Maximum Specific Binding Research Articles

Insulin action during pregnancy. Studies with the euglycemic clamp technique.

To assess the mechanisms responsible for the insulin resistance associated with both normal human pregnancy and gestational-onset diabetes, we have measured exogenous glucose disposal using sequential insulin infusions with the euglycemic glucose clamp technique and erythrocyte insulin binding. Three groups of women were studied: nonpregnant women with normal glucose tolerance (N = 7, mean age 32.9 +/- 2.1 yr), pregnant women with normal glucose tolerance (N = 5, mean age 24.8 +/- 3.5 yr), and pregnant women with gestational-onset diabetes (N = 5, mean age 34.6 +/- 2.6 yr). Despite normal plasma glucose levels obtained during a 100-g oral glucose tolerance test, plasma insulin levels were significantly elevated in the pregnant women compared with the nonpregnant control subjects, suggesting a state of insulin resistance. Insulin binding to erythrocytes was similar in all three groups (maximum specific binding being 5.0 +/- 0.6%, 5.5 +/- 1.1%, and 6.0 +/- 0.7% in nonpregnant, nondiabetic pregnant, and gestational-onset diabetic women, respectively). In vivo peripheral insulin action was measured using the euglycemic glucose clamp technique during an insulin infusion of 40 mU/m2 X min, with blood glucose clamped at a concentration of 75 mg/dl using a variable glucose infusion. Glucose infusion rates were 213 +/- 11 mg/m2 X min, 143 +/- 23 mg/m2 X min, and 57 +/- 18 mg/m2 X min in nonpregnant, nondiabetic pregnant, and gestational-onset diabetic women, respectively. This demonstrates that pregnant subjects display a state of insulin resistance, and that this appears to be more marked in gestational-onset diabetic subjects. To further define the possible mechanism of insulin resistance during pregnancy, the insulin infusion rate was increased to 240 mU/m2 X min and further euglycemic clamp measurements performed. Glucose infusion rates were 372 +/- 11 mg/m2 X min, 270 +/- 31 mg/m2 X min, and 157 +/- 26 mg/m2 X min, in nonpregnant, nondiabetic pregnant, and gestational-onset diabetic women, respectively. This demonstrates a shift to the right of the dose-response curve of insulin action and suggests that the insulin resistance of pregnancy may include a decrease in presumed "maximum" insulin responsivity. In four subjects, studies were repeated in the postpartum period, and these demonstrated that the insulin resistance of pregnancy is ameliorated shortly after delivery. These studies suggest that the insulin resistance of pregnancy results from a target cell defect in insulin action beyond the initial step of insulin binding to cellular receptors, a postreceptor (or postbinding) defect in insulin action.

Properties and compartmentalization of the testicular receptor for 1,25-dihydroxyvitamin D 3

Adult rat testis contains a specific, high-affinity, low-capacity binding protein for 1,25-dihydroxyvitamin D 3 (1,25-(OH) 2D 3) with properties similar to 1,25-(OH) 2D 3 receptors in other tissues. The receptor sediments at 3.5 ± 0.2 S 20,w in high-salt sucrose density gradients, but aggregates in low-salt gradients. Binding of 1,25-(OH) 2D 3 was abolished by trypsin, but not by DNase or RNase. Binding was also heavily reduced by the sulfhydryl alkylating agent, N-ethylmaleimide, and by the mercurial reagent, mersalyl, showing that free, reduced SH-groups are necessary for hormone-binding activity. The receptor shows high affinity for 1,25-(OH) 2D 3 ( K d = 3 × 10 −11 M), but low capacity ( N max = 8 fmol/mg protein) and is specific for 1,25-(OH) 2D 3 (Affinity: 1,25-(OH) 2D 3 > 1,24(R),25-(OH) 3D 3 > 25-OH-D 3 > 1α-OH-D 3 > 24(R),25-(OH) 2D 3 > 17β-estradiol, testosterone, dexamethasone, R5020, progesterone). With 0.6 nM [ 3H]1,25-(OH) 2D 3 and at 0°C, maximum specific binding was achieved after 4 h, and the occupied receptors were stable for more than 24 h. The dissociation of hormone-receptor complexes was temperature-dependent and very slow at low temperature ( t 1 2 (0°C) > 48 h). At 0°C, the second order association rate constant and the pseudo-first order dissociation rate constant were 2.7 × 10 7M −1 min −1 and 2 × 10 −5 min −1, respectively. Receptors for 1,25-(OH) 2D 3 are present in similar amounts in isolated seminiferous tubules and interstitial tissue of adult rats. No specific binding of [ 3H]1,25-(OH) 2D 3 could be detected in cultured immature Sertoli cells, cultured immature peritubular (myoid) cells or crude germ cells.

Binding of prostaglandin E 1 to human erythrocyte membrane

Prostaglandin E 1 is known to alter the structural and functional characteristics of red blood cells, yet, little is understood about the membrane receptors mediating this process. We therefore studied the binding of tritium-labeled prostaglandin E 1 to the intact human erythrocyte membrane and demonstrated that the interaction is highly specific, rapid, saturable and reversible. Scatchard analysis of prostaglandin E 1 binding to the membrane preparations showed the presence of two independent classes of prostaglandin E 1 binding sites which differed in their affinity for the autacoid. The high-affinity class had K d = 3.6 · 10 −9 M and the low-affinity class had K d = 5.6 · 10 −5M. The optimum pH for the binding of [ 3H]prostaglandin E 1 to the erythrocyte membrane was found to be around 7.5 and maximum specific binding occured at a concentration of 5 mM Mg 2+ in the incubation mixture. [ 3H]Prostaglandin E 1 bound to the membrane preparation could not be displaced by GTP or by its stable derivative Gpp[NH]p. However, prostaglandin E 1 bound to the erythrocyte membrane preparation could be rapidly displaced by cyclic AMP. The IC 50 (concentration of the nucleotide displacing 50% bound [ 3H]prostaglandin E 1 from the membrane) was 75 nM. Other adenine nucleotides or cyclic GMP could not substitute for cyclic AMP. Unlike the right-side-out erythrocyte membrane, the inside-out membrane preparations do not bind [ 3H]prostaglandin E 1. Treatment of right-side-out erythrocyte membrane preparation with neuraminidase markedly decreases the binding of prostaglandin E 1. Incubation of the erythrocyte membrane preparation with trypsin resulted in total loss of the binding activity. These results indicate that the prostaglandin E 1 binding sites located on the cell surface and sialic acid residues are required for prostaglandin E 1 binding to the human erythrocytes. These results also indicated that the binding sites are glycoprotein in nature.

Tumor promoter 12-O-tetradecanoylphorbol-13-acetate receptors in normal human transitional epithelial cells.

As a prelude to study the promotion with TPA of in vitro transformation of human urothelial cells (HUC) in culture, we characterized tumor promoter TPA receptors in primary cultures of HUC. [3H]TPA bound specifically to intact living HUC; maximum specific binding was attained in approximately 30 min at 37 degrees C. [3H]TPA bound to HUC in a saturable and competitive manner. Scatchard analysis of specific binding to intact cells displayed a single slope corresponding to an equilibrium dissociation constant (Kd) of 0.56 nM; at saturation TPA-binding capacity was 2.37 pmol/10(6) HUC (1.43 X 10(6) sites per cell). [3H]TPA bound specifically and with high affinity to the particulate fractions of HUC; binding was both saturable and reversible. Saturation of the specific binding of [3H]TPA occurred at approximately 1 nM at 4 degrees C. Scatchard analysis of specific binding to the particulate fraction displayed a single slope corresponding to a Kd of 1.08 nM; at saturation TPA-binding capacity was 2.05 pmol/mg protein (750 000 molecules per HUC). [3H]TPA binding was inhibited by the biologically active phorbol ester, phorbol didecanoate, whereas inactive phorbol did not compete for TPA binding. Binding was not affected by sodium saccharin, epidermal growth factor, retinoic acid or dexamethasone. [3H]TPA bound specifically to the HUC cytosolic fraction but only in the presence of calcium and phosphatidylserine. Calcium-activated and phospholipid-sensitive protein kinase activity was detected in HUC fractions. These results indicate the presence of high-affinity specific receptors for TPA in HUC.

Nuclear thyroxine and triiodothyronine binding in mononuclear cells in dependence of age.

Nuclear binding of thyroxine (T4) and triiodothyronine (T3) in mononuclear blood cells was investigated in 12 young (age 16-30 years) healthy subjects (group A), in 12 middle-aged (age 31-60 years) healthy subjects (group B) and in 12 elderly (61-90 years) healthy subjects. Serum free T3 was depressed in group C as compared to the younger age groups, whereas serum free T4 and TSH did not differ between the groups. Maximal specific nuclear binding capacity for both T4 and T3 decreased with increasing age, T4 group A: 1.2 fmol T4/100 micrograms DNA, group B: 1.2 fmol T4/100 micrograms DNA, group C: 0.7 fmol T4/100 micrograms DNA; T3 group A: 1.7 fmol T3/100 micrograms DNA, group B: 1.0 fmol T3/100 micrograms DNA, group C: 0.9 fmol T3/100 micrograms DNA. The equilibrium association constant (Ka) for T4 increased with age, group A: Ka = 3.3 X 10(9) l/mol, group B: Ka = 3.2 X 10(9) l/mol, group C: Ka = 6.4 X 10(9) l/mol, whereas Ka for nuclear binding of T3 decreased with age group A: Ka = 3.9 X 10(9) l/mol, group B: Ka = 5.9 X 10(9) l/mol, group C: Ka = 1.8 X 10(9) l/mol. We conclude that, whereas the opposite variations of nuclear capacity and binding affinity for T4 tend to preserve the nuclear T4 concentration, the nuclear T3 concentration definitely decreases with age. The unaltered serum levels of TSH suggest that the decrease of both serum levels of free T3 and the nuclear T3 concentration might represent physiologically changes in old age.

Sex steroid receptors in normal and hyperplastic human prostate.

The concentrations of sex steroid receptors (per unit DNA) were measured in normal periurethral and peripheral prostatic tissue samples from seven men (mean age 64 years; range 54-71 years) undergoing cystectomy for bladder cancer, and in hyperplastic nodules from 15 men with BPH (mean age 69 years; range 60-89). Occupied androgen (AR) and estrogen (ER) receptors were measured with an improved exchange procedure, where receptor-binding sites were stabilized by a combinatorial procedure involving careful washout of extracellular secretory products (including proteases) prior to homogenization, inclusion of 0.5 mM phenylmethyl sulfonylfluoride (PMSF) and 20 mM molybdate in the exchange medium, and long-term incubation at 0-4 degrees C. Bound radioligands were separated by a hydroxylapatite (HAP) batch adsorption procedure. Maximal specific exchange binding of 3H-R 1881 or 3H-estradiol in total homogenates of human prostate samples was achieved after incubation periods of about 72 h at 0-4 degrees C. In contrast, progestin receptors (PR) were readily available for binding 3H-R 5020; thus overnight binding at 0-4 degrees C was routinely used to measure PR. Binding specificities and equilibrium binding constants (calculated from 8-point Scatchard plots, correcting for nonsaturable binding) were found to be characteristic for AR, PR, and ER, respectively. The receptor results obtained in this study demonstrate that no significant differences existed in total AR per unit DNA between hyperplastic and either central or peripheral prostatic tissue samples; PR was present in both zones of normal prostatic tissue as often as in BPH samples, with PR concentrations significantly lower in hyperplastic samples; and ER was irregularly detected in both normal and hyperplastic tissue in low concentration relative to AR and PR; the frequency of ER detection was much lower in BPH than in normal prostate tissue. Studies of steroid receptor content relative to enzyme markers specific for epithelial and stromal cells in BPH samples showed a positive correlation between acid phosphatase activity (a specific marker for epithelial cells) and both AR and PR. No correlation was observed between AR or PR with either prolyl hydroxylase or myosin ATPase (specific markers for stromal cells). These observations suggest that PR, as well as AR, is primarily associated with the epithelial elements of prostate. Because of the relative infrequency of ER, similar correlation of ER with enzyme markers was not possible.

Asialo von Willebrand factor interactions with platelets. Interdependence of glycoproteins Ib and IIb/IIIa for binding and aggregation.

Asialo von Willebrand factor (AS-vWf) binds to and aggregates normal human platelets in the absence of ristocetin. Maximal specific binding of AS-vWf is 1-2 micrograms vWf protein/10(8) platelets. Despite the specificity of the binding, only 60% of the bound AS-vWf can be dissociated after equilibrium has been reached. We investigated the site of binding and the mechanism of aggregation of platelets by AS-vWf by (a) pre-incubating platelets with either of two monoclonal antibodies, one against glycoprotein Ib (GPIb) or a second against the glycoprotein IIb/IIIa complex (GPIIb/IIIa), and (b) varying the concentration of fibrinogen in the medium. The results of our studies indicate that AS-vWf binds initially to GPIb. This binding then results in the exposure of receptors for AS-vWf on GPIIb/IIIa. In the presence of plasma fibrinogen, both AS-vWf and fibrinogen bind to GPIIb/IIIa. In the presence of plasma fibrinogen, 50% more AS-vWf binds to the platelet, and this additional AS-vWf binds almost exclusively to GPIIb/IIIa. Despite this enhanced binding of AS-vWf in the absence of fibrinogen, platelet aggregation is much less than that which occurs in the presence of plasma fibrinogen. Comparative studies of AS-vWf binding to normal platelets and the platelets of patients with Glanzmann's thrombasthenia reveal decreased binding to the thrombasthenic platelets and a marked decrease in the extent of platelet aggregation. These studies indicate that AS-vWf binding to, and ensuing aggregation of, platelets is different from that observed with intact vWf protein when platelets are stimulated with either ristocetin or thrombin. The AS-vWf binds to GPIb which, in turn, makes additional AS-vWf receptors available on GPIIb/IIIa. If plasma fibrinogen is present, it competes with the AS-vWf for binding to GPIIb/IIIa and causes aggregation of platelets. In the presence of plasma fibrinogen, more of the AS-vWf binds to GPIIb/IIIa, but this AS-vWf is much less effective than fibrinogen in supporting platelet aggregation.

Displacement by α-Adrenergic Agonists and Antagonists of 3H-Prazosin Bound to the α-Adrenoceptors of the Dog Aorta and the Rat Brain

To characterize the α1-adrenoceptors in the dog aorta and the rat brain and to assess the antagonistic potencies of various chemicals inclusive of newly synthesized ones, radioligand binding assays were performed, and the potencies thus obtained were compared with those obtained from pharmacological observations. Reproducible binding to and displacement from the dog aorta of 3H-prazosin were observed. The rank orders of the inhibition of 3H-prazosin binding to the membrane preparations of the aorta expressed as IC50 (the concentration of drugs inhibiting 50% of maximal specific binding of 3H-prazosin) were: prazosin> YM09538 > phentolamine > yohimbine > phenoxybenzamine > labetalol > S-596 > dibenamine > K-351 > propranolol > hydralazine > N-696 for α-blockers and I-epinephrine > clonidine > I-norepinephrine > phenylephrine > I-isoproterenol for agonists. IC50 values of phenoxybenzamine, labetalol, dibenamine and K-351 obtained in the brain preparations were higher than those obtained in the preparation of the aorta. There was a good correlation (r=0.90) between the IC50 values obtained in the dog aorta and in the rat brain, suggesting that the α-adrenergic receptors in the dog aorta and the rat brain resemble each other. Good correlations (aorta, r=0.97 and brain, r=0.94) were also observed between IC50 values derived from the binding assay in the aorta or the brain and the pA2 values obtained as regards to the contractile response of the rat aorta to phenylephrine. Thus, this method could be useful for the assessment of newly synthesized chemicals as α-adrenergic antagonists.

Nuclear Thyroxine Receptors and Cellular Metabolism of Thyroxine in Obese Subjects before and after Fasting

Nuclear binding of thyroxine (T4), cellular deiodination of T4 and nuclear accumulation of endogenous triiodothyronine (T3) were investigated in mononuclear blood cells in 5 obese persons before and after 7 days of total fast. Serum-free T4, serum-free T3 and serum thyrotropin (TSH) did not differ between lean persons and obese fed subjects, but fasting induced a significant decrease of serum-free T4 and serum-free T3 (T4 from 16.6 to 14.4 pmol/l, T3 from 5.7 to 3.2 pmol/l). The maximal specific binding capacity (MBC) for T4 was decreased in fed obese subjects (MBC = 0.6 fmol T4/100 micrograms DNA, p less than 0.05) compared to normal weight persons (MBC = 1.3 fmol T4/100 micrograms DNA), whereas the equilibrium association constant (ka) did not differ. During complete fast, the MBC increased (MBC = 1.5 fmol T4/100 micrograms DNA, p less than 0.05). Neither total deiodination of T4 nor endogenous nuclear T3 accumulation differed between the groups suggesting that cellular T4 to T3 conversion is independent of the nutritional state. We suggest that the observed increase in nuclear T4 receptor capacity may compensate the decrease in serum thyroid hormone levels.

Stereospecific receptors for substance P on cultured human IM-9 lymphoblasts.

The neuropeptide substance P (SP), which has been demonstrated to bind specifically to human blood T lymphocytes and to stimulate their uptake of [3H]thymidine and [3H]leucine, now is shown to bind stereospecifically to cultured human lymphoblasts of the IM-9 line. The specific binding of [3H]SP by IM-9 lymphoblasts increases linearly with the concentration of IM-9 lymphoblasts, achieves a plateau after approximately 15 to 20 min at 4 degrees C and 4 to 6 min at 37 degrees C, and is rapidly reversible at both 4 degrees C and 37 degrees C. The binding of [3H]SP at steady-state conditions demonstrates a dissociation constant (KD) of 0.65 +/- 0.19 nM (mean +/- SD, n = 5) and 22,641 +/- 6143 receptors per IM-9 lymphoblast. Maximal specific binding of [3H]SP to IM-9 lymphoblasts is observed at pH 7.4 and is dependent on the presence of Mg2+, but not Ca2+, in the medium. The peptide structural determinants of the inhibition of binding of [3H]SP to IM-9 lymphoblasts by substituent peptides and homologs of SP indicate that the receptors recognize predominantly the carboxy-terminal portion of SP. The characteristics of the interaction of SP with IM-9 lymphoblasts suggests a receptor-directed mechanism by which neuropeptides may modulate specifically the contributions of lymphocytes to immunity.

Changes of [ 3H]spiroperidol binding in the renal artery of aged rabbits

Using mature (12 months), old (70 months) rabbits and 3H-labeled spiroperidol, the characteristics of renal artery dopamine receptors were studied. In old age the dissociation constant of [ 3H]spiroperidol was unchanged, while maximum specific binding was reduced more than 45%. The present data seem to indicate an impairment of peripheral dopaminergic function in ageing.

Identification of 2-tetradecylglycidyl coenzyme A as the active form of methyl 2-tetradecylglycidate (methyl palmoxirate) and its characterization as an irreversible, active site-directed inhibitor of carnitine palmitoyltransferase A in isolated rat liver mitochondria.

Methyl-2-tetradecylglycidic acid (MeTDGA) has been hypothesized to inhibit fatty acid oxidation by irreversible, active site-directed inactivation of carnitine palmitoyltransferase A after being converted to TDGA-CoA. Using synthetic TDGA-CoA, this hypothesis has been confirmed. Assessing enzyme inhibition in an isolated rat liver mitochondrial system, TDGA-CoA (synthetic or enzyme prepared) was more potent than TDGA or MeTDGA and retained activity in the absence of CoA or Mg2+-ATP. It inhibited palmitoyl-CoA but not palmitoyl carnitine oxidation. Enzyme inactivation was exponential, stereospecific, and fast (t0.5 = 38.5 s with 100 nM (R)-TDGA-CoA). TDGA-CoA was identified as a complexing type irreversible inhibitor (Ki approximately 0.27 microM) by the double reciprocal relationship between the pseudo-first order inactivation rate and its concentration, by the inverse dependence of the second order rate constant on its concentration, and by the independence of the first order rate from the enzyme concentration. Palmitoyl-CoA, CoA, and malonyl-CoA protected the enzyme, while L-carnitine and palmitoyl-L-carnitine were without effect. [3-14C] TDGA-CoA labeled a protein, Mr = 90,000, with a time course which paralleled that of enzyme inhibition; maximum specific binding was 16 pmol/mg of mitochondrial protein.

Measurement of specific [3H]-ouabain binding to different types of human leucocytes.

We have studied the specific binding of [3H]-ouabain to intact mononuclear leucocytes (82% lymphocytes) and polymorphonuclear leucocytes. In both types of cells [3H]-ouabain binding was saturable, confined to a single site of high affinity, slow to reach equilibrium, slow to reverse, temperature-dependent, competitively antagonized by potassium, and facilitated by the presence of divalent cations. The equilibrium dissociation constants were 2.4 +/- 0.7 nmol/l (polymorphs) and 2.4 +/- 0.4 nmol/l (mononuclear cells) (NS). The values of maximal specific ouabain binding, measured by Scatchard analysis of concentration vs binding curves (Bmax), were 33.9 +/- 6.0 fmol/10(6) cells (polymorphs) and 59.3 +/- 11.6 fmol/10(6) cells (mononuclear cells) (P less than 0.02). The corresponding numbers of sites per cell were 20415 +/- 3616 and 35712 +/- 6986 respectively (P less than 0.02). When the numbers of binding sites were expressed per square micron of cell surface area the difference between the two cell types was proportionately greater (83 and 186 sites per micron 2 respectively). We conclude that the [3H]-ouabain binding sites on mononuclear and polymorphonuclear leucocytes are similar in nature, but different in both number and density on the cell surface. Measurements of Bmax in mixed cell populations should therefore take account of cell type as well as cell size and number.

Characterization of low density lipoprotein binding to human adipocytes and adipocyte membranes.

125I-labeled low density lipoprotein (LDL) binding to purified plasma membranes prepared from freshly isolated human adipocytes was saturable, specific, and displaceable by unlabeled ligand. The maximum specific binding capacity measured at saturating concentrations of 125I-LDL was 1.95 +/- 1.17 micrograms of LDL bound/mg of membrane protein (mean +/- S.D., n = 16). In contrast to cultured fibroblasts, specific binding of LDL to adipocyte membranes was calcium-independent, was not affected by EDTA or NaCl, and was not destroyed by pronase. Plasma membranes purified directly from homogenized adipose tissue also showed calcium-independent LDL specific binding (0.58 +/- 0.33 micrograms of LDL bound/mg of membrane protein, mean +/- S.D. n = 11). Specific binding, internalization, and degradation of 125I-methylated LDL was demonstrated in isolated adipocytes and competition experiments showed that native and methylated LDL interacted with adipocytes through some common recognition mechanism(s). Compared to native LDL, specific binding of methylated LDL to adipocyte membranes was significantly reduced (43%), indicating that interaction of LDL with adipocyte was dependent in part on the lysine residues of apolipoprotein B. LDL binding to adipocyte plasma membranes was also competitively inhibited by human high density lipoprotein subfractions HDL2 and HDL3. Thus, LDL metabolism in mature adipocytes appears to be regulated by mechanisms distinctly different from a variety of cultured mesenchymal cells. In addition, the ability of adipocytes to bind, internalize, and degrade significant amounts of methylated LDL supports the view that adipose tissue is involved in the metabolism of modified lipoproteins in vivo.

Insulin degrading enzyme activity and insulin binding of erythrocytes in normal subjects and Type 2 (non-insulin-dependent) diabetic patients

Specific insulin degrading enzyme activity of erythrocytes was determined in relation to erythrocyte insulin binding in 16 healthy subjects, 14 Type 1 (insulin-dependent) and various groups of Type 2 (non-insulin-dependent) diabetic patients (n = 39). Degrading activity was increased in Type 2 diabetic patients on sulphonylureas, as well as in a subgroup with good metabolic control (p less than 0.001) and in patients with secondary failure to oral therapy (p less than 0.02); degrading activity returned to normal in the latter patients after 1 week of insulin treatment. Highest degrading activity was found in insulin-treated, yet insulin-insensitive patients (daily insulin dose greater than 80 U). Degrading activity was significantly correlated in healthy subjects both with circulating insulin concentrations and maximal specific insulin binding. In contrast, in Type 2 diabetic subjects, degrading activity was inversely correlated with serum insulin with no apparent association with maximal specific insulin binding except in those patients given 1 week of insulin treatment. High erythrocyte insulin degrading enzyme activity might be a common feature in the insulin-insensitive Type 2 diabetic patient and might occur subsequent to some aspect of insulin deficiency at the tissue level.

Characterization of a thyroid-hormone-binding site on nuclear envelopes and nuclear matrices of the male-rat liver.

Nuclear envelopes and nuclear matrices were isolated from the male-rat liver. Incubation of 125I-labelled 3,3',5-tri-iodothyronine (T3) with the nuclear-envelope fraction resulted in specific binding of T3 to the membranes. Maximum specific binding occurred at 30 degrees C after 2h incubation. Storage for 1 week at -80 degrees C resulted in no loss of binding. Scatchard analysis revealed a class of binding sites with KD 86 nM. 3,3',5'-Tri-iodothyronine was as effective a competitor of [125I]T3 binding to nuclear envelopes as was L-T3 itself, and tri-iodothyroacetic acid was 70% as potent as T3. L- and D-thyronine did not compete for [125I]T3 binding. Incubation of nuclear envelopes with 0.6 M-NaCl before addition of T3 resulted in the complete loss of specific binding sites, whereas exposure of the membranes to 2.0 M-NaCl after incubation with T3 did not extract binding sites. Nuclear matrices, after incubation with [125I]T3 under the same conditions, were shown to possess a class of binding sites with a similar KD but with approx. 30% of the maximum binding capacity. Nuclear envelopes from hypothyroid animals may possess slightly lower numbers of binding sites compared with nuclear envelopes from the intact animal, whereas nuclear matrices from hypothyroid animals have the same number of binding sites as do nuclear envelopes from the intact animal. In conclusion, nuclear envelopes and nuclear matrices have a class of binding sites with relatively high affinity for T3. It is distinct from nuclear and cytosolic binding sites.

Optimizing the Enzyme-Linked Immunosorbent Assay for Evaluating Immunity of Chickens to Newcastle Disease

Experiments employing the various steps and reagents used in the enzyme-linked immunosorbent assay (ELISA) were conducted to produce an ELISA with the highest sensitivity and specificity possible for detecting Newcastle disease antibodies in chicken sera. Of the four types of antigen tested, crude antigen gave inconsistent results. However, an alcohol-precipitated antigen prepared from infectious allantoic-amniotic fluids was as satisfactory as more highly purified virus preparations. Other factors found to be extremely important were a 0.5M concentration of NaCl in the diluent and wash solutions used in the procedure, and a pH of 13 for sensitizing solution for maximum specific binding of the antigen to the microplate plastic wells. A comparison was made between the hemagglutination-inhibition (HI) titers of 550 known chicken sera and the corresponding ELISA values. Although the ELISA is much more sensitive than the HI test, there was a general but not a direct correlation between the two tests. The ELISA did not give more information than the HI test concerning protection against an NDV-induced drop in egg production. Preliminary observations indicated that this ELISA procedure is also applicable for turkey sera.

Thyroxine binding and cellular metabolism of thyroxine in mononuclear blood cells from patients with anorexia nervosa.

Nuclear binding of thyroxine (T4), cellular deiodination of T4 and nuclear accumulation of endogenous tri-iodothyronine (T3) were investigated in mononuclear blood cells from eight patients with anorexia nervosa. Although the patients were euthyroid, serum T3 was depressed and serum reverse T3 increased. The nuclear maximal specific binding capacity (msbc) for T4 in cells from patients with anorexia nervosa was increased (2.4 X 10(-16) mol T4/10 micrograms DNA, P less than 0.01) as compared to that of normal subjects (msbc = 1.1 X 10(-16) mol T4/10 micrograms DNA), whereas the equilibrium association constant did not differ from that of normal subjects (Ka = 2.2 X 10(9) vs 3.0 X 10(9) l/mol). Cellular deiodination of T4 was significantly depressed (Vmax = 4.0 X 10(-17) mol iodine/10(6) cells per min) as compared to that of normal subjects (Vmax = 2.0 X 10(-16) mol iodine/10(6) cells per min), whereas nuclear accumulation of endogenous T3 was normal (Vmax = 3.0 X 10(-19) mol T3/10 micrograms DNA per min). Three patients started to gain weight and, concomitantly with the normalization of serum T3, the nuclear T4 capacity returned to normal. Cellular deiodination of T4 did not normalize within the observation period. In conclusion, patients with anorexia nervosa, in which serum T3 is depressed, have an increased capacity of nuclear T4 receptors. The cellular 5' deiodinase is inhibited but nuclear accumulation of endogenous T3 is normal.

Specific glucocorticoid binding in human uterine tissues, placenta and fetal membranes

The binding of [ 3H]dexamethasone to cytosol fractions of human myometrium, endometrium, decidua, chorion, amnion and placenta has been studied. All tissues examined contained high affinity, low capacity binding sites with high specificity for glucocorticoids. Maximum specific binding of [ 3H]dexamethasone was reached after about 10 h at 0–4°C and remained stable for at least the next 12 h. Sucrose density gradient analysis showed that the binding macromolecules sedimented at 7.9 S in hypotonie solutions and at 4.35 in solutions containing 0.4 M KCl. In the presence of sodium molybdate, the sedimentation coefficients shifted both in the absence and presence of 0.4 M KCl to 8.9 and 5.7 S, respectively. The apparent equilibrium dissociation constants ( K d ) of the glucocorticoid binding sites were similar in most tissues, ranging between 1 and 6 nM, with the exception of the placenta in which the binding sites showed a higher K d (13–22nM). In all tissues studied, the binding affinities were similar in nonpregnant and pregnant patients and in patients at different stages of pregnancy or in labor. The concentration of the binding sites in the different tissues ranged from 11 to 268 fmol/mg protein, higher concentrations being found in myometrium, placenta and amnion and lower concentrations found in endometrium, chorion and decidua. The number of binding sites was higher in the myometrium of nonpregnant than pregnant women, but was similar in the myometrium of women at term pregnancy before or during labor. In the placenta, the number of binding sites increased significantly from early pregnancy to midpregnancy, while in chorion, amnion and decidua the number of binding sites did not change during pregnancy. It is concluded that human uterine tissues, placenta and fetal membranes contain specific binding sites with properties characteristic of glucocorticoid receptors suggesting that these tissues may respond directly to glucocorticoids.

Erythrocyte insulin receptors remain intact through three days of storage.

The status of the erythrocyte insulin receptor was investigated prior to and during storage at 4C in acid-citrate dextrose (ACD) solution. The receptors on cells that were obtained from both resting and exercised fasting subjects and stored for up to three days in ACD were unchanged as evaluated by both maximal specific hormone binding and the concentration of insulin required to one half maximally inhibit specific binding. These findings indicate, therefore, that the binding of insulin to its receptor on erythrocytes can be assayed in samples of stored blood and that this assay reflects the status of the receptor at the time of sampling.