Studies on the non-activated and activated forms of the estrogen receptor.

The activation of the steroid receptor is a necessary process for the biological role of the receptor. Many factors are involved in this mechanism; in addition to time, temperature, and salt concentration, RNA and RNAase can also affect the transformation of the non-activated to the activated form of the receptor. Using as a model the estrogen receptor of fetal uterus of guinea-pig, the studies of the interaction with three different monoclonal antibodies (D547, H222 and H226) reveal structural transformation during the process of the receptor activation. These conformational transformations suggest that a change in the exposure of the functional domains of the estrogen receptor occurs during activation.     

Inactivation of rat liver glucocorticoid receptor by molybdate. Effects on both non-activated and activated receptor complexes.

When freshly prepared glucocorticoid-receptor complex from rat liver cytosol was incubated at 23 degrees C in the presence of sodium molybdate, its subsequent binding to isolated nuclei, DNA-cellulose and ATP-Sepharose was blocked. In addition, binding to these acceptors by cytosol receptor complex fractionated with (NH4)2SO4 was also blocked by incubation of the complexes with 50 mM-sodium molybdate. However, molybdate had no effect on the binding of activated receptor complexes to ATP-Sepharose. Molybdate was also effective in extracting the nuclear- and DNA-cellulose-bound glucocorticoid-receptor complexes in a dose-dependent manner. Molybdate appears to exert its effects directly on the receptor by interacting with both non-activated and activated receptor forms.     

Characterisation of non-activated and activated estrogen- and antiestrogen-receptor complexes by high performance ion exchange chromatography

The ionic species of cytosol estrogen receptors from mature rat uteri have been compared by HPIEC on a SynChrom AX-1000 column when complexed with either [125I]iodoestradiol, [3H]estradiol or [3H]4-hydroxy tamoxifen. Three species of receptors (isoforms) each suppressible by excess competitor were fractionated at identical salt concentrations regardless of ligand employed. One species eluted in the column void volume (10 mM) and the others at congruent to 90 mM and congruent to 155 mM phosphate. Activation of receptor complexes by increasing time of incubation with ligand from 1 to 24 h at 4 degrees C or addition of 10 mM GTP increased the proportion of the congruent to 90 mM species for all 3 ligands. The addition of 10 mM molybdate to homogenization and HPIEC buffers resulted in only two species being resolved at 10 and 110 mM phosphate. These species were again identical regardless of ligand employed. Increasing concentrations of estradiol (1-40 nM) tamoxifen (20 nM-4 microM) and 4-hydroxy tamoxifen (2-400 nM) were able to compete for binding of [125I]iodoestradiol to each of the three ionic species. Binding to each species was inhibited equally by each concentration of competitor. There was no preferential or unique association of estrogen or antiestrogen with any of the ionic species and all ligands gave identical ionic species of non-activated and activated receptor complexes.     

Three-dimensional structure of non-activated cGMP phosphodiesterase 6 and comparison of its image with those of activated forms

Cyclic GMP phosphodiesterase (PDE6) in rod photoreceptors, a key enzyme in vertebrate phototransduction, consists of two homologous catalytic subunits (Palpha and Pbeta) and two identical regulatory subunits (Pgammas). Pgamma regulates the PDE activity through its direct interaction with transducin. Here, using electron microscopy and image analysis of single particles, we show the three-dimensional organization of the basic form of bovine PDE, Palphabetagammagamma, and compare its average image with those of Pgamma-released PDE. The structure of Palphabetagammagamma appears to be a flattened bell-shape, with dimensions of 150 x 108 x 60A, and with a handle-like protrusion attached to the top of the structure. Except for the protrusion, the organization consists of two homologous structures arranged side by side, with each structure having three distinct regions, showing pseudo twofold symmetry. These characteristics are consistent with a model in which the overall structure of Palphabetagammagamma is determined by hetero-dimerization of Palpha and Pbeta, with each subunit consisting of one catalytic and two GAF regions. A comparison of the average image of Palphabetagammagamma with those of Pgamma-released PDE suggests that Pgamma release does not affect the overall structure of Palphabeta, and that the Palphabeta C-terminus, but not Pgamma, is a determinant for the Palphabeta orientation on carbon-coated grids. These observations suggest that the basic structure of PDE does not change during its regulation, which implies that Palphabeta is regulated by its regional interaction with Pgamma.     

Physicochemical properties of non-activated and activated renin from human amniotic fluid.

The main physicochemical and enzymic properties of non-activated and activated human amniotic renin (EC 3.4.99.19) were studied in order to clarify the relationships between the two enzymes. Human amniotic renin was activated by dialysis against acidic buffer (pH 3.3), direct acidification or trypsin treatment. All procedures produced similar activation. The physicochemical characteristics of non-activated and activated renin were compared to those of human renal renin. Non-activated renin had a molecular weight of 45,500. A similar molecular weight was obtained by gel eluate activation and by acid treatment of renin prior to gel filtration. Similar isoelectric points were also found for non-activated and activated renin. One major renin peak focused at pH 6.6, whereas no similar renin peak was detected in extracts from normal human kidney. In addition, non-activated and activated renin forms were found to have the same optimal pH, the same Km and the same inhibiting pepstatin concentrations.     

Intracellular flow of particulate steroid-receptor complexes in steroid target cells

The structural expression of nuclear transportation of receptor-bound oestradiol was investigated by immunoelectron microscopy on human breast cancer cells. In oestradiol-treated cells during transportation, oestradiol antibodies attached to cytoplasmic oestradiol-bearing particles which seemed to interact with the nuclear membrane. These particles, subserving the intracellular flow of steroid-receptor complexes, could constitute a special cellular system of macromolecular transport, communication and compartmentalisation.     

Testicular cytosol factors inhibit the binding of steroid-receptor complexes to chromatin

Testicular and prostatic androgen-receptor complexes as well as uterine estradiol-receptor complexes, partially purified by ammonium sulfate precipitation (15-37%), were bound to germ cell chromatin. At equivalent concentrations, less testicular androgen-receptor complexes bound to chromatin than did the other two steroid-receptor complexes. Addition of a partially purified testicular androgen-receptor preparation with prostatic androgen-receptor or uterine estradiol-receptor preparation to the binding interaction mixture reduced the binding of either of the latter two steroid-receptor complexes to chromatin. These data suggest the presence of inhibitory factor(s) in the testicular receptor preparations. Testicular cytosols were fractionated by ammonium sulfate precipitation into fractions A (15-37% saturation), B (37-50%) and C (50-75%). All fractions inhibit binding of these steroid-receptor complexes to chromatin. Fractions A and B appear to be heat labile, while fraction C was more stable. Further fractionation of A and C fractions on DEAE cellulose yielded A1 and C1 (filtrates) as well as A2 and C2 (0.3 M NaCl eluents), respectively. Subfractions A1, A2, and C2 contained inhibitory factors for the binding of steroid-receptor complexes to chromatin while C1 showed no effect. These data demonstrated that testicular cytosol contains a variety of inhibitory factors which affect the binding of both androgen-receptor and estradiol-receptor complexes to chromatin.     

Differential effects of molybdate on the hydrodynamic and DNA-binding properties of the non-activated and activated forms of the androgen receptor in calf uterus

Calf uterine cytosol contains an androgen receptor with a relative molecular mass of approx. 90,000. In this study we have analysed the structure and aggregation properties of the androgen receptor, using sucrose density gradient centrifugation on a vertical rotor (VTi65). In the presence of 10 mM NaCl the androgen receptor in whole cytosol sedimented at 8 S irrespective of the presence of molybdate. In 400 mM NaCl the receptor dissociated to a 4.3 S entity. In whole cytosol molybdate promoted a partial shift of the 4.3 S receptor into the aggregated 8 S state. The time of exposure of the receptor to molybdate and NaCl determined the proportion of receptor sedimentating at 8 S and 4.3 S. The DNA-binding form of the uterine androgen receptor when analysed under the conditions of the DNA-cellulose binding assay, sedimented at 6.5 S. Increasing concentrations of molybdate shifted its sedimentation coefficient gradually from 6.5 S to 4.5 S and in parallel reduced the DNA-binding capacity. Molybdate added to a partially purified, DNA-binding form of the androgen receptor did not promote receptor aggregation to faster sedimentating forms. This suggests that such preparations are devoid of an androgen receptor-aggregation factor. Indirect evidence for such a factor was obtained from reconstitution experiments with whole cytosol. Our results indicate that the DNA-binding form of the androgen receptor interacts with a cytosol factor to form the 8 S receptor complex. Molybdate has diverse effects: in the presence of the cytosol factor it stabilizes the 8S complex; in its absence molybdate prevents in a concentration-dependent way DNA-binding as well as reaggregation of the monomeric 4.3 S form.     

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.     

Sequence of formation of molecular forms of plasminogen and plasmin-inhibitor complexes in plasma activated by urokinase or tissue-type plasminogen activator.

Total cellular polyadenylated RNA [poly(A)+ RNA] was prepared after guanidinium thiocyanate extraction of frozen brain tissue from age-matched normal and Down's-syndrome (trisomy 21) human foetuses. Poly(A)+ RNA populations were analysed by translation in vitro, followed by two-dimensional gel analysis by using both isoelectric focusing (ISODALT system) and non-equilibrium pH-gradient electrophoresis (BASODALT system) as the first-dimension separation. The relative concentrations of poly(A)+ RNA species coding for seven translation products were significantly altered in Down's syndrome, as determined by both visual comparisons of translation-product fluorograms from normal and Down's-syndrome samples and by quantitative radioactivity determination of individual translation products. The relative concentrations of mRNA species coding for two proteins (68 kDa and 49 kDa) were increased in Down's syndrome and may represent genes located on chromosome 21. The relative concentrations of mRNA species coding for five proteins (37 kDa, 35 kDa, 25.5 kDa, 24.5 kDa, 23 kDa) were decreased in Down's syndrome, these probably representing secondary effects of the trisomy. Six Down's-syndrome-linked translation products (49 kDa, 37 kDa, 33 kDa, 25.5 kDa, 24.5 kDa, 23 kDa) did not migrate with appreciable amounts of cellular proteins on two-dimensional gels and hence may represent either proteins of high turnover rates or those that are post-translationally modified in vivo. One translation product (68 kDa) comigrated with a major cellular protein species, which was identified as a 68 kDa microtubule-associated protein by limited peptide mapping. The significance of these changes is discussed in relation to the mechanisms whereby the Down's-syndrome phenotype is expressed in the human brain.     

The cytotoxic action of activated and non-activated cyclophosphamide in yeast: Comparison of induced DNA damage

The cytotoxic and DNA-damaging effects of cyclophosphamide (CP) and its ‘activated’ derivative 4-OOH-CP were studied using a series of strains of S. cerevisiae which allow a phenotypical classification of genotoxic characteristics as well as direct physicochemical demonstration of key DNA lesions. The concurring results of biological and biochemical experiments indicate that (i) non-activated CP has a weak but detectable monofunctional alkylating potency, leading to DNA strand breaks and (ii) 4-OOH-CP has the ability to induce both DNA strand breaks and interstrand cross-links. The activity of CP is probably due to spontaneous decomposition in aqueous solution.     

Thermodynamic characterization of interactions between p27(Kip1) and activated and non-activated Cdk2: intrinsically unstructured proteins as thermodynamic tethers

The cyclin-dependent kinase inhibitor (CKI) p27Kip1 plays a critical role in cell cycle regulation by binding and inhibiting (or activating) various cyclin-dependent kinase (Cdk)/cyclin complexes. Thermal denaturation monitored by circular dichroism (CD) and isothermal titration calorimetry (ITC) were used to determine the relative stabilities and affinities of p27-KID (p27 kinase inhibitory domain) complexes with activated Cdk2 (phosphorylated at Thr160; P-Cdk2) and non-activated forms of Cdk2 and/or cyclin A. Phosphorylation of residue Thr160 only slightly increases the thermal stability of Cdk2, and its binary complexes with cyclin A and p27-KID. The p27-KID/P-Cdk2/cyclin A or p27-KID/Cdk2/cyclin A ternary complexes exhibited significantly higher thermal stabilities compared to the binary complexes (P-Cdk2/cyclin A or Cdk2/cyclin A). Differences in T(m) values between the binary and ternary complexes with P-Cdk2 and Cdk2 were +25.9 and +20.4 degrees C, respectively. These results indicate that the ternary complex with phosphorylated Cdk2 is stabilized to a larger extent than the non-phosphorylated complex. The free energy of association (deltaG(A)) for formation of the two ternary complexes was more favorable than for the binary complexes, indicating that a significantly smaller population of free components existed when all three components were present. These data indicate that p27-KID, which is intrinsically disordered in solution, acts as a thermodynamic tether when bound within the ternary complexes. It is proposed that thermodynamic tethering may be a general phenomena associated with intrinsically unstructured proteins (IUPs) which often function by binding to multiple partners in multi-protein assemblies.     

Immobilization of enzymes on crosslinked gelatin particles activated with various forms and complexes of titanium(IV) species

A number of methods of activating the surface of glutaraldehyde crosslinked gelatin beads with titanium(IV) compounds, for subsequent enzyme coupling, have been investigated. Glucoamylase (exo-1,4-α-d-glucosidase, EC 3.2.1.3) was so immobilized using titanium(IV)-urea, -acrylamide, -citric acid and -lactose complexes; however, immobilized enzyme preparations with low activities were obtained (0.36–1.28 U g^?1). Activation with uncomplexed titanium(IV) chloride, however, of both moist and freeze-dried crosslinked gelatin particles resulted in highly active immobilized glucoamylase preparations (1.74–26.6 U g^?1). Dual immobilized enzyme conjugates of glucoamylase and invertase (β-d-fructofuranosidase, EC 3.2.1.26) were also prepared using this method. Invertase was served on the entrapped enzyme while glucoamylase was coupled on the surface of titanium(IV)-activated gelatin pre-entrapped invertase particles. A dual gelatin coupled glucoamylase/gelatin entrapped glucoamylase was prepared (3.8 U g^?1) and ～72.5% of the total combined activity was due to the surface bound enzyme.     

Activated steroid-receptor complex. Comparison of assays using DNA-cellulose or homologous nuclei.

When soluble steroid-receptor complexes are exposed to DNA-cellulose only activated complexes bind. The specificity of the binding was shown by its dependence on the presence of hormone during activation. However, prolonged incubation of non-activated steroid-receptor complexes with DNA-cellulose led to a progressive activation of these complexes. When the same hepatic cytosol containing heat-activated [3H]triamcinolone acetonide-receptor complexes was titrated by high concentrations of nuclei or DNA-cellulose the former bound 75% of the complexes, the later only 40%. This decreased binding was due on the one hand to a lower initial interaction between DNA-cellulose and activated complexes than between nuclei and these complexes and on the other hand to increased losses during washes when DNA-cellulose was used. For these reasons nuclei and not DNA-cellulose should be used when accurate measurements of the concentration of activated complexes are required. When only comparative data are needed DNA-cellulose may, however, be employed.