In vitro modulation of rat liver glucocorticoid receptor by urea

We have examined the influence of urea on the properties of the rat liver glucocorticoid receptor (GR). A 1-h incubation of hepatic cytosol with 1-3 M urea at 0 or at 23 degrees C caused a progressive decrease in the steroid binding efficiency of GR. Urea treatment of cytosol incubated with 20 nM [3H]triamcinolone acetonide caused transformation of glucocorticoid-receptor complexes (GRc) and resulted in an increase in the binding of GRc to DNA-cellulose and ATP-Sepharose. The transforming effect was maximal with 2.5 M urea at 0 degrees C for 1 h, and it caused a shift in the rate of sedimentation of the 9 S untransformed GRc to a 4 S form, similar to that observed upon incubation of the cytosol GRc at 23 degrees C. This 9 to 4 S transformation could also be observed in the presence of Na2MoO4. The Stokes radii of the GRc eluted from a Bio-Gel-A-0.5m agarose column were determined to be 5.9 and 4.9 nm in the absence and presence of 2.5 M urea. The aqueous two-phase partitioning analysis revealed a significant change in surface properties of GR following urea treatment; the observed partition coefficient values (cpm upper phase/bottom phase) were 0.022, 0.208, and 0.60 for GRc, GRc + 23 degrees C, and GRc + 2.5 M urea, respectively. Furthermore, the urea treatment rendered the GRc less negatively charged, forcing their appearance in the flow-through fractions of a DEAE-Sephacel column. These results suggest that urea is a potent in vitro modulator of the physicochemical behavior of GR, influencing both the steroid binding and the process of receptor transformation.     

Chromatographic separation of nontransformed and transformed glucocorticoid-receptor complexes from rat liver cytosol. Use of tungstate in the purification, inactivation, and resolution of receptor forms

Aliquots of rat liver cytosol glucocorticoid-receptor complexes (GRc) were transformed by an incubation with 8-10 mM ATP at 0 degrees C and were compared with those transformed by an exposure to 23 degrees C. The extent of receptor transformation was measured by chromatography of the samples over columns of DEAE-Sephacel. The ATP-transformed complexes, like those which were heat-transformed, exhibited lower affinity for the positively charged ion-exchange resin and were eluted with 0.12 M KCl (peak-I): the nontransformed complexes appeared to possess higher affinity and required 0.21 M KCl (peak II) for their elution. As expected, the receptor in the peak-I exhibited the DNA-cellulose binding capacity and sedimented as 4S in sucrose gradients. Peak II contained an 8-9S glucocorticoid receptor (GR) form that showed reduced affinity for DNA-cellulose. Presence of sodium tungstate (5 mM) prevented both heat and ATP transformation of the GRc resulting in the elution of the complexes in the region of nontransformed receptors. When parallel experiments were performed, binding of the cytosol GRc to rat liver nuclei or DNA-cellulose was seen to increase 10-15 fold upon transformation by heat or ATP: tungstate treatment blocked this process completely. The transformed and nontransformed GRc were also differentially fractionated by (NH4)2SO4: tungstate-treated (nontransformed) receptor required higher salt concentration and was precipitated at 55% saturation. In addition, the GRc could be extracted from DNA-cellulose by an incubation of the affinity resin with sodium tungstate resulting in approximately 500-fold purification of the receptor with a 30% yield. These studies show that the nontransformed, and the heat-, salt-, and ATP-transformed GRc from the rat liver cytosol can be separated chromatographically, and that the use of tungstate facilitates the resolution of these different receptor forms. In addition, extraction of the receptor from DNA-cellulose by tungstate provides another new and efficient method of partial receptor purification.     

Transformation of calf uterine progesterone receptor: analysis of the process when receptor is bound to progesterone and RU38486

Effects of different transforming agents were examined on the sedimentation characteristics of calf uterine progesterone receptor (PR) bound to the synthetic progestin [3H]R5020 or the known progesterone antagonist [3H]RU38486 (RU486). [3H]R5020-receptor complexes [progesterone-receptor complexes (PRc)] sedimented as fast migrating 8S moieties in 8-30% linear glycerol gradients containing 0.15 M KCl and 20 mM Na2MoO4. Incubation of cytosol containing [3H]PRc at 23 degrees C for 10-60 min, or at 0 degrees C with 0.15-0.3 M KCl or 1-10 mM ATP, caused a gradual transformation of PRc to a slow sedimenting 4S form. This 8S to 4S transformation was molybdate sensitive. In contrast, the [3H]RU486-receptor complex exhibited only the 8S form. Treatment with all three activation agents caused a decrease in the 8S form but no concomitant transformation of the [3H]RU486-receptor complex into the 4S form. PR in the calf uterine cytosol incubated at 23 or at 0 degrees C with 0.3 M KCl or 10 mM ATP could be subsequently complexed with [3H]R5020 to yield the 4S form of PR. However, the cytosol PR transformed in the absence of any added ligand failed to bind [3H]RU486. Heat treatment of both [3H]R5020- and [3H]RU486-receptor complexes caused an increase in DNA-cellulose binding, although the extent of this binding was lower when RU486 was bound to receptors. An aqueous two-phase partitioning analysis revealed a significant change in the surface properties of PR following both binding to ligand and subsequent transformation. The partition coefficient (Kobsd) of the heat-transformed [3H]R5020-receptor complex increased about 5-fold over that observed with PR at 0 degrees C.(ABSTRACT TRUNCATED AT 250 WORDS)     

Transformation and phosphorylation of purified molybdate-stabilized chicken oviduct progesterone receptor

The non-transformed, molybdate-stabilized chick oviduct cytosol progesterone receptor was purified approx. 7000-fold using biospecific affinity resin (NADAC-Sepharose), DEAE-Sephacel chromatography and gel filtration on Bio-Gel A-0.5m agarose. The purified preparation contained progesterone receptor which sedimented as a 7.9S molecule, had a Stokes' radius of 7.5 nm, was composed of three major peptides corresponding to Mr 108,000, 90,000 and 79,000. Upon removal of molybdate, the purified [3H]progesterone-receptor complex could be transformed from the 8S form to a 4S form by exposure to 23 degrees C or by an incubation with 10 mM ATP at 0 degrees C. The purified thermally transformed receptor could be adsorbed to columns of ATP-Sepharose. No cytosol factor(s) appeared to be required for the 8S to 4S transformation of purified receptor or for its subsequent binding to ATP-Sepharose. Incubation of purified non-transformed receptor preparation with [gamma-32P]ATP and cAMP-dependent protein kinase led to incorporation of radioactivity in all the three major peptides at serine residues. The results of this study show for the first time that purified 8S progesterone receptor can be phosphorylated in vitro by a cAMP-dependent protein kinase, and that it can be transformed to a 4S form by 0 degrees C incubation with 10 mM ATP.     

Influence of phosphatase inhibitors and nucleotides on [3H]dexamethasone binding in cytosol of human placenta

The purpose of this investigation was to establish the properties of [3H]dexamethasone binding sites in cytosol of human placenta at term. Cytosol containing 20 mM sodium molybdate (MoO4Na2) was incubated for 120 min at 20 degrees C with 40 nM [3H]dexamethasone. The following properties were observed: (a) a single population of binding sites of high affinity and low capacity was measured by Scatchard analysis; (b) potent glucocorticoids such as dexamethasone and cortisol displaced the tritiated ligand, progesterone showed an intermediate activity, whereas cortisone, testosterone and 17 beta-estradiol were ineffective competitors; (c) ultracentrifugation on 16-41% glycerol gradients containing 20 mM MoO4Na2 yielded sedimentation values of 10.25 +/- 0.35 S (n = 4 placentas); (d) the binding sites could be differentiated from the enzyme 11 beta-hydroxysteroid dehydrogenase, as the activity of the former, but not that of the latter, was greatly dependent on the presence of MoO4Na2 in the incubation medium. Inactivation of binding sites labelled with [3H]dexamethasone by incubation at 20 degrees C was prevented by phosphatase inhibitors such as 20 mM MoO4Na2 (P less than 0.01), 20 mM sodium tungstate (WO4Na2) (P less than 0.01) and to a lower extent by 5 mM ATP and cAMP (P less than 0.05). 50 mM NaF, 5 mM GTP or cGMP had no effect. The protection afforded by MoO4Na2 and WO4Na2 was correlated with a significant inhibition of the activity of acid phosphatase, but not alkaline phosphatase. Neither ATP nor cAMP modified phosphatase activity. It is suggested that binding sites for [3H]dexamethasone in cytosol of human placenta showed properties similar to those described for glucocorticoid receptors in target cells, and that these binding sites are regulated by phosphorylation and dephosphorylation mechanisms.     

Resolution of non-activated and activated androgen receptors based on differences in their hydrodynamic properties

This study shows that cytosolic androgen receptor of rat ventral prostate sediments at 10-11 S on conventional low salt sucrose density gradients (SDG), and at 4.6 S on high salt SDG, whether it is activated or not; inclusion of 10 mM Na2MoO4 in all buffers does not alter these sedimentation coefficients. In the presence of 50 mM Na2MoO4 non-activated and activated androgen receptors sediment in high salt SDG at 7-8 S and 4.6 S, respectively. Thus the presence of high concentrations of molybdate during centrifugation inhibits the KCl induced disaggregation of receptor into subunits. Similar effects are observed on Sephacryl-S200 gel filtration; in 50 mM MoO2-4 and 0.4 M KCl non-activated receptor has an estimated Stokes radius of 67 A; this value decreases to 52 A upon activation in the presence of proteolysis inhibitors; omission of molybdate during chromatography yielded 52 A and 27 A entities. Estimated mol. wts are 198,000 Daltons for the non-activated 67 A form and 98,000 Daltons for the activated 52 A receptor. Sodium molybdate (50 mM) prevents temperature (18 degrees C) and high ionic strength (0.4 M KCl) induced receptor activation. This inhibition was overcome by removing molybdate by centrifugal gel filtration, or by increasing the KCl concentration to 0.8 M. The inhibitory effects of molybdate on salt induced receptor disaggregation into activated subunits are no longer observed at pH greater than 7.4 or after chemical modification of sulfhydryl groups. Once androgen receptor has been disaggregated into its activated subunits the activated state is maintained even upon reassociation to 10-11 S aggregates in low salt. The relative concentrations of KCl and molybdate are critical; thus, 10 mM Na2MoO4/0.4 M KCl and 50 mM Na2MoO4/0.8-1.2 M KCl did not differentiate activated from non-activated androgen receptor based on their hydrodynamic properties. In the presence of 0.4 M KCl and 50 mM molybdate, however, the hydrodynamic properties of androgen receptor can be correlated with receptor activation.     

Characterization of nontransformed and transformed androgen receptor from rat submandibular gland

Rat submandibular gland cytosol contained androgen receptor which had a single class of specific binding and an apparent dissociation constant of (1.1-1.2) X 10(-9) M. The process of transformation was investigated by a slightly modified minicolumn method in which the transformed receptor complexes were separated from the nontransformed receptor and meroreceptor. 10 mM ATP or pyrophosphate at 0 degrees C induced transformation of androgen receptor as did heat or salt treatment. 20 mM of sodium molybdate completely inhibited transformation that resulted from ATP, heat or salt treatment. The nontransformed androgen receptor complexes sedimented at 8 S and eluted at 250-260 mM KCl from DEAE-Sephacel, and its molecular weight was found to be 220 000 on Sephacryl S300 gel chromatography. On the other hand, the transformed androgen receptor complexes sedimented at 4.1-4.3 S (ATP or KCl treatment) or 3.5-3.8 S (heat treatment) and eluted at 60-80 mM KCl from DEAE-Sephacel. The molecular weight of the transformed androgen receptor complexes was 80 000-85 000 (ATP or KCl treatment) or 70 000-80 000 (heat treatment). These results suggest that the transformation of androgen-receptor complexes from rat submandibular gland was induced by the subunit dissociation and that salt bridges may be involved in the subunit interaction.     

Immunorecognition of the active form of the oestrogen receptor by using a monoclonal antibody.

In previous studies, two forms (alpha and beta) of the oestrogen receptor, with different immunological characteristics, were observed in the cytosol fraction of fetal guinea-pig uterus, by using a monoclonal antibody to the human oestrogen receptor (D547Sp gamma). Only the alpha form was recognized by the antibody, shifting its sedimentation coefficient in high-salt sucrose gradients. The present work investigated the effect of several factors (time, temperature, high salt concentrations and Na2MoO4) on the interconversion of these two forms. Only the beta form was observed when cytosol was incubated with oestradiol for only 2-3 h, but 20 h later, 40-60% of the total oestradiol-receptor complexes were found as the alpha form. The transformation from the beta to the alpha form was accelerated by temperature (25 degrees C, 15 min) and exposure to high salt concentrations (0.4 M-KCl). On the other hand, Na2MoO4 completely blocked the transformation induced by time and temperature, but had little effect on that induced by KCl. The appearance of the alpha form always correlated with an increase in receptor binding to nuclei and DNA-cellulose. Finally, it was found that the isolated beta form, recovered from the gradient, was transformed into the alpha form after overnight dialysis under reduced pressure. The present data suggest that the alpha form, which is recognized by the monoclonal antibody, is the activated form of the oestrogen receptor.     

Extracellular correction of the androgen-receptor transformation defect in two families with complete androgen resistance

We have characterized the cellular and extracellular phenotype of the mutant androgen receptor (AR) from two families who have complete androgen resistance despite a normal androgen-binding capacity (Bmax) in their genital skin fibroblasts (GSF). The cellular receptors fail to up-regulate their basal AR activity in response to prolonged incubation with 5 alpha-dihydrotestosterone (DHT), or with two synthetic androgens, methyltrienolone (MT) and mibolerone (MB), and form A-R complexes with increased equilibrium (Kd) and non-equilibrium (k) dissociation constants. In addition, they are thermolabile when recently dissociated, but not in their native state. A-R complexes made in normal or mutant cytosol at 4 degrees C elute from DEAE-Sephacel at approximately 0.25 M KCl (untransformed), with or without prior passage through Sephadex G-25; when made in cells at 37 degrees C, extracted with 0.4 M KCl in a buffer containing 10 mM Na2MoO4, and desalted by G-25, they elute at less than or equal to 0.1 M KCl. Normal KCl-extracted DHT- and MB-R complexes dissociate (37 degrees C) at the same slow, linear rate as their in-cell counterparts (transformed); the mutant ones dissociated more slowly than their rapidly-dissociating in-cell counterparts and, to a variable extent, nonlinearly-an early faster phase, a later slower (transformed). Thus, as judged by two conventional criteria of steroid-R complex transformation, the mutant A-R complexes can transform, possibly in two steps, under certain cell-free conditions. This behavior differentiates a class of structural AR mutations whose molecular definition awaits application of recombinant DNA techniques to the X-linked AR locus.     

Protein kinase activity of purified rat liver glucocorticoid receptor

Molybdate-stabilized nonactivated rat liver glucocorticoid receptor (GR) was purified to near homogeneity using a biospecific affinity adsorbent, Bio Gel A 0.5 m and DEAE-Sephacel. The purified GR sedimented in the 9-10S region in 5-20% sucrose gradients containing 0.10M KCl and 20mM Na2MoO4. SDS-polyacrylamide gel electrophoresis revealed a major single band with an apparent molecular weight of 90,000 +/- 2,000. Affinity labeling of GR with [3H]-dexamethasone mesylate showed association of the radioactivity with a peptide of 90,000 molecular weight. Purified receptor preparation was dialyzed to remove molybdate and was incubated with different protein substrates in the presence of 50 microM [gamma-32P]-ATP and divalent cations. Radioactive phosphate from [gamma-32P]-ATP was seen to be incorporated into calf thymus histones, turkey gizzard myosin light chain kinase and rabbit skeletal muscle kinase in the presence of Mg2+ and Ca2+ ions. Addition of steroid ligand exogenously to the reaction mixture appeared to increase the extent of protein phosphorylation. No autophosphorylation of GR was evident under the above conditions. The data suggest that purified rat liver GR displays protein kinase activity.     

Effects of calcium on the chick oviduct progesterone receptor

The chick oviduct cytosol progesterone receptor can be transformed to a small form (Rs = 21A, S20,w:2.9) denoted "mero-receptor" by incubation in the presence of Ca2+ [8]. In the molybdate-free cytosol all the progestin binding components could be completely transformed to mero-form by 1 h treatment with 100 mM Ca2+ at 0 degrees C. If EDTA was secondarily added, the ligand was rapidly released. If molybdate (20 mM) containing cytosol was incubated with Ca2+, no radioactivity was found in the meroposition on the Agarose A 0.5 m column, but the bound steroid sedimented at 2.9 S in sucrose gradients containing Ca2+ (and no molybdate). When 20 nM molybdate was added to cytosol containing receptor activated by 0.3 M KCl, complete mero-transformation by Ca2+ was obtained also by the gel filtration criterion, indicating that molybdate does not inhibit the mero-transforming factor. Ligand-free progesterone receptor could also be completely converted to mero-form by endogenous cytosolic transforming factor and calcium. The transforming factor was completely inactivated, when cytosol was run through Agarose A 0.5 m gel. Mero-transformation was found to be irreversible. The purified progesterone receptor subunit 110 K (B) was partially converted to smaller forms by calcium alone (100 mM, 0 degrees C, 1 h) whereas addition of a small amount of cytosol allowed complete conversion to mero-form.     

Temperature-Dependent Kinetic Correlates of the Activation of the Glucocorticoid-Receptor Complex

The effects of temperature on the kinetics of activation were studied in [3H]triamcinolone acetonide[( 3H]TA)-labeled cytosol preparations from mouse whole brain. After removal of unbound [3H]TA and molybdate (which prevents activation) from the unactivated steroid-receptor complex by gel exclusion chromatography, activation was initiated by incubation at 6-30 degrees C for 0.75-24 min and then rapidly quenched at -5 degrees C with Na2MoO4 (20 mM final concentration). The loss of the 9.2S (unactivated) form of the [3H]TA-receptor complex and the concomitant formation of the 3.8S (activated) form increased dramatically with increases in the activation temperature. These hydrodynamic changes were correlated directly with rapid time- and temperature-dependent increases in the binding of [3H]TA-labeled cytosol to DNA-cellulose (DNA-C). Further analyses of these data revealed a greater than 50-fold increase in the apparent first-order rate constant for the increased binding to DNA-C as the activation temperature was increased from 6 degrees C to 30 degrees C. An Arrhenius plot of these temperature-dependent kinetic constants revealed an energy of activation of 116 kJ. These data support a proposed model for activation of the glucocorticoid-receptor complex that includes the splitting of a 297 kDa, unactivated species into a 92 kDa, activated species.     

Activation of [3H]estradiol--and [3H]H1285-receptor complexes: effect of salt versus ATP on molybdate-stabilized estrogen receptors

The activation by salt or ATP of [3H]estradiol- and [3H]H1285-receptor complexes from rabbit uterus and their binding capacity to DNA-cellulose, phosphocellulose and ATP-Sepharose has been studied. The estrogen-receptor was prepared in 1 mM molybdate which stabilized the receptor; but both salt- and ATP-transformation of estrogen receptors occurred. The binding of molybdate-stabilized cytosol [3H]estradiol-receptor complexes to the various resins revealed that salt-activation by 0.3 M KCl caused the greatest binding (5-6-fold) to DNA-cellulose as compared to other resins. However, 5 mM ATP-dependent activation of receptor-complexes resulted in preferential binding to ATP-Sepharose. Activated cytosol [3H]H1285-receptor complexes bound all the resins to a lesser degree when compared to [3H]estradiol-receptor complexes. Partially purified receptor complexes also showed different resin-binding patterns for salt- and ATP-mediated activation. These findings suggest that salt-activation is different than ATP-activation. Further, the differential magnitude of [3H]estradiol- and [3H]H1285-receptor activation suggests that estrogen-receptor complexes are "fully" activated as compared to "partially" activated antiestrogen-receptor complexes.     

Transformation (4S to 5S) of the nuclear estrogen receptor is reversible but not accompanied by a change in the affinity for DNA

The nuclear estrogen receptor from calf uterus was used to investigate the possible relationship between receptor transformation (4S to 5S) and receptor activation (DNA binding). Receptors extracted from nuclei after exposure of uterine tissue tc [3H]estradiol sedimented at 5.2S, the characteristic value of the transformed receptor. After storage at -20 degrees C the receptor sedimented at 4.0S, indicating conversion of the 5S form into the non-transformed 4S form. Upon reincubation at 28 degrees C the 4S form transformed into the 5S form following second-order kinetics. The rate constant obtained was 4.3 x 10(7) M-1 min-1, a value identical to that reported for the cytosol receptor. These data show that receptor transformation is reversible. Molybdate (10-50 mM) was not able to prevent receptor transformation in the nuclear extract, but was inhibitory in cytosol. This suggests that molybdate does not prevent receptor transformation, but rather inhibits disaggregation of the 8S oligomer into the 4S monomer. In DNA-binding assays (DNA-cellulose or nuclei) the non-transformed (4S) and transformed (5S) states of the nuclear estrogen receptors displayed identical affinities for DNA. The present data show that 4S to 5S transformation of nuclear receptors follows a readily reversible process, but this process is not an essential step for the exposure of the receptors' DNA-binding site. Although the physiological function of the 5S form remains unclear it may be important for the recognition of specific gene regulatory sites.     

Characterization of 2',3'-O-(2,4,6-trinitrocyclohexadienylidine)adenosine 5'-triphosphate as a fluorescent probe of the ATP site of sodium and potassium transport adenosine triphosphatase. Determination of nucleotide binding stoichiometry and ion-induced changes in affinity for ATP

The fluorescent ATP derivative 2',3'-O-(2,4,6-trinitrocyclohexadienylidine) adenosine 5'-triphosphate (TNP-ATP) binds specifically with enhanced fluorescence to the ATP site of purified eel electroplax sodium-potassium adenosine triphosphatase, (Na,K)-ATPase. A single homogeneous high affinity TNP-ATP binding site with a KD of 0.04 to 0.09 microM at 3 degrees C and 0.2 to 0.7 microM at 21 degrees-25 degrees C was observed in the absence of ligands when binding was measured by fluorescence titration or with [3H]TNP-ATP. ATP and other nucleotides competed with TNP-ATP for binding with KD values similar to those previously determined for binding to the ATP site. Binding stoichiometries determined from Scatchard plot intercepts gave one TNP-ATP site/175,000 g of protein (range: 1.64 X 10(5) to 1.92 X 10(5) when (Na,K)-ATPase protein was determined by quantitative amino acid analysis. The ratio of [3H]ouabain sites to TNP-ATP sites was 0.91. These results are inconsistent with "half-of-sites" binding and suggest that there is one ATP and one ouabain site/alpha beta protomer. (Na,K)-ATPase maintained a high affinity for TNP-ATP regardless of the ligands present. K+ increased the KD for TNP-ATP about 5-fold and Na+ reversed the effect of K+. The effects of Na+, K+, and mg2+ on ATP binding at 3 degrees C were studied fluorimetrically by displacement of TNP-ATP by ATP. The results are consistent with competition between ATP and TNP-ATP for binding at a single site regardless of the metallic ions present. The derived KD values for ATP were : no ligands, 1 microM; 20 mM NaCl, 3-4 microM; 20 mM KCl, 15-19 microM; 20 mM Kcl + 4 mM MgCl2, 70-120 microM. These results suggests that a single ATP site exhibits a high or low affinity for ATP depending on the ligands present, so that high and low affinity ATP sites observed kinetically are interconvertible and do not co-exist independently. We propose that during turnover the affinity for ATP changes more than 100-fold owing to the conformational changes associated with ion binding, translocation, and release.     

Structure of the mouse glucocorticoid receptor: rapid analysis by size-exclusion high-performance liquid chromatography

Gel-exclusion high-performance liquid chromatography (HPLC) has been used to separate the untransformed from the transformed glucocorticoid receptor (GC-R) extracted from mouse AtT-20 cells. With 200 mM potassium phosphate as the eluent, an efficient separation of the forms of the GC-R is attained in 15-20 min. The untransformed cytosolic GC-R elutes from the column with a Stokes radius (Rs) of 8.2-8.6 nm, as do the molybdate-stabilized GC-R, the purified untransformed GC-R, and the cross-linked cytosolic GC-R. GC-R transformed in vitro by either ammonium sulfate precipitation, KCl treatment, or G-25 chromatography elutes with an Rs of 5.7-6 nm. Also, GC-R extracted from the nucleus with either 0.3 M KCl or 2 mM sodium tungstate, or purified by two cycles of DNA-cellulose chromatography, has an Rs of 5.5-6.3 nm. The data are identical either in the presence or in the absence of 20 mM Na2MoO4, suggesting that molybdate is not causing aggregation to produce a larger Rs value than that of the native receptor. Vertical tube rotor sucrose gradient ultracentrifugation of cytosol produces three forms of the GC-R: 9.1 S, 5.2 S, and 3.8 S. Sequential analysis of the GC-R forms by HPLC and vertical tube rotor ultracentrifugation and vice versa allows for the hydrodynamic determination of molecular weight within a very short time period (2-3 h total).(ABSTRACT TRUNCATED AT 250 WORDS)     

Kinetics of Na-ATPase activity by the Na,K pump. Interactions of the phosphorylated intermediates with Na+, Tris+, and K+

To determine the biochemical events of Na+ transport, we studied the interactions of Na+, Tris+, and K+ with the phosphorylated intermediates of Na,K-ATPase from ox brain. The enzyme was phosphorylated by incubation at 0 degrees C with 1 mM Mg2+, 25 microM [32P]ATP, and 20-600 mM Na+ with or without Tris+, and the dephosphorylation kinetics of [32P]EP were studied after addition of (1) 1 mM ATP, (2) 2.5 mM ADP, (3) 1 mM ATP plus 20 mM K+, and (4) 2.5 mM ADP plus Na+ up to 600 mM. In dephosphorylation types 2-4, the curves were bi- or multiphasic. "ADP-sensitive EP" and "K+-sensitive EP" were determined by extrapolation of the slow phase of the curves to the ordinate and their sum was always larger than Etotal. These results required a minimal model consisting of three consecutive EP pools, A, B, and C, where A was ADP sensitive and both B and C were K+ sensitive. At high [Na+], B was converted rapidly to A (type 4 experiment). The seven rate coefficients were dependent on [Na+], [Tris+], and [K+], and to explain this we developed a comprehensive model for cation interaction with EP. The model has the following features: A, B, and C are equilibrium mixtures of EP forms; EP in A has two to three Na ions bound at high-affinity (internal) sites, pool B has three, and pool C has two to three low-affinity (external) sites. The putative high-affinity outside Na+ site may be on E2P in pool C. The A leads to B conversion is blocked by K+ (and Tris+). We conclude that pool A can be an intermediate only in the Na-ATPase reaction and not in the normal operation of the Na,K pump.     

Characterization of the in vitro stability of the rat hepatic receptor for 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)

The in vitro stability of the Ah receptor from rat hepatic cytosol was evaluated by [3H]TCDD binding studies, gel filtration, and sucrose density gradient ultracentrifugation. Thermal inactivation of unoccupied receptor followed first-order kinetics between 5 and 40 degrees C, with an estimated Ea for inactivation of approximately 35 kcal/mol. Protease inhibitors did not reduce and dilution slightly increased the inactivation rate at 20 degrees C. Recovery and 20 degrees C stability decreased with increasing ionic strength. The TCDD-receptor complex was less susceptible to degradation at 20 degrees C, even in the presence of 0.4 M KCl. Specific binding was markedly pH dependent, with maximum recovery at 7.6. Analysis of the pH curve suggested that cysteine sulfhydryl groups may be involved in TCDD binding. Dithiothreitol (1 mM) maximized recovery and 20 degrees C stability, and addition of the thiol largely reactivated binding sites lost from cytosol prepared without it. Removal of low molecular weight components of cytosol by gel filtration resulted in a rapid 20 degrees C inactivation rate that could not be lessened by dithiothreitol. Glycerol (10% v/v) and EDTA (1.5 mM) maximized recovery of specific binding, but both decreased 20 degrees C stability in a concentration-dependent manner. Calcium chloride (4 mM) increased stability at 20 degrees C by approximately 20%, and retarded the characteristic shift in sedimentation coefficient from approximately 9 to approximately 6 S in high-salt sucrose gradients. The fact that sodium molybdate (20 mM) decreased recovery and 20 degrees C stability when dithiothreitol was present but slightly increased stability in its absence suggested an antagonism between the two compounds. Molybdate mitigated the inactivation induced by 0.4 M KCl, an effect which may be related to the observation of dual peaks in molybdate-containing high-salt sucrose gradients. These data indicate that thermal inactivation of the unoccupied rat hepatic Ah receptor primarily may be due to physical rather than enzymatic processes; (ii) sulfhydryl oxidation, removal of low molecular weight cytosolic components, and high ionic strength result in rapid rates of inactivation at 20 degrees C; and (iii) the large degree of protection conferred by TCDD binding implies a very tight ligand-receptor interaction, and as such accords with TCDDs extraordinary potency and persistence in producing its toxic and biochemical effects.     

Protective effect of sodium molybdate on the acute toxicity of mercuric chloride. V. Enhancement of renal regeneration after exposure to HgCl2

Pretreatment with Na2MoO4 protected rats from HgCl2-induced decreases in the renal concentration of amino acids, RNA, DNA, ATP and dry matter. It also reduced the mercury-induced increases in renal water, Ca and serum creatinine. Ma2MoO4 considerably elevated the RNA/DNA ratio in the renal cortex after treatment with HgCl2. In addition, subcellular distribution of mercury was markedly altered by pretreatment with Na2MoO4, specifically Na2MoO4 pretreatment decreased the mercury content in the particulate fractions such as the nuclei and mitochondria while increasing the mercury content of the cytosol. Sephadex G-75 gel filtration showed that the increase in mercury content in the cytosol of Na2MoO4-pretreated rats is due to an increase in the metal content of a metallothionein-like fraction. These results suggest that Na2MoO4-pretreatment protects against HgCl2 renal toxicity by stimulating mercury-mediated metallothionein induction in the renal cortex and renal regenerative processes.     

Differential roles of heat shock protein 70 in the in vitro nuclear import of glucocorticoid receptor and simian virus 40 large tumor antigen.

Nuclear import of glucocorticoid receptors (GRs) was analyzed in vitro with digitonin-permeabilized cells (S. A. Adam, R. Sterne-Marr, and L. Gerace, J. Cell Biol. 111:807-816, 1990). Indirect immunofluorescence methods were used to monitor the transport of GRs from rat hepatoma and fibroblast cell cytosol into HeLa nuclei. In vitro nuclear import of GRs was shown to be hormone dependent and to require ATP and incubation at ambient temperatures (i.e., 30 degrees C). Hormone-dependent dissociation of GR-bound proteins, such as the 90-kDa heat shock protein, hsp90, is part of an activation process that is obligatory for the expression of the receptor's DNA-binding activity. Inhibition of in vitro GR activation by Na2MoO4 blocked hormone-dependent nuclear import, demonstrating that receptor activation is required for nuclear import. The addition to GR-containing cytosol of antiserum directed against the cytosolic 70-kDa heat shock protein, hsp70, while effective in blocking the nuclear import of simian virus 40 large tumor antigen (SV40 TAg), did not affect hormone-dependent nuclear import of endogenous, full-length GRs or an exogenously added truncated GR protein (i.e., XGR556) that lacks a hormone-binding domain but possesses a constitutively active nuclear localization signal sequence (NLS). Depletion of hsp70 from HeLa cell cytosol did not affect the nuclear import of exogenously added XGR556 but led to inhibition of SV40 TAg nuclear import. Thus, two closely related NLSs, one contained within GRs and the other contained within SV40 TAg, are distinguished by their differential requirements for hsp70 in vitro.