Putative androgen receptors distinguished in wild-type and testicular-feminized (Tfm) mice.

The reduced level of putative androgen receptor in the mouse mutant, testicular feminization (Tfm), chromatographs on DNA-cellulose differently from the bulk of wild-type receptors. While the elution maximum for extracts of Tfm/Y kidney is in the 180–190 mM NaCl range, wild-type kidney extracts exhibit two maxima of elution at 140–150 mM NaCl and 180–190 mM NaCl, respectively. For hypothalamus-preoptic area, Tfm/Y has one elution maximum at approximately 180 mM NaCl, while the wild-type exhibits a major elution maximum at 140–150 mM NaCl, with a minor peak at approximately 180 mM NaCl. Mixing experiments between wild-type and Tfm/Y cytosols reveal that the different characteristic elution patterns are intrinsic to the binding complexes and are not conveyed simply by other soluble factors. The distinctive pattern for Tfm indicates that the mutation does not cause merely a reduced level of wild-type receptor. Rather the residual receptor of the mutant may be either an abnormal protein or a minor form of wild-type receptor, not readily seen in wild-type tissue due to the presence of more preponderant species. Differences in the elution profiles of androgen receptor species of wild-type kidney with the two bound androgens, testosterone and dihydrotestosterone, are also presented. A model of the androgen receptor system is proposed which includes several binding classes for androgen ligands and metabolites. In light of aromatization of androgens to estrogens and its probable role in some androgenic responses, we include the “estrogen receptor” in this mechanism.     

Purification and physical characterization of nucleic acid helix-unwinding proteins from calf thymus.

We have devised a general protein fractionation procedure which selects for eukaryotic DNA-binding proteins, some of which resemble DNA-unwinding proteins from prokaryotes. Proteins were selected which (a) pass through a native DNA-cellulose column, (b) bind to a denatured DNA-cellulose column, and (c) remain bound to the latter column during a rinse with a dilute solution of the sodium salt of the polyanion dextran sulfate. When this fractionation was applied to the soluble proteins fo calf thymus, three major protein species were recovered. The predominant one has an apparent molecular weight of about 24,000 in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, is isoelectric near neutrality, and elutes as a monomer from denatured DNA-cellulose at moderate NaCl concentrations. This protein, designated calf-unwinding protein 1 (UP1), has been purified to homogeneity. However, isoelectric focusing reveals four or five subspecies (apparently separated by single-charge differences) which differ appreciably in their affinities for DNA. Two other major proteins are obtained which have apparent molecular weights in sodium dodecyl sulfate of 33,000: the first, which elutes with low salt from DNA-cellulose as a homogeneous preparation, appears to be a basic protein (although it is clearly not a histone); the other, which elutes from DNA-cellulose as the major component of a "high salt eluting fraction," is an acidic protein which co-purifies with less prominent species of higher molecular weights. Proteins similar to each of these three major calf thymus proteins have been observed by us and others in tissue culture cells of mouse, hamster, monkey, and humans, suggesting their wide occurrence among eukaryotes.     

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.     

Interaction of vanadyl ribonucleoside complex with the androgen receptor

The addition of vanadyl ribonucleoside complex (VRC), a potent inhibitor of RNase, to the transformed 4.5S androgen receptor from rat submandibular gland caused an increase in the sedimentation coefficient to 7.0S. Moreover, VRC decreased the DNA-cellulose binding of the transformed receptor; 50% inhibition of the DNA-cellulose binding was achieved at 1.8 mM VRC. On the other hand, agents related to VRC and oxoanions of transient metals, such as ribonucleoside, vanadate, molybdate, tungstate and arsenate, exerted no effect on the DNA-cellulose binding ability of the receptor. These findings suggest that VRC binds to the transformed androgen receptor at the DNA-binding site and that both oxovanadium ion and ribonucleoside are indispensable for the binding of VRC to the transformed androgen receptor.     

Sedimentation velocity of sodium hyaluronate-lysozyme mixtures

A study of the sedimentation behaviour of lysozyme in sodium hyaluronate (Na-HA) solution and of the Na-HA medium itself, has been carried out to determine whether the strongly basic enzyme lysozyme forms complexes with Na-HA at physiological ionic strength. At typical physiological salt concentration, 0.146 m NaCl, and also in 0.100 M NaCl, lysozyme sedimentation in an Na-HA solution can be adequately described as independent sedimentation of a slightly associated protein through a three-dimensional network acting partially as a macromolecular sieve. The s_20,w of lysozyme when determined in 0.146 M NaCl, indicated partial aggregation of the enzyme at this salt concentration. Decreases in sedimentation coefficients of lysozyme with increase in Na-HA concentration show a pronounced sieving effect by the equality of observed sedimentation coefficient of lysozyme and Na-HA at higher Na-HA concentrations, but typically individual sedimentation coefficients when the macromolecular mixture was diluted approximately ten-fold.     

Glucocorticoid receptor from lactating goat mammary tissue comparison of native and activated forms in a cell free system

Physicochemical properties of native and activated (DNA-binding) forms of the glucocorticoid receptor in cytosol prepared from lactating goat mammary tissue have been examined. Under hypotonic conditions the cytosolic receptor sediments at 8.4 S or 9.9 S in the absence or presence of 10 mM molybdate, respectively. The receptor in cytosol, either with or without molybdate elutes from DEAE-cellulose at approximately 200 mM potassium phosphate concentration. Isoelectric focusing reveals that this form of the receptor focuses at pH 5.5. Further, the cytosolic form of the receptor exhibits minimal binding affinity for polyanions such as DNA-cellulose. Its Stokes radius is 77 A and the mol. wt is approximately 331,000. Following exposure to in vitro activating conditions (including elevated ionic strength or temperature), the liganded receptor exhibits much lower affinity for DEAE-cellulose (elution at 35-55 mM potassium phosphate concentration). Other alterations in properties of the activated receptor, after partial purification, include sedimentation at 3.9 S in hypotonic sucrose gradients, binding to polyanions (DNA-cellulose), and an isoelectric point at pH 7.2. This receptor has a Stokes radius of 58 A and a mol wt of 98,000. A degraded form, with a mol. wt of approximately 57,000 and high affinity for polyanions, was the major form of the receptor obtained if appropriate precautions to prevent or remove proteolytic activity were not observed during purification and/or characterization of the activated receptor.     

Chromatin receptors for thyroid hormones. Interactions of the solubilized proteins with DNA.

Thyroid hormone-responsive tissues contain chromatin "receptor" proteins that are concentrated in chromatin subfractions enriched in DNA. These receptors appear to be DNA-binding proteins. In the present study, we utilized a DNA-cellulose binding assay to further examine the interactions of solubilized receptors with DNA. [125I]Triiodothyronine associates with receptors bound to DNA-cellulose, whereas free [I]triiodothyronine and [125I]triiodothyronine associated with other proteins does not. The DNA-receptor interactions appear to be strong enough to exist at physiological ionic strength since binding is 50% maximal ag 0.175 M NaCl and is only partly inhibited by Ca2+ and Mg2+ in the 1 to 5 mM range. Most, if not all, of the receptors are capable of DNA binding, and there are at least 80,000 receptor binding sites/diploid DNA (assuming one triiodothyronine binding site/receptor). Binding of the receptor-[125I]triiodothyronine complexes to other DNAs and analogs was examined using a competition assay. There is similar binding by native and denatured DNA, gy eukaryotic DNA from different species and by prokaryotic DNA (Bacillus subtilis). Binding by natural DNAs is more avid than by cytoplasmic RNA, nuclear RNA, poly(dA-dT)-poly(dA-dT), or poly(dG-dC)-poly(dG-dC). Under these conditions, binding by tRNA and poly(dA) is insignificant, and the nucleotide monomers ATP and GTP have no detectable binding. These studies support the idea that the thyroid hormone receptor is a DNA-binding protein and that the interaction is a major determinant for receptor localization in chromatin. The competition studies suggest that the polynucleotide composition and/or conformation can have marked influences on the binding, and that multiple orders of binding affinity can exist. The presence of specific sequences cannot be excluded. However, the finding that receptors bind extensively and tightly to DNA suggests that receptors in chromatin may randomly bind to any available DNA, resulting in some of the receptors being at physiologically unimportant sites. If so, the several thousand hormone receptors present in each target cell may be required to enhance the possibility that some of the receptors are present at the actual sites of action.     

The antiglucocorticoid, cortexolone, fails to promote in vitro activation of cytoplasmic glucocorticoid receptors from the human leukemic cell line CEM-C7

Cortexolone functions as an antiglucocorticoid in the human leukemic cell line CEM-C7, since it blocks the growth inhibition and cell lysis mediated by the potent agonist triamcinolone acetonide (TA). At high concentrations (10(-5) M) cortexolone alone is inactive. The ability of cortexolone to block the TA-mediated biological effects is reflected in its ability (1000-fold molar excess) to effectively block the binding of [3H]TA to the cytoplasmic unactivated form of the receptors eluted from DEAE-cellulose at approx. 180 mM potassium phosphate (KP). Likewise a 1000-fold molar excess of TA inhibits the specific binding of [3H]cortexolone to the unactivated receptors and to a peak which elutes at low salt concentration (35 mM KP) but does not appear to represent activated [3H]cortexolone-receptor complexes. Thermal activation/transformation (25 degrees C for 30 min +/- 10 mM ATP) of the [3H]TA-receptor complexes significantly enhances the subsequent DNA-cellulose binding capacity of these complexes and also results in their elution from DEAE-cellulose at the low salt (50 mM KP) activated position. In contrast, exposure of the cytoplasmic [3H]cortexolone-receptor complexes to identical in vitro activating (transforming) conditions fails to enhance subsequent DNA-cellulose binding capacity or to result in the appropriate shift in DEAE-cellulose elution profile. This inability of [3H]cortexolone to facilitate activation/transformation of receptors was also verified using cytosol prepared from the glucocorticoid-resistant 'activation-labile' mutant, 3R7. Taken collectively the data suggest that cortexolone, unlike an agonist such as TA, fails to promote in vitro activation/transformation, a conformational change which also occurs in vivo under physiological conditions and is a prerequisite for nuclear binding.     

Purification of B-50 by 2-mercaptoethanol extraction from rat brain synaptosomal plasma membranes

Several methods have been described previously for the purification of the nervous-tissue specific protein kinase C substrate B-50 (GAP-43). In this paper we present a new purification method for B-50 from rat brain which employs 2-mercaptoethanol to release the protein from isolated synaptosomal plasma membranes. Most likely, 2-mercaptoethanol reduces disulfide bonds involved in the linkage of B-50 to the membrane. After washing the membranes with 100 mM NaCl to detach loosely bound proteins, B-50 is the major protein (and the only protein kinase C substrate) released by 0.5% 2-mercaptoethanol treatment. Further purification to apparent homogeneity is achieved by affinity chromatography on calmodulin sepharose. B-50 binds to calmodulin in the absence of calcium and specifically elutes from the column with 3 mM calcium. The procedures described is simple, rapid and highly suitable for large scale purification of B-50 from rat brain.     

Polyanionic-binding properties of the receptor for 2,3,7,8-tetrachlorodibenzo-p-dioxin. A comparison with the glucocorticoid receptor

The interaction of the rat hepatic receptor for 2,3,7,8-tetrachlorodibenzo-p-dioxin (dioxin) with immobilized heparin (heparin-Sepharose) or DNA (DNA-cellulose) has been compared to the polyanionic-binding properties of the rat hepatic glucocorticoid receptor. Both the nonoccupied and in vitro occupied forms of the receptors interacted with heparin-Sepharose but with varying strength, as determined by ligand binding assays or an enzyme-linked immunosorbent assay based on a monoclonal antibody against the steroid- and DNA-binding Mr approximately 94,000 glucocorticoid receptor protein. In the absence of ligand, both the dioxin and glucocorticoid receptors eluted from heparin-Sepharose at 0.1-0.2 M KCl, in contrast to the in vitro occupied receptor forms which eluted at 0.3-0.4 M KCl. Following elution of the in vitro occupied dioxin receptor from heparin-Sepharose, it was efficiently retained on DNA-cellulose and eluted at an ionic strength of approximately 0.2 M KCl. In the presence of 20 mM sodium molybdate which is known to inhibit the activation of steroid hormone receptors to a DNA-binding form, both the dioxin and glucocorticoid receptors eluted at 0.1-0.2 M KCl from heparin-Sepharose. In analogy to what has previously been shown for the glucocorticoid receptor, sodium molybdate stabilized a large dioxin-receptor complex with a sedimentation coefficient, S20,w, of 9-10 S, a Stokes radius of approximately 7.5 nm, and a calculated Mr of 290,000-310,000. Limited proteolysis of both the dioxin and glucocorticoid receptors with trypsin which is known to eliminate the DNA-binding property of both receptor forms also resulted in a decreased strength in the interaction of both in vitro occupied receptors with heparin-Sepharose (elution at 0.1-0.2 M KCl). In line with these data, calf thymus DNA in solution competed for receptor binding to heparin-Sepharose. In conclusion, the chromatographic properties of the dioxin receptor on heparin-Sepharose are indistinguishable from those of the glucocorticoid receptor, and both receptors appear to be structurally and functionally closely related proteins.     

DNA-binding protein from HeLa cells that binds preferentially to supercoiled DNA damaged by ultraviolet light or N-acetoxy-N-acetyl-2-aminofluorene

A DNA-binding protein was partially purified from extracts of HeLa cells by high-speed centrifugation and chromatography on DEAE-cellulose, phosphocellulose and ultraviolet light-irradiated DNA-cellulose columns. It eluted from the phosphocellulose column with 0.375 M potassium phosphate and from the ultraviolet light-irradiated DNA-cellulose column between 0.5 M and 1 M NaCl. The protein binds preferentially to supercoiled PM2 DNA treated with ultraviolet light or N-acetoxy-N-acetyl-2-aminofluorene, as compared to native supercoiled PM2 DNA. The binding is non-cooperative. Nicked or linear forms of PM2 DNA (damaged or untreated) are not efficient substrates, indicating a requirement of DNA supercoiling for DNA binding. The sedimentation coefficient of the protein estimated by glycerol gradient centrifugation is 2.0–2.5 S, corresponding to a molecular weight of about 20 000–25 000 if the protein is spherical. The binding to DNA irradiated with ultraviolet light or treated with acetoxyacetylaminofluorene is optimal at around 100–200 mM NaCl and is relatively independent of temperature and pH. MgCl₂ and MnCl₂ at concentrations between 1 and 5 mM do not markedly affect the binding, but it is inhibited by sucrose, ATP and caffeine. The biological significance of the DNA-binding protein remains to be determined. It does not possess significant glycosylase, endonuclease or exonuclease activities. The dissociation equilibrium constant for the binding reaction of the protein to the ultraviolet light or acetoxyacetylaminofluorene-induced binding sites on DNA is estimated to be 4·10^?11 M. There are at least 1·10⁵ DNA-binding protein molecules/HeLa cell.     

Factors influencing recombinant human lysozyme extraction and cation exchange adsorption

Human lysozyme has numerous potential therapeutic applications to a broad spectrum of human diseases. This glycosidic enzyme is present in tears, saliva, nasal secretions, and milk--sources not amendable for commercial development. Recently, a high expression level of recombinant human lysozyme (0.5% dry weight) was achieved in transgenic rice seed. This paper evaluates the effects of pH and ionic strength on rice protein and lysozyme extractability, as well as their interactions with the strong cation-exchange resin, SP-Sepharose FF. The extraction conditions that maximized lysozyme yield and the ratio of extracted human lysozyme to native rice protein were not optimal for lysozyme adsorption. The conditions that gave the highest extracted lysozyme to native protein ratio were pH 4.5 and 100 mM NaCl in 50 mM sodium acetate buffer. At pH 4.5, salt concentrations above 100 mM NaCl reduced the lysozyme-to-protein ratio. The best conditions for lysozyme adsorption were pH 4.5 and 50 mM sodium acetate buffer. Lysozyme extraction and subsequent adsorption at pH 4.5 and 50 mM NaCl was an acceptable compromise between lysozyme extractability, adsorption, and purity. The primary recovery of human lysozyme from pH 6 extracts, irrespective of ionic strength, was inferior to that using pH 4.5 with unacceptably low saturation capacities and lysozyme purity. High purity was achieved with a single chromatography step by adjusting the pH 4.5 extract to pH 6 before adsorption. The disadvantage of this approach was the drastically lower saturation capacity compared to adsorption at pH 4.5.     

A DNA-binding fraction of mouse kidney 3-ketosteroid reductase: Comparison with androgen receptors

Since approximately 1% of 3-ketosteroid reductase (which metabolizes dihydrotestosterone [17β-hydroxy-5α-androstan-3-one] to 5α-androstane-3α,17β-diol or 5α-androstane-3α,17β-diol) from mouse kidney cytosol adheres to DNA under conditions that allow virtually complete androgen receptor binding, these two DNA-binding activities were compared in cytosol extracts of mouse kidney and hypothalamus-preoptic area. This DNA-binding fraction of 3-ketosteroid reductase was distinguished from androgen receptor in several ways: (1) its pattern of elution from DNA-cellulose with steps of increasing NaC1 concentration differed from that for receptors from wild-type kidney; (2) it was influenced differently by the mutation Tfm, both in level and in DNA-cellulose elution pattern; (3) in mouse kidney cytosol it was relatively stable at moderate (25°C) temperatures which rapidly inactivated ligand-free androgen receptors in the same cytosols; (4) the DNA-binding was not proportional to androgen receptor levels between two wild-type tissues, the hypothalamus-preoptic area and kidney. By these criteria, a simple relationship of androgen receptors and a DNA-binding fraction of 3-ketosteroid reductase activity is unlikely.     

Removal of degradation products from calf thymus high mobility group non-histone chromatin proteins by chromatography on immobilized double-stranded DNA

The substantial protease activity in calf thymus chromatin inevitably produces some degradation of high mobility group (HMG) non-histone proteins in NaCl extracts of calf thymus chromatin. We have found that proteins considered to be degradation products can be conveniently and cleanly separated from intact high mobility group proteins 1 and 2 by chromatography on double-stranded DNA-cellulose in 0.2 M NaCl/1 mM Tris-HCl (pH 7.5). Under those conditions, only the presumptive degradation products are retained by the column.     

Association of the 90-kDa heat shock protein does not affect the ligand-binding ability of androgen receptor

An N-terminal truncated androgen receptor with putative DNA- and ligand- binding domains (AR438) and that with a ligand-binding domain (AR612) were expressed under control of the T7 promoter in E. coli or translated in vitro with rabbit reticulocyte lysate, and their ligand-binding properties and the interaction with HSP90 were investigated. Bacterially expressed AR438 and AR612 bound a synthetic androgen, [³H]R1881, with apparent dissociation constant of 2.6 ± 0.2 and 3.1 ± 0.7 nM, respectively, values which are comparable to those of androgen receptor in target tissues. The recombinant androgen receptors sedimented at the 4–5 S region irrespective of the presence of 10 mM tungstate, indicating that the receptor exists free from HtpG, which is the bacterial homolog of eukaryotic HSP90. The apparent dissociation constant of truncated androgen receptors translated in vitro was 0.1 nM for AR438 and 0.2 nM for AR612. Sedimentation coefficients of in vitro translated molecules were converted from 7–8 S in the presence of tungstate to 3 S in the absence of tungstate. Both AR438 and AR612 translated in vitro were retained by anti-rat HSP90 antibody-protein A Sepharose. Exposure to 0.3 M NaCl in the presence of ligand caused dissociation of AR438 and AR612 from HSP90, and concomitantly, the DNA-cellulose binding ability of AR438 was enhanced. Thus, we conclude that the androgen receptor associates with HSP90 through the ligand-binding domain and that this association prevents the interaction of the androgen receptor with DNA. However, HSP90 seems to have little effect on the ligand-binding characteristics of the androgen receptor.     

In vitro activation of rat cardiac glucocorticoid antagonist- versus agonist-receptor complexes

The synthetic antiglucocorticoid RU 38486 interacts with cardiac cytoplasmic glucocorticoid receptors and competes for in vitro binding with the potent agonist triamcinolone acetonide. In addition to binding to receptors with high affinity, RU 38486 also facilitates the in vitro conformational change in the receptor which is a consequence of the physiologically relevant activation step during which the receptor is converted from a non DNA- to a DNA-binding form. This ability of RU 38486 to promote receptor activation is reflected by both the appropriate shift in the elution profile of [3H]RU 38486-receptor complexes from DEAE-cellulose as well as by an increased binding of these complexes to DNA-cellulose. Although less effective than triamcinolone acetonide, RU 38486 promotes in vitro receptor activation under a variety of experimental conditions, including incubation of labeled cardiac cytosols at 25 degrees C for 30 min or at 15 degrees C for 30 min in the presence of 5 mM pyridoxal 5'-phosphate. Once thermally activated, the cardiac [3H]triamcinolone acetonide and [3H]RU 38486-receptor complexes bind to nonspecific DNA-cellulose with the same relative affinities, as evidenced by the fact that 50% of both activated complexes are eluted at approx. 215-250 mM NaCl. Thus, this pure antiglucocorticoid does promote, at least to some extent, many of the crucial in vitro events including high-affinity binding, activation, and DNA binding which have been shown to be required to elicit a physiological response in vivo.     

Characterization of HU-like protein from Bifidobacterium longum

A HU-like protein (HBl) of Bifidobacterium longum was purified and characterized. HBl is heat-stable and acid-resistant, and has a molecular weight of about 9.1 kDa as estimated by its mobility on electrophoresis. HBl is intermediate in basicity (pI 9.8) between the HU-1 and HU-2 proteins of Escherichia coli, and is dissociated from a calf thymus DNA-cellulose column at 300-400 mM NaCl. Its amino acid composition shows many similarities with that of E coli HU. The NH2-terminal amino acid sequence of HBl also shows significant similarities to the consensus sequence deduced from the sequences of eleven HU-like proteins from prokaryotic sources. Chemical crosslinking analysis indicated that the HBl protein predominantly forms a homotypic dimer.     

The role of sulfhydryl groups in permitting transformation and DNA binding of the glucocorticoid receptor

Treatment of rat liver cytosol containing temperature-transformed, [3H]dexamethasone-bound receptors at 0 degree C with the sulfhydryl-modifying reagent methyl methanethiosulfonate (MMTS) inhibits the DNA-binding activity of the receptor, and DNA-binding activity is restored after addition of dithiothreitol (DTT). When cytosol containing untransformed receptors is heated at 25 degrees C in the presence of MMTS, the 90-kDa heat shock protein dissociates from the receptor in the same manner as in the absence of MMTS, and the receptor will bind to DNA-cellulose if DTT is added subsequently at 0 degree C. These observations are consistent with the conclusion of Bodwell et al. (Bodwell, J. E., Holbrook. N. J. and Munck, A. (1984) Biochemistry 23, 1392-1398) that sulfhydryl moieties on the receptor are absolutely required for the receptor to bind to DNA, and they show that the sulfhydryl-modifying reagent does not inhibit the temperature-mediated dissociation of the heteromeric receptor complex that accompanies transformation to the DNA-binding state. When steroid-receptor complexes that are prebound to DNA-cellulose are exposed to MMTS, the steroid rapidly dissociates, but the receptor remains bound to DNA. Thus, the presence of steroid is not required for the receptor to remain bound to DNA in a high affinity manner. Treatment of cytosol containing transformed glucocorticoid-receptor complexes at 0 degrees C with 20 mM hydrogen peroxide also inactivates the DNA-binding activity of the receptor. The peroxide-induced inactivation is reversed by DTT. Incubation of rat liver cytosol containing untransformed glucocorticoid-receptor complexes at 25 degrees C with hydrogen peroxide prevents their transformation to the DNA-binding form as shown by their inability to bind to DNA-cellulose after addition of DTT. The presence of peroxide during heating of the cytosol also prevents dissociation of the receptor complex as assayed both by reduction in sedimentation value of the receptor and by dissociation of the 90-kDa heat shock protein from the steroid-binding protein. These results strongly suggest that critical sulfur moieties in the receptor complex must be in a reduced form for the temperature-mediated dissociation of the receptor to occur.     

Ionic effects on the structure of nucleoprotein cores from adenovirus

Nucleoprotein cores, prepared from adenovirus type 5 with a deoxycholate/heat treatment, consist of the viral DNA and two major internal proteins. The core particles exhibit structural characteristics that are highly reproducible and dependent on their ionic environment. In low-ionic-strength buffer, the cores had a sedimentation coefficient of 180 S and appeared in the electron microscope as homogeneous particles with distinct centers from which numerous arms and loops radiated. Condensation of the cores was induced by Mg2+ or Ca2+ over the range 0 to 1 mM. The sedimentation coefficient increased monotonically with divalent cation concentration, reaching a maximum of 405 S in 1 mM Mg2+. A corresponding condensation in the core structure was observed by electron microscopy. Increasing concentrations of NaCl also produced a conformational change in the cores, with an almost linear increase in sedimentation velocity up to 274 S in 0.04 M NaCl. Between 0.05 and 1.0 M NaCl, the cores were insoluble. In 2.0 M NaCl, the cores were again soluble with an s20,w of 228 S. Under all ionic strength conditions in which the cores were soluble, both core proteins remained bound to the DNA.     

Isolation of endonuclease from mammalian cells by DNA - cellulose chromatography.

By means of DNA-cellulose chromatography an enzyme with endonucleolytic activity has been isolated from nuclear acidic protein fraction of mammalian cells. The main active fraction, eluted at 0.7 M NaCl, effects the velocity sedimentation of UV-irradiated and alkylated DNA, resulting in a decrease of the molecular weight. The fraction is completely inactive using native as well as heat-denatured DNA.