High blood triiodothyronine and the euthyroid state

A high excess of circulating T3 was observed in an euthyroid woman. Agarose gel electrophoresis of serum preincubated with 125I-T3 revealed an abnormal T3-binding in gamma-globulin zone. This binding interfered with the hormone radioimmunoassay. Immunological characterization identified this protein as an IgG-K and IgG-lambda polyclonal antibody that bound T3 but not T4. Scatchard analysis of 125I-T3 binding to the gamma-globulin fraction isolated showed a single class of binding sites with a high affinity Ka = 0.4 X 10(9) L/M and maximal binding capacity of 5.2 X 10(-9) M.     

A case of hypothyroidism with simultaneous presence of stimulating type anti-thyrotropin (TSH) receptor antibodies and anti-thyroxine (T4) autoantibodies.

We have examined a hypothyroid patient with stimulating type anti-thyrotropin (TSH) receptor antibodies and without blocking type anti-TSH receptor antibodies. Although she had high serum TSH (240 microU/ml) and low free triiodothyronine (FT3, 0.49 pg/ml) concentrations, which agree with physical findings of hypothyroidism, she had an unusually high free thyroxine (FT4) concentration (3.56 ng/dl). Incubation of her serum with 125I-T4, followed by precipitation with 12.5% polyethylene glycol (PEG) disclosed a higher binding of 125I-T4 (34.4%) than in normal controls, being 5-7%. In addition, binding of 125I-T4 to her serum gamma-globulin was completely displaced by the addition of unlabelled T4. From these results it was concluded that her serum contained anti-T4 autoantibodies. Treatment with synthetic T4 was begun and her thyroid function was monitored by sensitive TSH radioimmunoassay (RIA) and RIA of FT4 after PEG treatment. Since both sensitive TSH RIA and FT4 RIA results after PEG treatment give results concordant with the physical findings, it was concluded that both of the RIA results are useful for the evaluation of thyroid function in patients with thyroid hormone autoantibodies.     

Evidence for reutilization of surface receptors for alpha-macroglobulin.protease complexes in rabbit alveolar macrophages

Rabbit alveolar macrophages internalize α-macroglobulin ¹²⁵I-trypsin complexes subsequent to binding of complexes to high affinity surface receptors. Cells were capable of accumulating a 5–10 fold greater amount of αM · ¹²⁵I-T at 37°C than at 0°C. At 0°C cell-bound αM · ¹²⁵I-T was bound solely to surface receptors, whereas at 37°C the majority (85%) of cell-bound radioactivity was intracellular. The temperature-dependent accumulation of αM · ¹²⁵I-T did not reflect a change in surface receptor number or ligand-receptor affinity. Rather, the greater rate of uptake reflected continued internalization of αM · ¹²⁵I-T complexes. At 37°C cells took up 5–9 fmole αMT per μg cell protein per hr, whereas binding to surface receptors accounted for 0.5–0.7 fmole per μg cell protein. Once bound to surface receptors internalized αM · ¹²⁵I-T was localized in lysosomes, where it was degraded at a rate of 35–45% per hr. Following binding of αM · T to receptors at 37°C, but not at 0°C, unoccupied receptors could be found on the cell surface. Using cycloheximide to probe receptor turnover, I calculated that receptors were replenished at a rate of 15% per hr. Cells incubated in the presence of cycloheximide exhibited unaltered ligand uptake and catabolism for hours. Thus the reappearance of receptor activity during ligand uptake was not primarily due to de novo receptor synthesis. The rate of ligand uptake was a function of the number of surface receptors. Measurement of αM¹²⁵I-T binding to subcellular fractions did not reveal the presence of any intracellular reservoir of receptors. These observations are consistent with the hypothesis that continued ligand uptake reflects receptor reutilization.     

Temperature shifts induce the selective loss of alveolar-macrophage plasma membrane components

A shift in the incubation temperature of rabbit alveolar macrophages (0 degree C leads to 37 degrees C leads to 0 degree C) resulted in a 40-60% reduction in the ability of cells to bind alphamacroglobulin. 125I-trypsin complexes (alphaM. 125I-T). The reduction in binding activity did not reflect a disruption of cell integrity since the levels of intracellular components (lactate dehydrogenase, beta-N-acetyl-hexosaminidase) or other plasma membrane components (alkaline phosphodiesterase) were unaltered. Analysis of receptor-ligand interaction indicated that the temperature shift effected a decline in receptor number rather than an alteration in ligand-receptor affinity. Studies indicated that a temperature shift resulted in the loss of unoccupied receptors, and that ligand bound to receptors was not lost. However, after ligand internalization, receptors were removed by the temperature shift. The rate of receptor loss was maximal when cells were incubated at temperatures greater than 24 degrees C. Receptor loss was not prevented by treatment of cells with colchicine, cytochalasin B, or N-ethylamaleimide, but was prevented by treatment with the cross-linking agent paraformaldehyde. Data indicate that the reduction in alphaM. 125I-T binding activity resulted from shedding of receptors into the media since media obtained from temperature-shifted cells contained material that competed with cell-bound receptors for alphaM. 125I-T. Additionally, binding of alphaM. 125I-T was diminished on membrane fragments obtained from temperature-shifted cells. Incubation with Triton X-100, of cells whose receptors were occupied with alphaM. 125I-T, led to the extraction of 40% of cell-bound activity. However, no radioactivity was extracted from cells labeled with alphaM. 125I-T after a temperature shift. Measurement of ligand accumulation by control and temperature-shifted cells incubated at 20 degrees C indicated that control cells exhibited a subpopulation of receptors capable of binding ligand but only slowly internalizing it. This subpopulation was not present on temperature-shifted cells. These results indicate that surface receptors for alphamacroglobulin . protease complexes are heterogeneous and that the temperature shift resulted in the selective loss of membrane components.     

Identification of two structurally and functionally distinct sites on human platelet membrane glycoprotein IIb-IIIa using monoclonal antibodies

Platelet membrane glycoprotein IIb-IIIa exists as a calcium-dependent complex of two large peptides (designated IIb and IIIa) in Triton X-100 solutions, but it remains unknown if these peptides are subunits of one glycoprotein or are actually two individual glycoproteins in the intact platelet membrane. We used crossed immunoelectrophoresis to define the epitopes of two monoclonal antibodies to IIb-IIIa, then used these antibodies to study the structural and functional organization of IIb and IIIa in the platelet membrane. Human platelets solubilized in Triton X-100 were electrophoresed through an intermediate gel containing 125I-monoclonal IgG, then into an upper gel containing rabbit anti-human platelet antibodies. Our previously characterized antibody. Tab, and a new monoclonal antibody, T10, both bound to the immunoprecipitate corresponding to the IIb-IIIa complex. When platelets were electrophoresed after solubilization in 5 mM EDTA, 125I-Tab bound to the dissociated IIb polypeptide, but not to IIIa. In contrast, 125-I-T10 did not react with either IIb or IIIa. Thus, Tab recognizes a determinant on IIb, while T10 recognizes a determinant created only after the association of IIb and IIIa. Gel-filtered platelets from six normal donors bound 50,600 +/- 5,600 125I-T10 molecules/platelet and 47,800 +/- 11,200 125I-Tab molecules/platelet, consistent with IIb-IIIa being a heterodimer. 125I-T10 binding was identical in unactivated platelets and platelets stimulated with 10 microM ADP. However, platelets did not aggregate or bind 125I-fibrinogen until ADP was added. T10, but not Tab or nonimmune mouse antibody, inhibited ADP-induced platelet aggregation and 125I-fibrinogen binding. Our findings suggest that IIb and IIIa exist as subunits of a single membrane glycoprotein in unstimulated platelets. Fibrinogen binding appears to require not only the interaction of IIb and IIIa, but also some additional change occurring after platelet activation.     

Type II iodothyronine deiodinase provides intracellular 3,5,3'-triiodothyronine to normal and regenerating mouse skeletal muscle

The FoxO3-dependent increase in type II deiodinase (D2), which converts the prohormone thyroxine (T(4)) to 3,5,3'-triiodothyronine (T(3)), is required for normal mouse skeletal muscle differentiation and regeneration. This implies a requirement for an increase in D2-generated intracellular T(3) under these conditions, which has not been directly demonstrated despite the presence of D2 activity in skeletal muscle. We directly show that D2-mediated T(4)-to-T(3) conversion increases during differentiation in C(2)C(12) myoblast and primary cultures of mouse neonatal skeletal muscle precursor cells, and that blockade of D2 eliminates this. In adult mice given (125)I-T(4) and (131)I-T(3), the intracellular (125)I-T(3)/(131)I-T(3) ratio is significantly higher than in serum in both the D2-expressing cerebral cortex and the skeletal muscle of wild-type, but not D2KO, mice. In D1-expressing liver and kidney, the (125)I-T(3)/(131)I-T(3) ratio does not differ from that in serum. Hypothyroidism increases D2 activity, and in agreement with this, the difference in (125)I-T(3)/(131)I-T(3) ratio is increased further in hypothyroid wild-type mice but not altered in the D2KO. Notably, in wild-type but not in D2KO mice, the muscle production of (125)I-T(3) is doubled after skeletal muscle injury. Thus, D2-mediated T(4)-to-T(3) conversion generates significant intracellular T(3) in normal mouse skeletal muscle, with the increased T(3) required for muscle regeneration being provided by increased D2 synthesis, not by T(3) from the circulation.     

3,5,3'-triiodo-L-thyronine binding sites in nuclei of human trophoblastic cells

Nuclear binding sites of T3 in human trophoblastic cells were biochemically characterized. Nuclei were isolated by a combination procedure with mild homogenization of the freshly obtained trophoblastic tissue aged term gestation, centrifugations and Triton X-100 treatment. The isolated nuclei were incubated with various concentrations of 125I-T3 at 20 degrees C for 3 h. The total number of T3 binding sites per nucleus was approximately 650. The apparent association constant (Ka) was 6.0 X 10(9)M-1. Nuclear proteins extracted from purified nuclei with 0.4M KCl were able to bind T3 giving rise to nuclear thyroid hormone binding protein-T3 complexes and they were precipitated with bovine IgG, as a carrier protein, by 12.5% polyethylene glycol. Binding was maximum in 3 h incubation at 20 degrees C or in 18 h at 0 degrees C, while it dropped quickly at 37 degrees C. The binding characteristics were analyzed by Scatchard plots. In nuclear proteins obtained from 8 term placentae there was a single set of high affinity-low capacity T3 binding sites with Ka of 7.0 X 10(9)M-1. The capacity is about 62.7 fmol T3/mg DNA. The binding sites were found to be specific for L-T3, while L-T4 was about 100-fold less effective, rT3 ineffective, and D-T3 and D-T4 were roughly 1/8 and 1/5 as active as L-T3 and L-T4, respectively in displacing 125I-T3 from the binding sites. These data confirmed that human placenta is a target organ of thyroid hormones; trophoblastic cells contain T3 nuclear receptors which are biochemically similar to those isolated from liver, although the capacity is low.     

Binding and endocytosis of apo- and holo-lactoferrin by isolated rat hepatocytes.

We characterized binding and endocytosis of 125I-bovine lactoferrin by isolated rat hepatocytes. Iron-depleted (apo-Lf), approximately 30% saturated (Lf), and iron-saturated (holo-Lf) lactoferrin were used. At 4 degrees C, cells bound 125I-apo-Lf and 125I-holo-Lf with nearly identical apparent first order kinetics (t1/2 = approximately 42 min). Holo-Lf and apo-Lf competed with each other for binding. Hepatocytes bound lactoferrin optimally at pH greater than or equal to 7 but poorly at pH less than or equal to 6. Ca2+ (greater than or equal to 100 microM) enhanced Lf binding to cells, and holo-Lf remained monomeric with Ca2+ present as determined by gel filtration chromatography. With Ca2+, cells exhibited approximately 10(6) high affinity sites (Kd approximately 20 nM) and approximately 10(7) low affinity sites (Kd approximately 700 nM) for both apo- and holo-Lf. Without Ca2+, cells bound 125I-holo-Lf by the low affinity component only. EGTA and dextran sulfate together released greater than or equal to 90% 125I-Lf prebound at 4 degrees C, but individually removed separate populations of surface-bound 125I-Lf. Cells bound 125I-Lf in a Ca(2+)-dependent manner with dextran sulfate present. We conclude that the high affinity but not the low affinity sites require Ca2+; only the low affinity sites are dextran sulfate-sensitive. Neither transferrin nor asialo-orosomucoid blocked lactoferrin binding to hepatocytes. Some cationic proteins but not others inhibited lactoferrin binding. At 37 degrees C, hepatocytes endocytosed 125I-apo-Lf and 125I-holo-Lf similarly, and hyperosmolality (greater than 500 mmol/kg) blocked uptake by approximately 90%. These data support the proposal that hepatocytes regulate blood lactoferrin concentration by receptor-mediated endocytosis.     

Characteristics of the transport of structural analogs of thyroid hormone through hemato-encephalic barrier

The kinetics of competitive inhibition of 125I-T3 transport across the blood-brain barrier by two groups of thyroid hormone analogs (THA) was studied using the carotid injection technique. We confirm previous data that the transport of 125I-T3 was saturable; the Km of T3 transport was 1.2 microM. Our results also indicate that the mechanism of cross inhibition of T3 transport by THA with alanine side chain is competitive inhibition. The replacement of alanine side chain by an acetic acid group greatly reduced the affinity of the transport system. Consequently, the development of THA with selective tissue effects must take into account differential penetration rates of THA in the brain, when correlating biological effects with nuclear binding.     

Thyroid hormones inhibit the Ca2+ calmodulin-induced activation of myosin light chain kinase

L-Thyroxine (T4) and L-triiodothyronine (T3) specifically, inhibited myosin light chain kinase (MLC-kinase) from various tissues whereas inhibitory effects of T4 and T3 on other protein kinases such as protein kinase C, cAMP-dependent protein kinase, casein kinase I, casein kinase II and calmodulin kinase II were much weaker. T4 was a more potent inhibitor of MLC-kinase than T3. Kinetic studies showed that T4 behaved as a competitive inhibitor of MLC-kinase toward calmodulin (CaM) and that Ki value was 2.5 microM. The activity of the catalytic fragment of MLC-kinase, which is active without CaM, was not inhibited by T4. 125I-T4 gel overlay revealed that CaM did not bind T4 but MLC-kinase had 125I-T4 binding activity. These observations suggest that T4 binds at or near CaM binding domain of MLC-kinase and inhibits CaM-induced activation of MLC-kinase.     

Receptor-mediated uptake of hypertriglyceridemic very low density lipoproteins by normal human fibroblasts

Our previous studies showed that very low density lipoproteins, Sf 60-400 (VLDL), from hypertriglyceridemia subjects, but not VLDL from normolipemic subjects, suppress HMG-CoA reductase activity in normal human fibroblasts. To determine if this functional abnormality of hypertriglyceridemic VLDL resulted from differences in uptake of the VLDL by the low density lipoprotein (LDL) receptor pathway, we isolated VLDL subclasses from the d less than 1.006 g/ml fraction of normal and hypertriglyceridemic plasma by flotation through a discontinuous salt gradient for direct and competitive binding studies in cultured human fibroblasts. VLDL from the plasma of subjects with hypertriglyceridemia types 4 and 5 were at least as effective as normal LDL in competing for 125I-labeled LDL binding, uptake, and degradation when compared either on the basis of protein content or on a particle basis. By contrast, normolipemic Sf 60-400 VLDL were ineffective in competing with the degradation of 125I-labeled LDL, and Sf 20-60 VLDL (VLDL3) were less effective in reducing specific 125I-labeled LDL degradation than were LDL, consistent with their effects on HMG-CoA reductase activity. In direct binding studies, radiolabeled VLDL from hypertriglyceridemic but not normolipemic subjects were bound, internalized, and degraded with high affinity and specificity by normal fibroblasts. Uptake and degradation of iodinated hypertriglyceridemic VLDL Sf 100-400 showed a saturable dependence on VLDL concentration. Specific degradation plateaued at approximately 25 micrograms VLDL protein/ml, with a half maximal value at 6 micrograms/ml. The most effective competitor of hypertriglyceridemic VLDL uptake and degradation was hypertriglyceridemic VLDL itself. LDL were effective only at high concentrations. Uptake of normal VLDL by normal cells was a linear rather than saturable function of VLDL concentration. By contrast, cellular uptake of the smaller normal VLDL3 was greater than uptake of larger VLDL and showed saturation dependence. After incubation of normal VLDL with 125I-labeled apoprotein E, reisolated 125I-E-VLDL were as effective as LDL in suppression of HMG-CoA reductase activity, suggesting that apoE is involved in receptor-mediated uptake of large suppressive VLDL. We conclude that 1) hypertriglyceridemic VLDL Sf 60-400 are bound, internalized, and degraded by normal fibroblasts primarily by the high affinity LDL receptor-mediated pathway; 2) by contrast, normal VLDL, Sf 60-400 are bound, internalized, and degraded by normal fibroblasts primarily by nonspecific, nonsaturable routes; and 3) of the normal VLDL subclasses, only the smallest Sf 20-60 fraction is bound and internalized via the LDL pathway.     

Comparison of the nuclear and cytosolic forms of the Ah receptor from Hepa 1c1c7 cells: charge heterogeneity and ATP binding properties.

2-[125I]iodo-7,8-dibromo-p-dioxin ([125I]Br2DpD) and 2-[125I]iodo-3-azido-7,8-dibromo-p-dioxin ([125I]N3Br2-DpD) are both capable of binding to the Ah receptor (AhR) with a high degree of specificity in cultured Hepa 1c1c7 cells. After incubation with either [125I]N3Br2DpD or [125I]Br2DpD Hepa 1c1c7 cytosolic and high salt nuclear extracts were analyzed by sucrose density gradient analysis with the following results: (i) With both radioligands an approximately 9 S form of the AhR was observed in cytosolic extracts. (ii) Nuclear extracts labeled with [125I]N3Br2DpD revealed both approximately 6 S and approximately 9 S forms of the AhR. (iii) In contrast, analysis of nuclear extracts labeled with [125I]Br2DpD revealed only an approximately 6 S form of the AhR. The approximately 9 S [125I]N3Br2DpD-labeled AhR was preferentially extracted with 100 mM KCl from a nuclear fraction and mixed with monoclonal antibody 8D3, an anti-90-kDa heat shock protein antibody. Monoclonal antibody 8D3 was able to bind to the approximately 9 S nuclear form of the AhR and caused the receptor to sediment as a heavier complex on sucrose density gradients. This would indicate that the AhR can reside in the nucleus bound to 90-kDa heat shock protein. The [125I]N3Br2DpD-labeled approximately 6 S peak fractions were collected and subjected to denaturing two-dimensional gel electrophoresis. A comparison of [125I]N3Br2DpD-labeled cytosolic (9 S) AhR preparations with the nuclear (6 S) AhR by 2-D gel electrophoresis was performed. The cytosolic form of the AhR was present in the apparent pI range of 5.2-5.7; the nuclear form focused between 5.5 and 6.2. The [125I]N3Br2DpD-labeled nuclear extracts were incubated with ATP-agarose and 43% of the photoaffinity-labeled AhR bound to the affinity gel. In contrast, approximately threefold lower binding of [125I]N3Br2DpD-labeled receptor was obtained when GTP-, AMP-, or ADP-agarose was used. Only 2% of the [125I]N3Br2DpD-labeled cytosolic AhR was able to bind to ATP-agarose. These results suggest that after the AhR translocates into the nucleus the following biochemical changes occur: (i) The sedimentation value for the AhR changes from an approximately 9 S to an approximately 6 S species. (ii) The AhR attains the ability to bind with specificity to ATP. (iii) The AhR undergoes a shift to a more basic pI.     

Suppression of plasma thyroid hormone concentrations by cortisol in the European eel Anguilla anguilla

Female European eels, Anguilla anguilla, were given a single intra-arterial injection via a catheter of cortisol hemisuccinate at doses ranging from 3.5 to 35 micrograms (15 to 150 micrograms/kg body wt), yielding mean plasma cortisol levels of 87-410 ng/ml 2 hr after injection. Cortisol treatment (17.5 and 35 micrograms) significantly decreased plasma levels of thyroxine (T4) and triiodothyronine (T3) within 24 hr relative to those in control fish. Cortisol treatment (35 micrograms) appeared to increase the clearance rate of 125I-T3 from plasma and the proportionate uptake of radioactivity in certain tissues after injection of 125I-T3. Cortisol treatment had no apparent effect on the plasma clearance of 125I-T4 or tissue distribution of radioactivity after injection of 125I-T4.     

Subcellular localization and binding of 125I-triiodothyronine in calf thyroid

The subcellular distribution of 125I-T3 was studied in calf thyroid slices, under the same experimental conditions where T3 inhibits protein and RNA synthesis, labelled hormone was found mainly in the 20,000 X g supernatant. The specificity of each subcellular localization was determined by incubating the slices with 10(-5)M T3. Only in the purified nuclei a significant decrease was found, indicating a specific localization of the labelled hormone. When slices were incubated with 125I both labelled T3 and T4 were found in purified nuclei, indicating that endogenously synthesized hormones can reach thyroid nuclei. Purified thyroid nuclei were incubated with labelled T3 and increasing amounts of cold hormone. Specific binding reached a plateau after 90 min of incubation at 20 degrees C. When the displacement curves were analysed by a Scatchard plot a binding site with a Ka of 5.2 X 10(7) M-1 and a capacity of 3.0 X 10(-15) moles/microgram DNA was observed. Digestion of nuclei with trypsin and protease abolished completely the binding of 125I-T3 thus indicating the protein nature of the receptor. The hormone-receptor complex could be extracted with 0.4M KCI and eluted in the void volume after Sephadex G-25 column chromatography, similar to peripheral tissues nuclear T3 receptors. The present studies provide the first evidence for the existence of nuclear receptors for T3 in the thyroid, an event probably related to the autoregulatory mechanism.     

An improved and simplified method for the detection of thyroid hormone autoantibodies (THAA) in serum

We have developed and evaluated a new and simplified method for the detection of thyroid hormone autoantibodies (THAA) in serum. The method includes acidification of serum followed by adsorption of liberated thyroid hormones onto dextran-coated charcoal and then alkalinisation of the serum in assay buffer prior to performing a binding study. Using our method, specific binding of 125I-T4 to serum THAA in two patients with Hashimoto's thyroiditis was almost the same regardless of whether or not the sera had been preincubated with a large amount of cold T4. On the other hand, without the acid treatment, preincubation with cold T4 considerably inhibited the binding of 125I-T4 to serum THAA in both cases. These results indicate that serum THAA can be easily detected under conditions in which circulating thyroid hormones hardly affect the binding study by using our new sensitive method.     

A soluble binding protein specific for interleukin 1 beta is produced by activated mononuclear cells

Soluble interleukin 1 (IL 1) binding proteins were identified by gel filtration and covalent cross-linking of 125I IL 1 in normal human serum and inflammatory exudate. High molecular weight 125I IL 1 protein complexes occurred with both IL 1 alpha and IL 1 beta, however, high molecular weight binding appeared to be non-specific. One specific IL 1 beta binding protein was observed to elute at approximately 100 kDa on gel filtration when bound to 125I IL 1 beta. This complex migrated as a broad band at 60 kDa when covalently cross-linked and analyzed by SDS-PAGE. The protein did not bind 125I IL 1 alpha and 125I IL 1 beta binding was only displaceable by excess cold IL-1 beta. The production of the specific IL 1 beta binding protein was assessed in a number of cell populations. Unstimulated peripheral blood mononuclear cells (PBMNC) did not produce the binding protein, but stimulation with phytohemagglutinin (PHA) caused production within 24 hr and binding protein levels remained elevated for up to 7 days. Stimulation with lipopolysaccharide (LPS) and IL 1 alpha did not consistently induce synthesis of the binding protein. Ligand-binding studies were performed to compare solubilized EL 4 NOB.1 cell membrane IL 1 receptor (sIL 1R) with semi-purified IL 1 beta binding protein from pooled synovial fluid. The sIL 1R preparation bound ligand with an affinity of 168 pM while the IL 1 beta binding protein bound 125I IL 1 beta with an affinity of 370 pM. This protein may function as an important carrier molecule for IL 1 beta and determine its distribution and kinetics in vivo.     

Characterization of an intracellular hyaluronic acid binding site in isolated rat hepatocytes.

125I-HA, prepared by chemical modification at the reducing sugar, specifically binds to rat hepatocytes in suspension or culture. Intact hepatocytes have relatively few surface 125I-HA binding sites and show low specific binding. However, permeabilization of hepatocytes with the nonionic detergent digitonin results in increased specific 125I-HA binding (45-65%) and a very large increase in the number of specific 125I-HA binding sites. Scatchard analysis of equilibrium 125I-HA binding to permeabilized hepatocytes in suspension at 4 degrees C indicates a Kd = 1.8 x 10(-7) M and 1.3 x 10(6) molecules of HA (Mr approximately 30,000) bound per cell at saturation. Hepatocytes in primary culture for 24 h show the same affinity but the total number of HA molecules bound per cell at saturation decreases to approximately 6.2 x 10(5). Increasing the ionic strength above physiologic concentrations decreases 125I-HA binding to permeable cells, whereas decreasing the ionic strength above causes an approximately 4-fold increase. The divalent cation chelator EGTA does not prevent binding nor does it release 125I-HA bound in the presence of 2 mM CaCl2, although higher divalent cation concentrations stimulate 125I-HA binding. Ten millimolar CaCl2 or MnCl2 increases HA binding 3-6-fold compared to EGTA-treated cells. Ten millimolar MgCl2, SrCl2, or BaCl2 increased HA binding by 2-fold. The specific binding of 125I-HA to digitonin-treated hepatocytes at 4 degrees C increased greater than 10-fold at pH 5.0 as compared to pH 7.(ABSTRACT TRUNCATED AT 250 WORDS)     

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).     

Association between human erythrocyte calmodulin and the cytoplasmic surface of human erythrocyte membranes

This report describes Ca2+-dependent binding of 125I-labeled calmodulin (125I-CaM) to erythrocyte membranes and identification of two new CaM-binding proteins. Erythrocyte CaM labeled with 125I-Bolton Hunter reagent fully activated erythrocyte (Ca2+ + Mg2+)-ATPase. 125I-CaM bound to CaM depleted membranes in a Ca2+-dependent manner with a Ka of 6 x 10(-8) M Ca2+ and maximum binding at 4 x 10(-7) M Ca2+. Only the cytoplasmic surface of the membrane bound 125I-CaM. Binding was inhibited by unlabeled CaM and by trifluoperazine. Reduction of the free Ca2+ concentration or addition of trifluoperazine caused a slow reversal of binding. Nanomolar 125I-CaM required several hours to reach binding equilibrium, but the rate was much faster at higher concentrations. Scatchard plots of binding were curvilinear, and a class of high affinity sites was identified with a KD of 0.5 nM and estimated capacity of 400 sites per cell equivalent for inside-out vesicles (IOVs). The high affinity sites of IOVs most likely correspond to Ca2+ transporter since: (a) Ka of activation of (Ca2+ + Mg2+)-ATPase and KD for binding were nearly identical, and (b) partial digestion of IOVs with alpha-chymotrypsin produced activation of the (Ca2+ + Mg2+)-ATPase with loss of the high affinity sites. 125I-CaM bound in solution to a class of binding proteins (KD approximately 55 nM, 7.3 pmol per mg of ghost protein) which were extracted from ghosts by low ionic strength incubation. Soluble binding proteins were covalently cross-linked to 125I-CaM with Lomant's reagent, and 2 bands of 8,000 and 40,000 Mr (Mr of CaM subtracted) and spectrin dimer were observed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis autoradiography. The 8,000 and 40,000 Mr proteins represent a previously unrecognized class of CaM-binding sites which may mediate unexplained Ca2+-induced effects in the erythrocyte.     

Binding sites for tachykinin peptides in the brain and stomach of the dogfish, Scyliorhinus canicula.

In membranes of dogfish brain and stomach, two binding sites for tachykinins were identified. One site specifically bound [125I]-Bolton-Hunter substance P (BH-SP) and the rank potency of tachykinins to compete for BH-SP binding revealed similarities with the rank potency of an NK1 receptor. The pharmacology of the other site, which specifically bound [125I]-Bolton-Hunter scyliorhinin II (BH-Scy II), did not resemble any of the mammalian tachykinin receptors. The rank potency to inhibit BH-Scy II binding to this second site was: scyliorhinin II approximately scyliorhinin I greater than eledoisin approximately substance P approximately neurokinin A greater than phyllomedusin approximately physalaemin greater than [Sar9Met(O2)11]substance P. Neurokinin B and senktide did not displace BH-Scy II binding. In addition, nucleotide analogues inhibited BH-SP binding but not BH-Scy II binding. Our binding data suggest the existence of a mammalian-like NK1 receptor and of a nonmammalian tachykinin receptor in the dogfish.