative cooperativity was evaluated from the rates of association and dissociation at pH 7.4. The kinetically determined binding constants (Kd = 1.7 X 10(-11) M; Ro = 2 pmol/mg of protein) were more similar to those determined for the high-affinity component than to those predicted from the negative cooperativity model. Dissociation of bound TSH was independent of initial site occupancy over a 40-fold range, corresponding to a 100-fold range of free TSH concentration. The dissociation rate of 125I-TSH was enhanced by unlabeled TSH to a similar degree, irrespective of initial binding site occupancy. Because the negative cooperativity model does not accommodate these data, it is concluded that TSH receptors in human thyroid behave kinetically and at equilibrium as a single class of high-affinity sites up to TSH concentrations well above the physiological range.