Hormone and DNA binding activity of a purified human thyroid hormone nuclear receptor expressed in Escherichia coli

Using a T7 expression system, large amounts of the human placental c-erbA protein (h-TR beta 1) were expressed. From 1 liter of Escherichia coli culture, approximately 50-100 micrograms of purified h-TR beta 1 were obtained. Analysis of the binding data indicated that the purified h-TR beta 1 binds to 3,3',5-triiodo-L-thyronine (T3) with a Ka = 2.8 x 10(9) M-1. It binds to 3,3',5-triiodo-L-thyropropionic acid, 3,3',5-triiodo-L-thyroacetic acid, D-T3, L-thyroxine (T4), and 3',5',3-triiodo-L-thyronine with 475, 120, 39, 7, and 0.1%, respectively, of the activity of L-T3. This order of binding activity to T3 analogs is similar to that reported for the T3 nuclear receptor identified in tissues or cultured cells. Furthermore, the purified h-TR beta 1 binds to the T3 response element of the rat growth hormone gene. Thus, the purified h-TR beta 1 is active. To identify the hormone binding domain, the purified h-TR beta 1 was affinity labeled with underivatized [3',5'-125I]T4. A partial digestion by trypsin yielded a 125I-labeled 25-kDa fragment which was identified to be the domain Phe240-Asp456 by amino acid sequencing. Thus, the purified h-TR beta 1 appears suitable for other structural and functional studies.     

Characterization of a gain of function mutation of integrin alpha IIb beta 3 (platelet glycoprotein IIb-IIIa).

Integrin alpha IIb beta 3 (platelet glycoprotein IIb-IIIa) is a prototype of integrins involved in cellular adhesive functions. As part of a structure-function analysis of this molecule, we constructed a mutant, designated alpha IIb beta 3 (beta 1-2), by replacing 6 amino acids within a putative ligand binding domain of the beta 3 subunit with sequences derived from beta 1. The alteration did not affect the capacity of beta 3(beta 1-2) to combine with transfected alpha IIb, nor did it cause it to combine with endogenous alpha 5. Integrin alpha IIb beta 3(beta 1-2) was in a "resting" state on Chinese hamster ovary cells as judged by minimal binding of an activation-specific anti-alpha IIb beta 3, PAC1. Nevertheless, cells expressing alpha IIb beta 3(beta 1-2) spontaneously bound fibrinogen with low affinity (Ka = (4.85 +/- 0.84) x 10(6) M-1). Activation with an anti-beta 3 antibody (monoclonal antibody 62) resulted in a 10-fold increase in fibrinogen binding affinity (Ka = (4.55 +/- 0.77) x 10(7) M-1), which was 3-fold greater than fibrinogen binding to activated wild type alpha IIb beta 3 (Ka = (1.66 +/- 0.33) x 10(7) M-1, F = 7.46, p = 0.008). The mutant receptor also bound fibrinogen mimetic peptide ligands with enhanced affinity as measured by the conformation-specific antibody, anti-LIBS1. This indicates that the increased affinity for fibrinogen was caused by enhanced interaction of alpha IIb beta 3(beta 1-2) with known recognition sequences in fibrinogen. Thus, this gain of function mutant augments ligand binding function, supporting a role for this region of the beta subunit in ligand binding to integrins.     

Functional properties of human thyroid hormone receptor beta 1 overexpressed using baculovirus

We have overexpressed the human beta 1 thyroid hormone receptor in insect cells using a recombinant baculovirus to a level of 5-10% of total cellular protein. The recombinant protein migrates as a 50 kDa band by SDS-PAGE and Western blot analysis. The expressed receptor binds to L-T3 with a Kd of 1.3 +/- 0.4 x 10(-10) M and to thyroid hormone analogues with an affinity hierarchy of TRIAC greater than L-T3 greater than L-T4 greater than rT3. Gel retardation assays show highly specific receptor binding to a TRE which is modified by the presence of ligand and avidin-biotin complex DNA analysis shows a Kd of 6.2 +/- 2.0 x 10(-10) M for this interaction. These results indicate high level expression of hTR beta with authentic hormone and DNA binding properties.     

Novel type of very high affinity calcium-binding sites in beta-hydroxyasparagine-containing epidermal growth factor-like domains in vitamin K-dependent protein S

Vitamin K-dependent protein S is shown to contain four very high affinity Ca2(+)-binding sites. The number of sites and their affinities were determined from Ca2+ titration in the presence of the chromophoric chelator Quin 2. In 0.15 M NaCl, pH 7.5, the four macroscopic binding constants are K1 greater than or equal to 1 x 10(8) M-1, K2 = 3 +/- 2 x 10(7) M-1, K3 = 4 +/- 2 x 10(6) M-1, and K4 = 9 +/- 4 x 10(5) M-1. At low ionic strength, the corresponding values are K1 greater than or equal to 2 x 10(9) M-1, K2 = 9 +/- 4 x 10(8) M-1, K3 = 2 +/- 1 x 10(8) M-1, and K4 = 9 +/- 4 x 10(7) M-1. To localize the Ca2(+)-binding sites, protein S was subjected to proteolysis using lysyl endopeptidase. This yielded a 20-21-kDa fragment which comprised the third and fourth epidermal growth factor (EGF)-like domains and remained high affinity Ca2(+)-binding site(s). The susceptibility of the EGF-like domains to proteolysis increased when Ca2+ was removed from protein S indicating that the Ca2+ binding is important for the stability and/or conformation of the EGF domains. Three of the four EGF-like domains in protein S contain beta-hydroxyasparagine. In each of these domains there is a cluster of three or four negatively charged amino acid residues which are likely to contribute to the extraordinary high Ca2+ affinity. From sequence homology it is suggested that this novel type of high affinity Ca2(+)-binding site is present in several other proteins, e.g. in the EGF-like domains in the low sensity lipoproteins receptor, thrombomodulin, the Notch protein of Drosophila melanogaster, and transforming growth factor beta 1-binding protein.     

Multi-component system of estrogen binding proteins from the liver: characterization of binding properties of high molecular weight protein from rat liver similar to uterine estradiol receptors]

A comparative study of the hormonal specificity of the affinity, the equilibrium association constant (Ka) and the kinetics of [3H]-estradiol (3H-E2) interaction with high molecular weight specifically binding E2 proteins from liver cytosol of male and female rats and with uterine estrogen receptors was carried out. The hormonal specificity of the affinity for the E2-binding proteins from the three sources was found to be similar, i.e. only the compounds possessing the estrogen activity competed with 3H-E2 for the binding sites. The values of the apparent equilibrium constants (Ka) for the proteins from male rat liver and female rat liver and uterus were equal to (6,6 +/- 1,2) . 10(9) M-1, (7,4 +/- 0,9) . 10(9) M-1 and (11,2 +/- 2,3) . 10(9) M-1, respectively. The dissociation kinetics of the 3H-E2--protein complexes from the three tissues at 0--4 degrees were two-phase: during the first 8--12 hours the dissociation processes were characterized by the dissociation rate constants (k-1) equal to (4--5) . 10(-5) S-1; then the k-1 values were decreased approximately by one order of magnitude. The kinetics of 3H-E2 association with the three types of proteins are presumably two-phase as well. During the first 10--15 min the association process can be characterized by association rate constants equal to (8--27). .10(5 M-1 S-1; then these values decreased about 4-fold. The data obtained suggest that the high molecular weight estrogen--binding proteins from different tissues are similar in their E2-binding properties on the one hand, and may be interpreted as evidence for the heterogeneity of the populations of E2-binding proteins in various tissues, on the other.     

Androgen and estrogen binding in rat skeletal and perineal muscles.

Specific in vitro binding of [3H]testosterone (T), 5ALPHA[3H]dihydrotestosterone (DHT), and [3H[estradiol (E2) was demonstrated in the 30 000 X g supernatant (cytosol) of thigh muscles (TM) and of the levator ani - bulbocavernosus muscle complex (LA-BC) by gel filtration through Sephadex G-25 columns. In TM cytosol, T and E2 [are bound with high affinity (Ka = 1.1 X 10(9) M-1, and 2.3 X 10(9) M-1 respectively) whereas DHT binding is of lower affinity (Ka = 5.0 X 10(7) M-1).] In LA-BC cytosol, T, E2, and DHT are bound with high affinity (Ka = 1.9 X 10(9) M-1, 0.3 X 10(9) M-1, and 0.5 X 10(9) M-1, respectively). Competition experiments suggest that the binding of the three hormones (T, E2, and DHT) is due to different proteins. In addition to TM and LA-BC, T and E2 binding was found in other muscles of male and female rats, including gastrocnemius, the pectoralis, diaphragm, and heart.     

Modulation of gonadotropin-releasing hormone receptor concentration in cultured female rat pituitary cells by estradiol treatment

Short-term (0.5-4 h) treatment of rat pituitary cells in culture with estradiol (E2) results in a significant decrease of Gonadotropin-Releasing Hormone (GnRH) induced LH-release. We studied whether changes in the concentrations of GnRH-receptors (GnRH-R) might account for this phenomenon: pituitary cells from adult female rats were incubated for 4 or 24 h in the presence or absence of 10(-9) M E2. Then saturation curves of D-Ala6-des-Gly10-GnRH ethylamide binding were obtained. In addition, binding studies were carried out in cultures incubated for 0.5, 1, 2 or 4 h with or without 10(-9) M E2 using a near saturating concentration of GnRH-analog. No changes of GnRH-R affinity occurred (4 h experiments: Ka in vehicle treated cells: 0.94 +/- 0.2 x 10(9) M-1, Ka in E2 treated cells: 1.06 +/- 0.3 x 10(9) M-1; 24 h experiments: Ka vehicle: 0.95 +/- 0.2 x 10(9) M-1, Ka E2: 0.82 +/- 0.3 x 10(9) M-1). The GnRH-R concentrations, however, were significantly reduced (44 +/- 3%; P less than 0.001) by 4 h E2 treatment and increased (by 68 +/- 8%; P less than 0.01) by 24 h of E2 treatment. The GnRH induced LH-release in aliquots of the same cell preparations was significantly reduced after 4 h and markedly increased after 24 h of E2 treatment. The experiments on the time-course of the reduction of D-Ala6-GnRH-binding by E2 treatment showed that the number of GnRH-R was significantly decreased (24 +/- 1%; P less than 0.05) already after 0.5 h of exposure to the estrogen. This is also the time period after which the negative E2-effect on GnRH-induced LH-release becomes significant. These data provide first evidence that the short-term negative E2-effect on GnRH induced LH-release by rat pituitary cells in culture could be mediated via a reduction of available GnRH-R.     

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.     

Prostatic cytosol binding of 5 alpha-androstane-3 beta, 17 beta-diol and estradiol-17 beta

The goal of the present research was characterization of the interaction of 5 alpha-androstane-3 beta, 17 beta-diol (3 beta-diol) with prostatic estradiol-17 beta(E2) binding sites to address the role of this 5 alpha-dihydrotestosterone(DHT)a metabolite in prostatic regulation. Using dextran-charcoal assay we demonstrated specific 3 beta-diol and E2 binding sites in rat ventral prostate cytosol (RVPC) and dog prostate cytosol (DPC). In both cytosols, E2 binding is of high affinity (Ka congruent to 10(9) M-1; RVPC:68 fmol/mg protein), DPC:170 fmol/mg protein), and 3 beta-diol binding is of moderate affinity (Ka congruent to 10(8) M-1; RVPC:62 fmol/mg protein, DPC:165 fmol/mg protein). Unlabeled 3 beta-diol competes effectively for cytosolic 3H-E2 binding sites, whereas unlabeled DHT, 5 alpha-androstane-3 alpha, 17 beta-diol (3 alpha-diol) and testosterone (T) are poor competitors for 3H-E2 binding sites. Using DNA-cellulose column chromatography, we separated prostatic androgen and estrogen binding activities. The E2 binding activity which adhered to DNA-cellulose was displaced by 100-fold excess 3 beta-diol but not by DHT. Thus data from two assay procedures show competition of 3 beta-diol for 3H-E2 binding sites in rat and dog prostate.     

High-affinity binding of thyroid hormones to neuroblastoma plasma membranes

The binding of thyroid hormones to isolated plasma membranes was studied in NB41A3 neuroblasts. Saturable binding of L-T3, D-T3 and L-T4 was observed. Binding was time-dependent, with equilibrium reached in less than 60 min and maximal binding occurring between pH 7.4 and 7. Saturation experiments demonstrated two classes of sites for L-T3: a high-affinity site with Ka 8.4 X 10(9) M-1 and a low-affinity site with Ka 7.3 X 10(6) M-1.L-T3 and D-T3 inhibited each other's binding, L-T3 being several-times more potent. Affinity labeling of isolated membranes with bromoacetylated thyroid hormones disclosed stereospecific binding to SDS-PAGE bands with approximate molecular masses of 27 kDa (preferentially labeled by BrAc-L-T3), 32 kDa (preferentially labeled by BrAc-D-T3), and 48 and 87 kDa (preferentially labeled by BrAc-L-T4). Binding of BrAc-L-T3 to the 27 kDa band accounted for 3.4% of total binding, was selectively inhibited by excess L-T3, and may be involved in intracellular transport of L-T3.     

Characterization of a third human thyroid hormone receptor coexpressed with other thyroid hormone receptors in several tissues

A cDNA that encodes a third type of human thyroid hormone receptor (hTR alpha 1) has been isolated from a skeletal muscle library. The cDNA encodes a 410 amino acid protein, Mr = 46,820. When expressed and translated in vitro, hTR alpha 1 binds T3 with an association constant (ka) of 1.8 x 10(9) M-1. Comparison of the DNA sequence of hTR alpha 1 and a previously identified alpha type thyroid hormone receptor (hTR alpha 2) suggests that they could be transcribed from the same gene, and that alternative RNA splicing results in the synthesis of either hTR alpha 1 or hTR alpha 2. Two mRNA (3.2 kilobases and 6 kilobases) of hTR alpha 1 have been detected in several tissues. At least three types of thyroid hormone receptors (hTR alpha 1, alpha 2, beta), which possess similar affinities for hormone ligands, can be expressed in the same tissue.     

Prostromelysin-1 (proMMP-3) stimulates plasminogen activation by tissue-type plasminogen activator.

Matrix metalloproteinase-3 (MMP-3 or stromelysin-1) specifically binds to tissue-type plasminogen activator (t-PA), without however, hydrolyzing the protein. Binding affinity to proMMP-3 is similar to single chain t-PA, two chain t-PA and active site mutagenized t-PA (Ka of 6.3 x 106 to 8.0 x 106 M-1), but is reduced for t-PA lacking the finger and growth factor domains (Ka of 2.0 x 106 M-1). Activation of native Glu-plasminogen by t-PA in the presence of proMMP-3 obeys Michaelis-Menten kinetics; at saturating concentrations of proMMP-3, the catalytic efficiency of two chain t-PA is enhanced 20-fold (kcat/Km of 7.9 x 10-3 vs. 4.1 x 10-4 microM-1.s-1). This is mainly the result of an enhanced affinity of t-PA for its substrate (Km of 1.6 microM vs. 89 microM in the absence of proMMP-3), whereas the kcat is less affected (kcat of 1.3 x 10-2 vs. 3.6 x 10-2 s-1). Activation of Lys-plasminogen by two chain t-PA is stimulated about 13-fold at a saturating concentration of proMMP-3, whereas that of miniplasminogen is virtually unaffected (1.4-fold). Plasminogen activation by single chain t-PA is stimulated about ninefold by proMMP-3, whereas that by the mutant lacking finger and growth factor domains is stimulated only threefold. Biospecific interaction analysis revealed binding of Lys-plasminogen to proMMP-3 with 18-fold higher affinity (Ka of 22 x 106 M-1) and of miniplasminogen with fivefold lower affinity (Ka of 0.26 x 106 M-1) as compared to Glu-plasminogen (Ka of 1.2 x 106 M-1). Plasminogen and t-PA appear to bind to different sites on proMMP-3. These data are compatible with a model in which both plasminogen and t-PA bind to proMMP-3, resulting in a cyclic ternary complex in which t-PA has an enhanced affinity for plasminogen, which may be in a Lys-plasminogen-like conformation. Maximal binding and stimulation require the N-terminal finger and growth factor domains of t-PA and the N-terminal kringle domains of plasminogen.     

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.     

Thyroid hormone and DNA binding properties of a mutant c-erbA beta receptor associated with generalized thyroid hormone resistance

We have previously reported a family, Kindred A, with autosomal dominant generalized thyroid hormone resistance in which affected members were found to have a mutation in the carboxy-terminal domain of the c-erbA beta thyroid hormone receptor. In the current study, the thyroid hormone and DNA-binding properties of this mutant receptor were determined using c-erbA beta protein synthesized in vitro. Both the wild-type human placental c-erbA beta and Kindred A receptors bound [125I]-triiodothyronine, although the Kindred A receptor had decreased affinity for the hormone. The affinity for triiodothyronine was 4.5 x 10(9) M-1 and 2.3 x 10(10) M-1 for the mutant and wild-type receptors, respectively. No abnormality of DNA-binding was detected with the Kindred A receptor using a sensitive avidin-biotin DNA-binding assay with DNA fragments containing thyroid hormone response elements. The Kindred A mutant receptor which displays abnormal triiodothyronine-binding but normal DNA-binding activities in vitro acts as a dominant negative inhibitor of thyroid hormone action in man.     

Purification and characterization of NADPH-dependent cytosolic 3,5,3'-triiodo-L-thyronine binding protein in rat kidney

The NADPH-dependent cytosolic 3,5,3'-triiodo-L-thyronine(T3)-binding protein (CTBP) has been purified over 30,000-fold from rat kidney by using charcoal extraction, Mono Q-Sepharose, Blue Sepharose CL-6B, and Sephacryl S-200 column chromatography. Purified CTBP had a sedimentation coefficient of 4.7 S, Stokes radius of 32.5A, and calculated molecular weight of 58,000. The apparently homogeneous protein consisted of a single polypeptide chain with Mr of 58,000 as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Scatchard analysis of T3 binding showed that NADPH increases maximal binding capacity without changes in the affinity constant (Ka = 2.43 X 10(9) M-1). Double reciprocal analysis of NADPH and binding capacity gave maximal binding capacity of 16,400 pmol/mg of CTBP, Mr = 58,000. The order of affinity of iodothyronine analogues to purified CTBP was as follows: L-T3 = D-T3 greater than triiodothyroacetic acid greater than L-thyroxine. [125I]T3 bound to purified CTBP spontaneously dissociated from CTBP at 20 degrees C (t 1/2 = 22 min) in the absence of NADPH, whereas the dissociation was not observed in the presence of NADPH. The optimal pH for T3 binding was 7.2-7.5 Na+, K+, Ca2+, and Mg2+ (0-200 mM) did not influence T3 binding to CTBP. The purified CTBP did not bind to DNA and was not adsorbed to concanavalin A-Sepharose.     

Molecular basis of the isoform-specific ligand-binding affinity of inositol 1,4,5-trisphosphate receptors

Three isoforms of the inositol 1,4,5-trisphosphate (IP(3)) receptor (IP(3)R), IP(3)R1, IP(3)R2, and IP(3)R3, have different IP(3)-binding affinities and cooperativities. Here we report that the amino-terminal 604 residues of three mouse IP(3)R types exhibited K(d) values of 49.5 +/- 10.5, 14.0 +/- 3.5, and 163.0 +/- 44.4 nm, which are close to the intrinsic IP(3)-binding affinity previously estimated from the analysis of full-length IP(3)Rs. In contrast, residues 224-604 of IP(3)R1 and IP(3)R2 and residues 225-604 of IP(3)R3, which contain the IP(3)-binding core domain but not the suppressor domain, displayed an almost identical IP(3)-binding affinity with a K(d) value of approximately 2 nm. Addition of 100-fold excess of the suppressor domain did not alter the IP(3)-binding affinity of the IP(3)-binding core domain. Artificial chimeric proteins in which the suppressor domain was fused to the IP(3)-binding core domain from different isoforms exhibited IP(3)-binding affinity significantly different from those of the proteins composed of the native combination of the suppressor domain and the IP(3)-binding core domain. Systematic mutagenesis analyses showed that amino acid residues critical for type-3 receptor-specific IP(3)-binding affinity are involved in Glu-39, Ala-41, Asp-46, Met-127, Ala-154, Thr-155, Leu-162, Trp-168, Asn-173, Asn-176, and Val-179. These results indicate that the IP(3)-binding affinity of IP(3)Rs is specifically tuned through the intramolecular attenuation of IP(3)-binding affinity of the IP(3)-binding core domain by the amino-terminal suppressor domain. Moreover, the functional diversity in ligand sensitivity among IP(3)R isoforms originates from at least the structural difference identified on the suppressor domain.     

Human autoantibody to vasoactive intestinal peptide: increased incidence in muscular exercise

Specific autoantibodies to vasoactive intestinal peptide were present in plasma from 29.6% healthy human subjects who habitually performed muscular exercise, compared to 2.3% healthy subjects who did not. Saturation analysis of VIP binding by the plasmas suggested the presence of 1 and 2 classes of autoantibodies in 5 and 3 high exercise subjects, respectively, distinguished by their equilibrium affinity constants (Ka). The mean Ka values for the high and low affinity autoantibody classes were, respectively, 1.3 x 10(8) M-1 and 0.8 x 10(7) M-1. These values are lower than the Ka range reported previously for tissue VIP receptors (G. Rosselin. Peptides 7, Suppl. 1, 89, 1986) but are larger than the inverse Michaelis constant (1/Km) for VIP-degrading proteases (T.N. Keltz, E. Straus, R.S. Yalow. Biochem. Biophys. Res. Commun. 92, 669, 1980). The autoantibodies may not interfere with VIP-receptor binding but are potential inhibitors of the proteolytic inactivation of VIP.     

The monomer of pyruvate kinase, subtype M1, is both a kinase and a cytosolic thyroid hormone binding protein

Using a T7 expression system, the monomer of rat pituitary pyruvate kinase, subtype M1 (PKM1), was overexpressed in Escherichia coli and purified to homogeneity. The monomeric p58-M1 has intrinsic enzymatic activity with a Vmax of 79 +/- 20 units/mg and Km's for ADP and PEP of 1.43 +/- 0.76 and 0.14 +/- 0.07 mM, respectively. The monomer binds 3,3',5-triiodo-L-thyronine (T3) with Ka = 1.5 x 10(7) M-1. The order of analog specificity is L-T3 greater than L-thyroxine greater than D-T3 greater than 3'-isopropyl-3,5-diiodo-L-thyronine greater than or equal to 3',5',3-triiodo-L-thyronine. In contrast, tetrameric PKM1 lacks T3 binding activity. The kinase activity of p58-M1 is inhibited by T3 and its analogs in a concentration-dependent manner with the order of inhibitory activity similar to that of binding activity. This inhibition, however, is reversed by the addition of fructose 1,6-bisphosphate. p58-M1 is the second PK isoenzyme monomer to be identified as having thyroid hormone binding activity.     

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.