Cyclic oxidation-reduction reactions regulate thromboxane A2/prostaglandin H2 receptor number and affinity in human platelet membranes

The radiolabeled thromboxane A2/prostaglandin H2 (TXA2/PGH2) agonist 125I-BOP bound to the TXA2/PGH2 receptor on human platelet membranes. Scatchard analysis showed that pretreatment of platelet membranes with the reducing agent dithiothreitol (DTT) (10 mM) for 10 min decreased maximal 125I-BOP binding (Bmax) from 1.51 +/- 0.11 pmol/mg to 0.51 +/- 0.05 pmol/mg (p = 0.001) and increased the affinity of the remaining binding sites (Kd = 647 +/- 64 pM (untreated), 363 +/- 46 pM (treated), p = 0.006). Prolonged incubation of membranes with DTT (10 mM) for 40 min further reduced the Bmax to 0.23 +/- 0.08 pmol/mg (p = 0.001 from untreated), and the binding affinity remained elevated (Kd = 334 +/- 117 pM, p = 0.035 from untreated). Kinetic analysis of 125I-BOP binding indicated that the apparent increase in binding affinity after DTT treatment was due exclusively to an increase in the rate of ligand-receptor association with no change in dissociation rate. The effects of DTT on 125I-BOP binding were dose-dependent with an EC50 of 8.1 +/- 0.2 mM. DTT inactivation of TXA2/PGH2 receptors was time-dependent with a second order rate constant (k2) of 0.123 M-1 s-1 at 20 degrees C. The platelet membrane 125I-BOP binding site was partially protected from DTT inactivation by prior occupation with the ligand. TXA2/PGH2 receptor protection by I-BOP was dose-dependent and linearly related (r = 0.97, p = 0.002) to the proportion of receptors occupied, but was incomplete since agonist occupation of 89% of the total number of receptors resulted in only a 38% protective effect. Inhibition of 125I-BOP binding after reduction with DTT could be made permanent by addition of the sulfhydryl alkylating agent N-ethylmaleimide (25 mM), but was completely reversed by reoxidation with dithionitrobenzoic acid (DTNB) (5 mM). Oxidation of untreated receptors with DTNB resulted in a 64% increase in 125I-BOP binding sites from 1.65 +/- 0.12 pmol/mg to 2.70 +/- 0.08 pmol/mg (p = 0.013) without affecting binding affinity. DTNB-induced increases in 125I-BOP binding were concentration-dependent with an EC50 of 668 +/- 106 microM and occurred in less than 1 min at 37 degrees C. In the absence of DTT, alkylation of free sulfhydryl groups with N-ethylmaleimide reduced 125I-BOP Bmax in platelet membranes to 0.85 +/- 0.08 pmol/mg (p = 0.003), but did not change the affinity of the remaining receptors. The EC50 for N-ethylmaleimide inactivation of TXA2/PGH2 receptors was 139 +/- 8 mM, and the k2 in time course experiments was 0.067 M-1 s-1 at 20 degrees C.(ABSTRACT TRUNCATED AT 400 WORDS)     

Thioredoxin and glutaredoxin enhance the binding of L-triiodothyronine to its hepatic nuclear receptors

The influence of thioredoxin and glutaredoxin on binding of L-triiodothyronine (T3) to the rat hepatic nuclear T3 receptor was compared with that of the exogenous activator dithiothreitol. Specific [125I]T3 binding, the affinity constant, Ka, and the maximal binding capacity, MBC, were measured using whole nuclei, solubilized preparations of receptor, and chromatographed nuclear receptor. Both the thioredoxin system (thioredoxin, thioredoxin reductase, and NADPH) and the glutaredoxin system (glutaredoxin, glutathione reductase, glutathione, and NADPH) increased specific binding of T3 to nuclei, solubilized receptor, and chromatographed receptor significantly. Compared with the values obtained in the absence of added thiol (Ka = 1.6 +/- 0.1 x 10(9) M-1, MBC = 1.7 +/- 0.06 pM), the thioredoxin and glutaredoxin systems increased Ka by 147 and 112%, respectively, while decreasing MBC by 51 and 45%, respectively, when chromatographed receptor was used. The same tendency was observed with solubilized receptor. However, dithiothreitol increased Ka without affecting MBC when solubilized receptor was used. These results, the first demonstration of endogenous disulphide reductant systems enhancing binding of T3 to its receptor, suggest that the thioredoxin and (or) glutaredoxin systems may modulate the physiological effects of thyroid hormone.     

Evidence for the presence of two active forms of cytosolic 3,5,3'-triiodo-L-thyronine (T3)-binding protein (CTBP) in rat kidney. Specialized functions of two CTBPs in intracellular T3 translocation

Cytosolic NADPH-dependent 3,5,3'-triiodo-L-thyronine (T3)-binding protein (CTBP) purified from rat kidney was further characterized in its T3 binding and its interaction with nuclei. Pretreatment of the CTBP with NADP induced dithiothreitol (DTT)-dependent T3 binding. The DTT-dependent T3 binding was increased by NADP in a concentration-dependent manner, and the maximal binding was obtained by 0.1 microM NADP. Higher concentrations of NADP (more than 0.1 microM), however, reduced T3 binding. NAD also induced DTT-dependent T3 binding, but was very low compared to that induced by NADP. NADPH and NADH did not produce DTT-dependent T3 binding. This NADP-activated, DTT-dependent T3 binding was characterized as follows: Ka for T3 binding was 1.8 x 10(9) M-1, and the maximal binding capacity was 15,000 pmol/mg of protein in the CTBP activated by 0.1 microM NADP. The molecular weight of the CTBP was 58,000 (4.7 S). A complex of [125I]T3 and CTBP (NADP.DTT.CTBP.[125I]T3), which was made from the CTBP pretreated with NADP and DTT, did not bind to DNA. However, the complex bound to the nuclei prepared from rat kidney. Treatment of the nuclei with 0.38 M KCl and with DNase I did not lead to loss of the binding activity for the complex. Treatment of nuclei with 0.5 M NaCl led to the loss of the activity for binding the complex. A complex of [125I]T3 and NADPH-activated CTBP did not bind these nuclear preparations. These results suggested that the active form of CTBP is present in two different forms: one is NADPH-activated, which plays a role as a reservoir for cytoplasmic T3, and the other is NADP-activated, which plays a role as a T3 carrier protein that transfers T3 from cytoplasm to nucleus.     

Increased thyrotropin binding in hyperfunctioning thyroid nodules

The object of this study was to investigate TSH receptors in hyperfunctioning thyroid nodules (HFN). In HFN, obtained from seven patients, 125-I-TSH binding as determined by equilibrium binding analysis on particulate membrane preparations, was found to be significantly increased as compared with normal thyroid tissues (five patients; P less than 0.001). Scatchard analysis of TSH-binding revealed two kinds of binding sites for both normal thyroid tissue and HFN, and displayed significantly increased association constants of high- and low-affinity binding sites in HFN (Ka = 11.75 +/- 6.8 10(9) M-1, P less than 0.001 and Ka = 2.1 +/- 1.0 10(7) M-1, P less than 0.025; x +/- SEM) as compared with normal thyroid tissue (Ka = 0.25 +/- 0.06 10(9) M-1, Ka = 0.14 +/- 0.03 10(7) M-1; x +/- SEM). The capacity of the high-affinity binding sites in HFN was found to be decreased (1.8 +/- 1.1 pmol/mg protein, x +/- SEM) in comparison with normal thyroid tissue (4.26 +/- 1.27 pmol/mg protein; x +/- SEM). TSH-receptor autoradiography applied to cryostatic tissue sections confirmed increased TSH binding of the follicular epithelium in HFN. These data suggest that an increased affinity of TSH-receptor sites in HFN in iodine deficient areas may be an important event in thyroid autonomy.     

Ontogenetic changes in porcine adrenocortical adrenocorticotropic hormone receptors.

1. Binding of [125I]ACTH(1-38) analog to adrenal receptors was measured in fetal pigs (Sus domesticus) at 15-day intervals from midpregnancy (60 days) to near term (105 days; pregnancy length 114 days). 2. Binding was greatest at day 60 (0.42 +/- 0.03 fmol/200 micrograms protein or 0.50 +/- 0.08 fmol/50 micrograms DNA), and least at day 105 (0.13 +/- 0.03 fmol/200 micrograms protein or 0.16 +/- 0.04 fmol/50 micrograms DNA). Total adrenal binding was constant (0.61 +/- 0.02 fmol/paired adrenals). 3. Scatchard analyses at day 60 and day 105 showed comparable apparent affinities of ACTH receptors (Ka day 60 = 1.51 +/- 0.72 x 10(9) M-1 vs Ka day 105 = 1.94 +/- 0.78 x 10(9) M-1). 4. DNA per paired adrenals and membrane-associated protein increased 1.6-fold, providing a constant protein: DNA ratio. Concentrations of adrenal cortisol were constant from 60 to 90 days of gestation age but increased dramatically by day 105. 5. These data suggest that during 60-105 days of gestation age the number of ACTH receptors per cell is reduced.     

A selenium-containing catalytic antibody with Type I deiodinase activity

Acting as a mimic of type I deiodinase (DI), a selenium-containing catalytic antibody (Se-4C5) prepared by converting the serine residues of monoclonal antibody 4C5 raised against thyroxine (T4) into selenocysteines, can catalyze the deiodination of T(4) to 3,5,3'-triiodothyronine (T(3)) with dithiothreitol (DTT) as cosubstrate. Investigations into the deiodinative reaction by Se-4C5 revealed the relationship between the initial velocity and substrate concentration was subjected to Michaelis-Menten equation and the reaction mechanism was ping-pong one. The kinetic properties of the catalytic antibody were a little similar to those of DI, with Km values for T(4) and DTT of approximately 0.8 microM and 1.8 mM, respectively, and V(m) value of 270 pmol per mg protein per min. The activity could be sensitively inhibited by PTU with a Ki value of approximately 120 microM at 2.0 microM of T(4) concentration, revealing that PTU was a competitive inhibitor for DTT.     

Effects of adrenaline pretreatment on in vitro binding of 125I-triiodothyronine to nuclear receptor, intracellular distribution of endogenous triiodothyronine and activities of alfa-glycerophosphate dehydrogenase and malic enzyme in rat liver

Especially coated adrenaline tablets (A) or placebo tablets (P) which release linearly the hormone were implanted in male Wistar rats. Six hours later animals were sacrificed and kinetic parameters of T3-125I binding to nuclear receptor, intracellular distribution of endogenous T3 and activities of alfa-GPD and ME were investigated. The association constant values (Ka) of nuclear receptor were increased after pretreatment with 7.5, 15 and 45 mg A tablets and were 1.07, 1.35 and 1.48 X 10(9) M-1 vs 0.85 X 10(8) M-1 value seen after P. The maximal binding capacity (MBC) values decreased after pretreatment with the same doses of A and were 0.044, 0.036 and 0.025 pmol T3/100 micrograms DNA vs. 0.065 pmol T3/100 micrograms DNA in P pretreated. Adrenaline pretreatment significantly increased the amount of endogenous T3 present in liver nuclei while the amount of T3 present in cytosol decreased. Activity of mitochondrial alfa-GPD was increased after 15 and 45 mg of A. Significant rise of activity of cytosol ME was seen only after pretreatment with 45 mg of A.     

An essential role of domain D in the hormone-binding activity of human beta 1 thyroid hormone nuclear receptor

By analogy with steroid receptors, human placental thyroid hormone nuclear receptor (hTR beta 1) could be divided into four functional domains: A/B (Met1-Leu101), C (Cys102-Ala170), D (Thr171-Lys237), and E (Arg238-Asp456). The E domain was thought to bind thyroid hormone. To evaluate whether domain E alone is sufficient to bind T3 or requires the presence of other domains for functional T3-binding activity, a series of deletion mutants was constructed. The mutants were expressed in Escherichia coli, and the expressed proteins were purified. Analysis of the T3-binding affinity and analog specificity of the purified truncated hTR beta 1 indicated that domain E alone did not have T3-binding activity. Extension of the amino-terminal sequence of domain E to include part of domain D yielded a mutant (Lys201-Asp456) with a Ka for T3 of 0.5 +/- 0.2 x 10(9) M-1. Further extension to include the entire domain D (Met169-Asp456) yielded a mutant with T3-binding activity with a Ka of 0.8 +/- 0.1 x 10(9) M-1. Further extension of the amino-terminal sequence to include domain C increased the affinity for T3 by nearly 2-fold (Ka = 1.5 +/- 0.4 x 10(9) M-1). The Ka for the wild-type hTR beta 1 is 1.5 +/- 0.2 x 10(9) M-1. Furthermore, mutant (Met169-Asp456) binds to 3',5',3-triiodo-L-thyropropionic acid, D-T3, L-T4, and L-T3 with 307%, 37%, 7%, and 0.1%, respectively, of the activity of L-T3. This order of analog affinity is similar to that of the wild-type hTR beta 1.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of the asialoglycoprotein receptor in a continuous hepatoma line

The asialoglycoprotein receptor has been identified on a continuous human hepatoma cell line, HepG2. This receptor requires Ca2+ for ligand binding and is specific for asialoglycoprotein. There are approximately 150,000 ligand molecules bound/cell at 4 degrees C. These receptors represent a homogeneous population of high affinity binding sites with Kd = 7 X 10(-9) M. From the rate of 125I-ASOR binding at 4 degrees C, kon was 0.95 X 10(6) M-1 min-1. Uptake of 125I-ASOR at 37 degrees C was approximately 0.02 pmol/min/10(6) cells.     

Hepatic uptake of asialoglycoprotein is different among mammalian species due to different receptor distribution

Isolated hepatocytes of rat, rabbit and guinea pig were found to take up and degrade 125I-labelled asialoorosomucoid at different rates with the rank order: rabbit greater than rat greater than guinea pig. Measurement of 125I-asialoorosomucoid binding at 4 degrees C to these hepatocytes revealed that all these cells had two classes of receptors with a major difference occurring in the number of high-affinity binding sites. The average binding affinity constants (K) and receptor concentration (N) calculated from a least-square analysis of the Scatchard plots were K1 = 1.15.10(9) M-1, K2 = 0.93.10(7) M-1, N1 = 0.049 pmol/mg cell protein and N2 = 0.27 pmol/mg cell protein for the rat; K2 = 3.16.10(7) M-1, N1 = 0.027 pmol/mg cell protein and N2 = 0.13 pmol/mg cell protein for the guinea pig and K1 = 0.74.10(9) M-1, K2 = 3.85.10(7) M-1, N1 = 0.205 pmol/mg cell protein and N2 = 0.37 pmol/mg cell protein for the rabbit hepatocytes, respectively. Measurement of the total number of cellular receptors after solubilization with Triton X-100 also revealed the same receptor concentration rank order of rabbit (5.8 pmol/mg cell protein) greater than rat (0.55 pmol/mg cell protein) greater than guinea pig (0.18 pmol/mg cell protein). Intravenous injection of 125I-asialoorosomucoid into anesthetized animals of matched body weight also indicated that the rate of plasma clearance and the rate of appearance of the degraded product of the tracer were different among these species with the same rank order as that observed with isolated hepatocytes. Thus there is a fundamental difference in the number of asialoglycoprotein receptors both on the cell surface and inside hepatocytes of these mammalian species.     

Studies on the T3 suppression test by the 24-hour thyroidal 131I uptake in patients with Graves' disease: comparison of the effects of propylthiouracil and methylmercaptoimidazole

The T3 suppression test by the 24-hr thyroidal 131I uptake was reevaluated in patients with Graves' disease before and after withdrawal of antithyroid drug. Fifty patients had been treated with propylthiouracil (PTU) or methylmercaptoimidazole (MMI) for 12 to 70 months. They were prescribed a maintenance dose of antithyroid drug (PTU, 50 mg/day; MMI, 5 mg/day) at the time of investigation and regarded as euthyroid on the basis of serum T3, T4 and TSH levels. Each patient was given 75 micrograms T3 daily for 8 days in conjunction with PTU or MMI. The 24-hr thyroidal 131I uptake was then measured (post T3 uptake). In 30 patients whose post T3 uptake was below 35%, treatment was stopped and the T3 suppression test was repeated at one and 3 months later. During the two-year follow up, 24 remained well, while 6 relapsed within 4 to 12 months. In patients with sustained remission, the post T3 uptake was significantly lower in the MMI-treated group (13 cases, 7.7 +/- 1.0%) than in the PTU-treated group (11 cases, 18.6 +/- 1.9%). MMI withdrawal produced a marked rebound in the post T3 uptake, whereas none of the patients showed the rebound after PTU withdrawal. In patients who relapsed later, there was no difference in the post T3 uptake during treatment and the rebound occurred in the both groups following goitrogen withdrawal. Serum T3, T4 and TSH levels were within normal ranges at one and 3 months after cessation of antithyroid drug. From the results of the present study, it is concluded that criteria for T3 suppressibility by the 24-hr uptake should be determined by the antithyroid drug employed and by the time of investigation. There is a dissociation in the post T3 uptake values following withdrawal of the two different antithyroid drugs.     

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.     

Interaction of di-iodinated 125I-labelled alpha-bungarotoxin and reversible cholinergic ligands with intact synaptic acetylcholine receptors on isolated skeletal-muscle fibres from the rat.

1. Intact synaptic acetylcholine receptors on freshly isolated rat skeletal-muscle fibres were characterized by their interaction with di-iodinated 125I-labelled alpha-bungarotoxin, acetylcholine and other cholinergic ligands at room temperature (22 deggrees C). 2. The time course and concentration dependence of 125I-labelled alpha-bungarotoxin association conformed to a bimolecular mechanism. In time-course experiments with different concentrations of 125I-labelled alpha-bungarotoxin (1.4--200 nM) the bimolecular-association rate constant, k + 1, was (2.27 +/- 0.49) x 10(4)M-1.S-1 (mean +/- S.D., N = 10). In concentration-dependence experiments, k + 1 was 2.10 x 10(4)M-1.S-1 and 1.74 x 10(4) M-1.S-1 with 10 and 135 min incubations respectively. In association experiments the first-order rate constant was proportional to the 125I-labelled alpha-bungarotoxin concentration. 125I-Labelled alpha-bungarotoxin dissociation was first order with a dissociation constant, k-1, less than or equal to 3 x 10(-6)S(-1) (half-life greater than or equal to 60 h.) The results indicated a single class of high-affinity toxin-binding sites at the end-plate with an equilibrium dissociation constant, Kd, equal to or less than 100 pM. The number of toxin-binding sites was (3.62 +/- 0.46) x 10(7) (mean +/- S.D., n = 22) per rat end-plate. 3. The apparent inhibitor dissociation constants, Ki, for reversible cholinergic ligands were determined by studying their effect at equilibrium on the rate of 125I-labelled alpha-bungarotoxin binding. There was heterogeneity of binding sites for cholinergic ligands, which were independent and non-interacting with antagonists. In contrast agonist affinity decreased with increasing receptor occupancy. Cholinergic ligands in excess inhibited over 90% of 125I-labelled alpha-bungarotoxin binding. 4. Cholinergic ligand binding was accompanied by an increase in entropy, which was greater for the agonist carbachol (delta So = +0.46 kJ.mol-1.K-1) than the antagonist tubocurarine (delta So = +0.26 kJ.mol-1.K-1). 5. The entropy and affinity changes that accompanied agonist binding suggested that agonists induced significant conformational changes in intact acetylcholine receptors. 6. The affinity and specificity of 125I-labelled alpha-bungarotoxin and tubocurarine binding to synaptic acetylcholine receptors from slow and fast muscle fibres were the same. 7. The study of binding only requires milligram amounts of tissue and may have application to other neurobiological studies and to the study of human neuromuscular disorders.     

Ontogeny of the liver nuclear T3-receptor during the last days of incubation and posthatch in the chick embryo.

The characteristics of the nuclear T3 receptor in the liver of the chick embryo were studied from incubation day 18 until day 1 posthatching. Treatment of the nuclei with 3 mol.l-1 MgCl2, which removed the endogenously bound hormone, was used in order to determine the total amount of receptors. The affinity constant Ka decreased between incubation day 18 (0.996 +/- 0.276.10(9) M-1) and day 19 (0.247 +/- 0.072.10(9) M-1), remained the same thereafter until hatching and increased again on day 1 posthatching (1.846 +/- 0.928.10(9) M-1). The total amount of receptors tended to increase from incubation day 18 to day 20 non-pipping (np) (from 4.40 to 11.55 fmol/micrograms DNA) and decreased thereafter to 2.38 fmol/micrograms DNA on day 1 posthatching. The amount of free binding sites reached a maximum on day 19 (6.91 fmol/micrograms DNA) and then decreased drastically until posthatching (0.19 fmol/micrograms DNA). The maximal specific binding was found on day 20 (np), just prior to penetration of the air chamber. During the time at which the level of T3 remains high in the plasma, a reduction in the amount of receptor was observed, which may be the consequence of a down-regulation by T3 itself.     

Epidermal growth factor receptors in cultured human trophoblast cells from first- and third-trimester placentas

Epidermal growth factor (EGF) receptors were studied during the in vitro differentiation of human trophoblast cells from first- and third-trimester placentas. Cytotrophoblasts were isolated by enzymatic digestion and purified on a discontinuous Percoll gradient. As analyzed by flow cytometry, 5% of the cells are in the G2M phase in the early placenta and 0% in the term placenta. In culture, the cytotrophoblasts at both gestational ages flatten out, aggregate, and fuse together to form syncytiotrophoblasts. This in vitro morphological differentiation is associated with a threefold increase in the ability to bind specifically 125I-EGF. Trophoblastic cells from the term placenta have a significantly (p less than 0.005) higher receptor number (68.6 +/- 9.5 fmol/mg protein) for EGF after 2 days of culture than first-trimester cytotrophoblasts (35.8 +/- 2.3 fmol/mg protein). Scatchard plot analysis revealed two classes of binding sites with a similar affinity in both first-trimester and term placentas (9.5 x 10(9) M-1 for the high-affinity, 0.5 x 10(9) M-1 for the low affinity site). When 125I-EGF was affinity cross-linked to cytotrophoblasts, the receptors appeared as a specific band with a molecular weight of 180 kD in SDS-PAGE. This study demonstrates that the culture of cytotrophoblasts offer an appropriate model to study the modulation of EGF receptors.     

Specificity of siderophore receptors in membrane vesicles of Bacillus megaterium.

Membrane vesicles of Bacillus megaterium strains SK11 and Ard1 bound the ferrischizokinen and ferriferrioxamine B siderhores (iron transport cofactors). An approximately equimolar uptake of both labels of [3H, 59Fe]ferrischizokinen indicated binding of the intact chelate. Binding reached equilibrium in 2 to 5 min, was temperature independent, and was unaltered by the addition of several energy sources. A 91% dissociation of bound [Fe]ferrischizokinen was achieved in 60 s by the addition of excess ferrischizokinen. Ferriaerobactin, a siderophore which is structurally related to ferrischizokinen, caused no detectable release of bound [59Fe]ferrischizokinen. Of several other ferrigydroxamates tested, only ferriferrichrome A achieved the release (11%) of [Fe]ferrischizokinen. Rapid dissociation (92%) of bound [59Fe]ferriferrioxamine B by the addition of ferriferrioxamine B was observed, and a 67% release of [59Fe]ferriferrioxamine B was caused by ferriA2265, its structural relative. Ferrischizokinen, ferriferrichrome A, and ferrirhodotorulic acid produced a 6, 25, and 29% dissociation, respectively, of [59Fe]ferriferrioxamine B; ferriaerobactin caused no dissociation. [59Fe]ferriaerobactin was bound by the membranes, but its dissociation was not effected by unlabeled ferriaerobactin, suggesting no specific receptors for this chelate. The respective binding affinity constants and maximal binding capacities of membrane vesicles of strain SK11 were 2 x 10(7) M-1 and 280 pmol per mg of protein for ferrischizokinen and 7 x 10(7) M-1 and 37 pmol per mg of protein for ferriferrioxamine B. These values in strain Ard1 were, respectively, 1.4 x 10(7) M-1 and 186 pmol per mg of protein for ferrischizokinen and 11 x 10(7) M-1 and 23 pmol per mg of protein for ferriferrioxamine B. Separate, specific binding sites (receptors) for ferrischizokinen and ferriferrioxamine B exist on the vesicles. The ferrischizokinen receptors have a lower affinity but a higher binding capacity (eightfold) than that shown by the ferriferrioxamine B receptor. These receptors may be components of independent transport systems.     

In vitro demonstration of putative nuclear 3,5,3'-triiodothyronine receptors in isolated liver nuclei of Singi fish, Heteropneustes fossilis (Bloch)

Putative thyroid hormone (TH) receptors have been demonstrated in the isolated liver nuclei of Singi fish, Heteropneustes fossilis (Bloch), and their binding characteristics have been examined. Nuclear T3 saturation analyses were carried out in vitro at 27 degrees C in a sucrose-Tris-HCl buffer (pH 7.5) containing calcium (2 mM), magnesium (3 mM) and 2-mercaptoethanol (5 mM). After incubation the bound and free hormones were separated by centrifugation and the nuclei were treated with Triton X-100 (final concentration 0.25%) to reduce the non-specific binding. The binding was saturable and reached equilibrium by 20 minutes of incubation and was also stable for 2 hours. The binding was reversible and the rate of dissociation was more or less equal to the rate of association. The binding was linearly increased with the increased concentrations of the DNA (nuclei). Scatchard analyses of the equilibrium binding data revealed that only one class of binding sites for T3 did exist in the hepatic nuclei of Singi fish. The affinity of these sites or the mean dissociation constant (Kd = 0.20 +/- 0.07 x 10(-10) M) and the mean maximum binding capacity (MBC = 0.17 +/- 0.04 pmol/mg DNA) were in reasonable agreement with the values reported for other teleost fishes.     

Modes of mononucleotide binding to ribonuclease T1.

The binding of the mononucleotide inhibitors 2'-GMP, 3'-GMP, and 5'-GMP to genetically engineered ribonuclease T1 has been investigated by conventional inhibition kinetics, fluorimetric titrations, molecular modeling, and fast relaxation techniques. The fluorimetric titrations in conjunction with molecular modeling revealed that apart from the already known primary binding site, three to four additional sites are present on the enzyme's surface. The association constants obtained from the fluorimetric titrations and the temperature jump experiments range between 3.1 x 10(6) M-1 and 4.3 x 10(6) M-1, indicating that the binding of the mononucleotides to the specific binding site of ribonuclease T1 is at least one order of magnitude tighter than has been anticipated so far. The kinetics of binding are nearly diffusion controlled with a kon determined for 2'-GMP and 3'-GMP, as (5.0 +/- 0.5 x 10(9) and 6.1 +/- 0.5 x 10(9) M-1, s-1 and koff as 1.2 +/- 0.2 x 10(3) and 2.0 +/- 0.3 x 10(3) s-1, respectively. Molecular modeling studies indicate that all three nucleotides are able to bind via their phosphate group to a positively charged array of surface amino acids including His27, His40, Lys41, and most probably Lys25 without obvious stereochemical hindrance. We propose that RNA wraps around RNase T1 in a similar fashion via phosphate binding when enzymatic hydrolysis occurs.     

2[125I]iodomelatonin binding sites in spleens of guinea pigs.

2-[125I]Iodomelatonin was found to bind specifically to the membrane preparations of the spleens of guinea pigs with high affinity. The binding was rapid, stable, saturable and reversible. Scatchard analysis of the binding assays revealed an equilibrium dissociation constant (Kd) of 49.8 +/- 4.12 pmol/l and binding site density (Bmax) of 0.69 +/- 0.082 fmol/mg protein at mid-light (n = 10). There was no significant change in the Kd (41.8 +/- 3.16 pmol/l) or the Bmax (0.58 +/- 0.070 fmol/mg protein) at mid-dark (n = 10). Kinetic analysis showed a Kd of 23.13 +/- 4.81 pmol/l (mean +/- SE, n = 4), in agreement to that derived from the saturation studies. The 2-[125I]iodomelatonin binding sites have the following order of potency: 2-iodomelatonin greater than melatonin greater than 6-chloromelatonin much greater than N-acetylserotonin, 6-hydroxymelatonin greater than 5-methoxytryptamine, 5 methoxytryptophol greater than serotonin, 5-methoxyindole-3-acetic acid greater than 5-hydroxytryptophol, 3-acetylindole, 1-acetylindole-3-carboxyaldehyde, L-tryptophan greater than tryptamine, 5-hydroxyindole-3-acetic acid. Differential centrifugation studies showed that the binding sites are localized mainly in the nuclear fraction (65.5%), the rest are distributed in the microsomal fraction (17.4%), mitochondrial fraction (14.7%) and cytosolic fraction (0.3%). The demonstration of 2-[125I]iodomelatonin binding sites in the spleen suggests the presence of melatonin receptors and a direct mechanism of action of melatonin on the immune system.     

Binding of ellipticine base and ellipticinium cation to calf-thymus DNA. A thermodynamic and kinetic study

The acid-basic properties of ellipticine have been re-estimated. The apparent pK of protonation at 3 microM drug concentration is 7.4 +/- 0.1. The ellipticine free base (at pH 9, I = 25 mM) intercalates into calf-thymus DNA with an affinity constant of 3.3 +/- 0.2 X 10(5) M-1, and a number of binding sites per phosphate of 0.23. The ellipticinium cation (pH 5, I = 25 mM) binds also to DNA with a constant of 8.3 +/- 0.2 x 10(5) M-1 and at a number of binding sites (n = 0.19). It is postulated that the binding of the drug to DNA at pH 9 is driven by hydrophobic and/or dipolar effects. Even at pH 5, where ellipticine exists as a cation, it is thought that the hydrophobic interaction is the main contribution to binding. The neutral and cationic forms share common binding within DNA sites but yield to structurally different complexes. The free base has 0.04 additional specific binding sites per phosphate. As determined from temperature-jump experiments, the second-order rate constant of the binding of the free base (pH 9) is 3.4 x 10(7) M-1 s-1 and the residence time of the base within the DNA is 8 ms. The rate constant for the binding of the ellipticinium cation is 9.8 x 10(7) M-1 s-1 when it is assumed that drug attachment occurs via a pathway in which the formation of an intermediate ionic complex is not involved (competitive pathway).