Physical-chemical properties of the estrogen receptor released by deoxyribonuclease I

The physical-chemical properties of the nuclear estrogen receptor released by DNase I were characterized. Nuclei were isolated from MCF-7 cells previously exposed to 10-nM-[3H]estradiol. The parameters determined were: sedimentation coefficients (S) on a sucrose gradient, Stokes radii (Rs) by gel filtration on a Sephadex G-200 column and the binding ability to a DNA-cellulose column. The molecular weights (Mr) and frictional ratios (f/fo) were calculated from the S and Rs values. The properties of the receptor released by DNase I obtained from Worthington were compared to the properties of the receptor released by DNase I obtained from Sigma. Digestion with DNase I (Worthington) excised a receptor form which could be solubilized from nuclei by EDTA. This form sedimented at 5.2S with a Rs = 7.08 nm and a calculated Mr = 152.000. About 40% of this receptor form bound to a DNA-cellulose column. 0.4 M KCl dissociated this receptor form into a smaller form sedimenting at 4.2S with Rs = 4.64 nm and a calculated Mr = 80.000. The properties of the receptor solubilized by micrococcal nuclease followed by DNase I (Worthington) digestion were identical to the properties of the DNase I (Worthington) released receptor. Digestion with DNase I (Sigma) released a 3.2S receptor form, which diffused through the nuclear membrane and a 4-5S form which could be extracted from nuclei by EDTA. The 3.2S receptor had a Rs = 2.41 nm, a calculated Mr = 32.000 and less than 5% of it bound to a DNA-cellulose column. Digestion with micrococcal nuclease followed by DNase I (Sigma) solubilized a receptor form with identical properties to the 3.2S receptor. These results suggest that DNase I (Worthington) released a receptor form still associated with some molecules, probably chromatin proteins, which complexed it to DNA, while DNase I (Sigma) released the estradiol binding fragment of the receptor (meroreceptor) as a result of a proteolytic activity present in this preparation.     

Effect of ribonuclease on the physico-chemical properties of estrogen receptor

Estrogen receptors (ER) from rat and rabbit uterine cytosol were examined for their sensitivity to ribonuclease (RNase). After RNase treatment, a major part of rabbit uterine ER was converted from the 7S to 3-4S form, and its binding to DNA-cellulose was increased by 40%. Similar treatment on rat uterine ER showed a shift from 7S to 4.5S, and the DNA-cellulose binding was stimulated by 20%. Measurement of endogenous RNase levels showed that lower RNase concentration in rabbit uterine cytosol coincided with larger stimulation of DNA-cellulose binding by exogenous RNase. These results indicate that a major part of 7S ER is susceptible to RNase, and cleavage of bound RNA seems to uncover additional binding sites for DNA. In contrast to the general thinking that 4S to 5S transformation is essential for nuclear binding, we have observed that RNase-treated rat uterine ER did not undergo such a transformation by warming at 25 degrees C, while DNA-cellulose binding of the receptors increased. Thus, temperature activation could occur independent of 4S to 5S transformation.     

Ribonuclease-induced transformation of progesterone receptor from rabbit uterus

The effect of RNase on the transformation of progesterone receptor from rabbit uterus was studied by density-gradient centrifugation and DNA-cellulose binding assay. The 7S form of the receptor in crude cytosol was RNase sensitive, and converted to the 4S form after RNase treatment. This reaction was prevented by an RNase inhibitor and reversed by the addition of ribosomal RNA. RNase treatment also caused a two-fold increase in the DNA binding of cytosolic receptor, and reduced the time required for heat-induced transformation. However, sucrose-gradient-purified progesterone receptor (7S) did not undergo transformation by warming unless exogenous RNase was added, thereby suggesting that a cytosolic factor, which might be endogenous RNase, is necessary for the heat-induced transformation of progesterone receptor. Furthermore, degradation of the receptors which occurred after prolonged warming at 25 degrees C in the presence of RNase could be prevented by the addition of DNA-cellulose to the reaction mixture. These results indicate that RNA is associated with the 7S form of progesterone receptor, and that its hydrolysis by RNase might be involved in the transformation of this receptor.     

Ribonucleic acid association with androgen receptor from rat submandibular gland

The transformed androgen receptor from rat submandibular gland converts to a faster sedimenting form (6-8S) on a glycerol gradient centrifugation after withdrawal of a transformation-inducing reagent (KCl or ATP). In this report, the association of cytosolic RNA with the transformed androgen receptor was investigated as a possible mechanism of molecular conversion of the androgen receptor. When the transformed and converted androgen receptors were treated with RNase A, these receptors sedimented at 4.5S in a low-salt glycerol gradient. Addition of RNA from rat submandibular gland to the RNase-Sepharose-treated transformed receptor caused a shift of receptor peak from 4.5S to 5.8S. RNA from rat submandibular gland, yeast RNA and E. coli rRNA inhibited DNA-cellulose binding of a RNase-treated transformed receptor in the absence of molybdate. These observations suggest that conversion from the transformed 4S androgen receptor to a 6-8S form resulted from the association of RNA(s) with the transformed receptor.     

Sodium molybdate converts the RNA-associated transformed, oligomeric form of the glucocorticoid receptor into the transformed, monomeric form

The glucocorticoid receptor from rat liver cytosol prepared in 2 ml buffer/g tissue sedimented at approximately 10 S in low salt density gradient centrifugation without molybdate. When the receptor was heated at 25 degrees C, both approximately 10 S and approximately 7 S forms were seen in low salt gradient. The approximately 10 S form was not capable of binding to DNA-cellulose and was stabilized by sodium molybdate, namely it corresponded to untransformed receptor. The approximately 7 S form was capable of binding to DNA-cellulose and regarded as transformed receptor. On the other hand, partially-purified transformed receptor labeled with [3H]dexamethasone-21-mesylate sedimented at approximately 5 S, which migrated as a approximately 94 kDa species in SDS-polyacrylamide gel electrophoresis. The reconstitution analysis of this partially-purified approximately 5 S receptor and liver cytosol, showed the shift to approximately 7 S form. RNase A or T1 converted approximately 7 S transformed form into approximately 5 S but it did not affect approximately 10 S untransformed form. 5-20 mM sodium molybdate also shifted approximately 7 S to approximately 5 S. These results indicate that the approximately 7 S transformed form of the glucocorticoid receptor observed in low salt conditions might be an oligomer, probably including both approximately 5 S steroid-binding component and RNA/ribonucleoprotein, and that molybdate dissociates these interactions in a specific manner.     

Physical-chemical properties of the estrogen receptor solubilized by micrococcal nuclease

The physical-chemical properties of the nuclear estrogen receptor from MCF-7 cells were determined. The receptor was solubilized by micrococcal nuclease. Nuclei were isolated from cells previously exposed to 10 nM [3H]estradiol. The amount of receptor released was parallel to the extent of chromatin solubilized, which suggested that the receptor is homogeneously distributed on the chromatin. Following mild nuclease digestion the excised receptor sedimented as an abundant 6-7 S form and as a less abundant approximately 12 S species. The 6-7 S form represented the receptor excised in association with linker DNA, while the approximately 12 S may represent receptor bound to linker DNA which remained associated with the nucleosome. Increasing the extensiveness of digestion resulted in one receptor form sedimenting at 5.6 S. Additional digestion with DNase I did not affect the sedimentation coefficient of the receptor. Sedimentation of the micrococcal nuclease hydrolysate in a 0.4 M KCl sucrose gradient resulted in a 4.2 S receptor form. The same receptor form was extracted from undigested nuclei with 0.4 M KCl. Using Sephadex G-200 column chromatography we have determined the Stokes radii (Rs), molecular weight (Mr) and frictional ratio (f/fo) for the 5.6 S and 4.2 S receptor forms. For the 5.6 S form: Rs = 7.04 nm, Mr = 163,000 and (f/fo) = 1.80. For the 4.2 S receptor, Rs = 4.45 nm, Mr = 77,000 and (f/fo) = 1.46. The ability of the nuclease solubilized 5.6 S receptor to bind DNA was tested using DNA-cellulose column and highly polymerized DNA. About 40% of the applied receptor bound to the column and could be eluted by high salt concentrated buffer. The 5.6 S receptor form was sedimented on sucrose gradient by the highly polymerized DNA. These results suggested that the receptor is bound in chromatin as a dimer or as a monomer in association with other protein(s) which complexed it with DNA.     

Characterization of the progesterone receptor solubilized by micrococcal nuclease and DNase I digestion

In order to investigate the functional organization of the progesterone receptor in chromatin we characterized the physical-chemical properties of the receptor bound chromatin fragments released by micrococcal nuclease and DNase I digestion. The crude nuclear fraction was isolated from T 47 D cells, previously exposed to 0.1 microM [3H]ORG 2058. The parameters determined in low and high salt concentrated buffers were: sedimentation coefficients (S) on a sucrose gradient, Stokes radii (Rs) by gel filtration on a Sephadex G-200 column and the binding abilities to a DNA-cellulose column. The molecular weights (Mr) and frictional ratios (f/fo) were calculated from the S and Rs values. Micrococcal nuclease digestion solubilized a receptor form sedimenting as a single peak at 4.4 S with a Rs = 7.78 nm and an estimated Mr = 144,000. About 53% of the applied receptor bound to a DNA-cellulose column could be eluted by high salt concentrated buffer. 0.4 M KCl dissociated this receptor form into a smaller receptor sedimenting at 3.3 S with Rs = 5.53 nm and a calculated Mr = 76,000. A similar receptor form was extracted by 0.6 M KCl from the undigested crude nuclear fraction. DNase I digestion solubilized a receptor form sedimenting at 3.3 S with a Rs = 6.87 nm and a calculated Mr = 94,000. About 26% of the applied receptor bound to a DNA-cellulose column could be eluted by high salt concentrated buffer. Dissociation of this receptor form by 0.4 M KCl resulted in a receptor sedimenting at 2.8 S with a Rs = 6.53 nm and an estimated Mr = 76,000. These results suggest: The progesterone receptor in chromatin is associated with several molecules probably proteins which complexed it to DNA. Some of these molecules still associated with the progesterone receptor could be released by nucleases digestion. Micrococcal nuclease releases a larger portion of these molecules than those release by DNase I.     

Effects of bovine pancreatic ribonuclease A, S protein, and S peptide on activation of purified rat hepatic glucocorticoid-receptor complexes

Bovine pancreatic ribonuclease (RNase) A and S protein (enzymatically inactive proteolytic fragment of RNase A which contains RNA binding site) stimulate the activation, as evidenced by increasing DNA-cellulose binding, of highly purified rat hepatic glucocorticoid-receptor complexes. These effects are dose dependent with maximal stimulation of DNA-cellulose binding being detected at approximately 500 micrograms (50 units of RNase A/mL). RNase A and S protein do not enhance DNA-cellulose binding via their ability to interact directly with DNA or to increase nonspecific binding of receptors to cellulose. Neither S peptide (enzymatically inactive proteolytic fragment which lacks RNA binding site) nor cytochrome c, a nonspecific basic DNA binding protein, mimics these effects. RNase A and S protein do not stimulate the conformational change which is associated with activation and is reflected in a shift in the elution profile of receptor complexes from DEAE-cellulose. In contrast, these two proteins interact with previously heat-activated receptor complexes to further enhance their DNA-cellulose binding capacity and thus mimic the effects of an endogenous heat-stable cytoplasmic protein(s) which also function(s) during step 2 of in vitro activation [Schmidt, T. J., Miller-Diener, A., Webb, M. L., & Litwack, G. (1985) J. Biol. Chem. 260, 16255-16262]. Preadsorption of RNase A and S protein to an RNase affinity resin containing an inhibitory RNA analogue, or trypsin digestion of the RNA binding site within S protein, eliminates the subsequent ability of these two proteins to stimulate DNA-cellulose binding of the purified receptors.(ABSTRACT TRUNCATED AT 250 WORDS)     

Activation of the human glucocorticoid receptor: evidence for a two-step model

The relationship between glucocorticoid receptor subunit dissociation and activation was investigated by DEAE-cellulose and DNA-cellulose chromatography of monomeric and multimeric [3H]triamcinolone acetonide ([3H]TA)-labeled IM-9 cell glucocorticoid receptors. Multimeric (7-8 nm) and monomeric (5-6 nm) complexes were isolated by Sephacryl S-300 chromatography. Multimeric complexes did not bind to DNA-cellulose and eluted from DEAE-cellulose at a salt concentration (0.2 M KCl) characteristic of unactivated steroid-receptor complexes. Monomeric [3H]TA-receptor complexes eluted from DEAE-cellulose at a salt concentration (20 mM KCl) characteristic of activated steroid-receptor complexes. However, only half of these complexes bound to DNA-cellulose. This proportion could not be increased by heat treatment, addition of bovine serum albumin, or incubation with RNase A. Incubation of monomeric complexes with heat inactivated cytosol resulted in a 2-fold increase in DNA-cellulose binding. Unlike receptor dissociation, this increase was not inhibited by the presence of sodium molybdate. Fractionation of heat inactivated cytosol by Sephadex G-25 chromatography demonstrated that the activity responsible for the increased DNA binding of monomeric [3H]TA-receptor complexes was macromolecular. These results are consistent with a two-step model for glucocorticoid receptor activation, in which subunit dissociation is a necessary but insufficient condition for complete activation. They also indicate that conversion of the steroid-receptor complex to the low-salt eluting form is a reflection of receptor dissociation but not necessarily acquisition of DNA-binding activity.     

Purification and preliminary characterization of a macromolecular inhibitor of glucocorticoid receptor binding to DNA

Rat liver cytosol contains a heat-labile macromolecule that inhibits the binding of the transformed glucocorticoid-receptor complex to nuclei or DNA-cellulose (Milgrom, E., and Atger, M. (1975) J. Steroid Biochem. 6, 487-492; Simons, S. S., Jr., Martinez, H. M., Garcea, R. L., Baxter, J. D., and Tomkins, G. M. (1976) J. Biol. Chem. 251, 334-343. We have developed a quantitative assay for the inhibitor and have purified it 600-700-fold by ammonium sulfate precipitation, ethanol precipitation, and phosphocellulose and Sephacryl S-300 chromatography. The inhibitory activity copurifies with a Mr = 37,000 protein doublet. Under low salt conditions, both the inhibitory activity and the 37-kDa protein doublet behave as high Mr aggregates that subsequently dissociate in the presence of salt. The inhibitor is positively charged at physiological pH, and it is not affected by digestion with several serine proteases or RNase. The inhibitor does not affect the transformation process, and it does not cause the release of steroid-receptor complexes that have been prebound to DNA-cellulose. The inhibitor preparation does not cleave receptors in L-cell cytosol that are covalently labeled with the site-specific affinity steroid [3H]dexamethasone 21-mesylate. If the steroid-receptor complex is first separated from the great majority of cytosol protein by transforming it and binding it to DNA-cellulose, addition of the inhibitor preparation results in receptor cleavage. Under these conditions, cleavage can be blocked with 1-chloro-3-tosylamido-7-amino-L-2-heptanone and antipain, but protease inhibitors do not affect the inhibition of DNA binding that occurs in whole cytosol. The inhibitor acts through an interaction with the receptor, not with DNA. We suggest that the inhibitor may prove to be a useful tool for studying the interaction of the steroid-receptor complex with DNA or nuclei and speculate that it may be important in determining normal events of the receptor cycle as they occur in the intact cell.     

Single-strand-specific nuclease from rat liver endoplasmic reticulum: characterization and mode of action

1. The characteristics and mode of action of a single-strand-specific nuclease isolated from rat liver endoplasmic reticulum are investigated with respect to its DNA and RNA substrates. 2. The RNase activity of the enzyme is slightly influenced by the presence of divalent cations but the DNase activity is enhanced by divalent cations particularly Mn2+. 3. Activity is partially inhibited by the presence of EGTA; this effect is reversed most efficiently by the addition of Mn2+. 4. The enzyme exhibits small pH dependence between pH 6-9 and maximum activity is observed at pH 7-7.5 for both DNase and RNase activities. 5. Sulfhydryl group reagents do not affect its action but histidyl group reagents exert a small but definite effect. 6. The enzyme degrades DNA and RNA endonucleolytically producing fragments which possess 3'-OH and 5'-phosphate termini. 7. Monomers are not produced even after prolonged degradation. 8. The end product of poly(U)degradation ranges between two and four building blocks but the DNA product is longer probably due to considerable percentage of secondary structure.     

Salt-induced activation of 1,25-dihydroxyvitamin D3 receptors to a DNA binding form

In this report we examine the DNA-cellulose binding and sedimentation properties of 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) receptors from rat intestine and cultured human mammary cancer cells (MCF-7) extracted in nonactivating (low salt) buffers. Receptors prepared in hypotonic buffer had low DNA binding (13%) compared to receptors extracted with 0.3 M KCl (50%). Treatment of low salt receptor preparations with KCl significantly increased (approximately 3-fold) DNA-binding (activation), demonstrating that receptors can be "activated" in vitro. Activated receptors eluted from DNA-cellulose at 0.18 M KCl. Sedimentation analysis followed by DNA-cellulose binding indicated that activated receptors are approximately 3.2 S and unactivated receptors 5.5 S in size. These results suggest that dissociation of an aggregated moiety may lead to receptor activation. Treatment of unactivated receptor with RNase did not alter DNA binding or sedimentation properties of the aggregated receptor. Treatment of unactivated receptor complexes with heat did not increase DNA binding, and molybdate did not block subsequent salt activation. In summary these results suggest that 1,25(OH)2D3 receptors undergo a salt-induced activation step similar to that described for other steroid receptor systems. However, 1,25(OH)2D3 receptors differ from other steroid receptors in not exhibiting heat activation nor having salt activation blocked by molybdate.     

Isolating and sequencing the infrequent 3'-ends of a specific mRNA

Procedures are described for identification of very infrequent in vivo 3'-ends of RNA. After purification by filter hybridization, the 3'-ends were labeled with [5'-32P] cytosine-3'-P in the RNA ligase reaction. Significantly fewer counts were incorporated in the ligase reaction than in the polynucleotide kinase reaction to label 5'-ends. The incorporation was increased by increasing the RNA concentration 5-10 fold by using only one round of filter hybridization. Non-specific RNA binding could be eliminated by RNase A treatment of the filter if a great excess of denatured heterologous DNA was immobilized along with the DNA probe. Significant amounts of DNA were released when eluting the hybrid RNA from such filters. DNA inhibited the ligase reaction, while its DNase products were even more inhibitory. Treatment of the DNase products with alkaline phosphatase completely eliminated the inhibition. We detected no spurious 5'- or 3'-ends generated in the hybrid RNA by RNase A activity used to reduce the non-specific RNA. Also, RNase T1 could be used in place of RNase A to eliminate non-specific RNA binding, but about 25 times more RNase T1 (microgram/microgram) was needed. We used partial alkali digestion to sequence 3'-ends. A major (one hit) and minor (two hit) set of products were produced which could be distinguished from each other by alkaline phosphatase treatment and homochromatography of the products.     

Temperature-dependent inactivation of nucleic acid binding and aggregation of the 1,25-dihydroxyvitamin D3 receptor

The interaction of the 1α,25-dihydroxyvitamin D₃ receptor with immobilized calf thymus DNA has been compared with its sedimentation properties on hypotonic sucrose gradients. Forty to sixty percent of total hormone:receptor complexes formed at 4 °C were retained by DNA-cellulose and could be eluted by 0.18 to 0.2 m KCl. In contrast, heating preparations to 25 °C rapidly and irreversibly converted receptor to a form which bound hormone and DEAE-cellulose normally, but was unable to associate with DNA. Similarly, the ability of receptor to aggregate to a 6 S species was labile at 25 °C. Stabilization of receptor in the DNA binding aggregating form was accomplished using Ca²⁺, Mg²⁺, Mn²⁺, or Na₂MoO₄ while several protease and phosphatase inhibitors were ineffective. An examination of DNA binding properties of aggregating and nonaggregating receptor forms revealed that only receptor competent to enter into aggregates could bind DNA suggesting that a functional nucleic acid binding site, and, hence, a nucleic acid interaction is necessary for aggregate formation. Consistent with this view, an RNA:receptor interaction appears to be involved in formation of the 6 S complex since removal of RNA by ribonuclease treatment or purification of receptor reduced aggregation, an effect that could be reversed by addition of purified RNA.     

Structural and biochemical characterization of CRN-5 and Rrp46: An exosome component participating in apoptotic DNA degradation

Rrp46 was first identified as a protein component of the eukaryotic exosome, a protein complex involved in 3′ processing of RNA during RNA turnover and surveillance. The Rrp46 homolog, CRN-5, was subsequently characterized as a cell death-related nuclease, participating in DNA fragmentation during apoptosis in Caenorhabditis elegans. Here we report the crystal structures of CRN-5 and rice Rrp46 (oRrp46) at a resolution of 3.9 Å and 2.0 Å, respectively. We found that recombinant human Rrp46 (hRrp46), oRrp46, and CRN-5 are homodimers, and that endogenous hRrp46 and oRrp46 also form homodimers in a cellular environment, in addition to their association with a protein complex. Dimeric oRrp46 had both phosphorolytic RNase and hydrolytic DNase activities, whereas hRrp46 and CRN-5 bound to DNA without detectable nuclease activity. Site-directed mutagenesis in oRrp46 abolished either its DNase (E160Q) or RNase (K75E/Q76E) activities, confirming the critical importance of these residues in catalysis or substrate binding. Moreover, CRN-5 directly interacted with the apoptotic nuclease CRN-4 and enhanced the DNase activity of CRN-4, suggesting that CRN-5 cooperates with CRN-4 in apoptotic DNA degradation. Taken together all these results strongly suggest that Rrp46 forms a homodimer separately from exosome complexes and, depending on species, is either a structural or catalytic component of the machinery that cleaves DNA during apoptosis.     

Characterization of glucocorticoid receptor in HeLa-S3 cells

Glucocorticoid receptor of the human cell line HeLa-S3 has been characterized and has been compared to rat and to mouse glucocorticoid receptors. If HeLa cells were lysed in the absence of glucocorticoid, glucocorticoid receptor was isolated in a nonactivated form, which did not bind to DNA-cellulose. If HeLa cells were preincubated with glucocorticoid, glucocorticoid receptor was isolated in an activated, DNA-binding form. HeLa cell glucocorticoid receptor bound [3H]triamcinolone acetonide with a dissociation constant (KD = 1.3 nM at 0 degrees C) that was similar to those of mouse and rat glucocorticoid receptors. Similarly, the relative binding affinities for steroid hormones decreased in the order of triamcinolone acetonide greater than dexamethasone greater than promegestone greater than methyltrienolone greater than aldosterone greater than or equal to moxestrol. Nonactivated and activated receptors were characterized by high-resolution anion-exchange chromatography (FPLC), DNA-cellulose chromatography, and sucrose gradient centrifugation. Human, mouse, and rat nonactivated glucocorticoid receptors had very similar ionic and sedimentation properties. Activated glucocorticoid receptors were eluted at similar salt concentrations from DNA-cellulose columns but at different salt concentrations from the FPLC column. A monoclonal mouse anti-rat liver glucocorticoid receptor antibody [Westphal, H.M., Mugele, K., Beato, M., & Gehring, U. (1984) EMBO J. 3, 1493-1498] did not cross-react with HeLa cell glucocorticoid receptor. Glucocorticoid receptors of HeLa, HTC, and S49.1 cells were affinity labeled with [3H]dexamethasone and with [3H]dexamethasone 21-mesylate. The molecular weights of [3H]dexamethasone 21-mesylate labeled glucocorticoid receptors (MT 96 000 +/- 1000) were undistinguishable by polyacrylamide gel electrophoresis.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization and quantification of the androgen and glucocorticoid receptors in cytosol from rat skeletal muscle

The binding of the radioactive synthetic hormonal steroids [3H]dexamethasone (9 alpha-fluoro-11 beta, 17 alpha, 21-trihydroxy-16 alpha-methyl-1,4-pregnadiene-3,20-dione) and [3H]methyltrienolone (17 beta-hydroxy-17 alpha-methyl-4,9,11-estratien-3-one) to cytosol from rat skeletal muscle was studied using dextran-coated charcoal to separate unbound and receptor-bound steroid. The rates of association, dissociation, and degradation of the complexes of dexamethasone and methyltrienolone with receptor were highly dependent on temperature. The temperature dependence of association was greater for dexamethasone, and that of degradation was greater for methyltrienolone. Dissociation rates were insignificant for both steroid-receptor complexes compared to association and degradation rates. The apparent equilibrium dissociation constants for the binding of dexamethasone and methyltrienolone to their receptor binding sites were about 7 and 0.3 nM, respectively, regardless of temperature (0. 15 or 23 degrees C). The lack of influence of temperature on the equilibrium constants indicate that the binding was of hydrophobic character, and the corresponding free energy changes upon binding of dexamethasone and methyltrienolone to their respective binding sites were -41 and -49 kJ mol-1 under equilibrium conditions at 0 degrees C. The apparent maximum number of binding sites determined from Scatchard plots under these conditions was about 1900 fmol/g of tissue, 3500 fmol/mg of DNA or 30 fmol/mg of protein in the case of the dexamethasone receptor, and the corresponding figures for the methyltrienolone were about 100 fmol/g of tissue, 200 fmol/mg of DNA or 2 fmol/mg of protein. The ligand specificities of the binding sites for dexamethasone and methyltrienolone were typical of a glucocorticoid and an androgen receptor, respectively. Both steroid-receptor complexes were retained on DNA-cellulose columns, and were eluted by NaCl at an ionic strength of 0.1. The DNA-cellulose step purified about 20 times, and was used to allow gel exclusion chromatography and electrofocusing. Both steroid-receptor complexes were excluded from a column of Sephadex G-150. Electrofocusing in preparative columns gave reproducible patterns consisting of three peaks for each receptor. The apparent isoelectric points were 5.4, 5.6 and 6.2 for the glucocorticoid receptor, and 5.9, 6.2 and 8.5 for the androgen receptor.     

Mechanism of action of Moloney murine leukemia virus RNA-directed DNA polymerase associated RNase H (RNase H I)

The mechanism of action of the ribonuclease H (RNase H) activity associated with Moloney murine leukemia virus RNA-directed DNA polymerase (RNase H I) and the two-subunit (alpha beta) form of avian myeloblastosis virus DNA polymerase were compared by utilizing the model substrate (A)n.(dT)n and polyacrylamide gel electrophoresis in 7 M urea to analyze digestion products. Examination on 25% polyacrylamide gels revealed that a larger proportion of the RNase H I oligonucleotide products generated by limited digestion of [3H](A)(1100).(dT)n were acid insoluble (15-26 nucleotides long) than acid soluble (less than 15 nucleotides long), while the opposite was true for products generated by alpha beta RNase H. RNase H I was capable of attacking RNA in RNA.DNA in the 5' to 3' and 3' to 5' directions, as demonstrated by the use of [3H,3'- or 5'-32P](A)(380).(dT)n and cellulose--[3H](A)n.(dT)n. Both RNase H I and alpha beta RNase H degraded [3H]-(A)n.(dT)n with a partially processive mechanism, based upon classical substrate competition experiments and analyses of the kinetics of degradation of [3H,3'- or 5'-32P](A)(380).(dT)n. That is, both enzymes remain bound to a RNA.DNA substrate through a finite number of hydrolytic events but dissociate before the RNA is completely degraded. Both RNase H I and alpha beta RNase H were capable of degrading [14C](A)n in [3H](C)n-[14C](A)n-[32P](dA)n.(dT)n, suggesting that retroviral RNase H is capable of removing the tRNA primer at the 5' terminus of minus strand DNA at the appropriate time during retroviral DNA synthesis in vitro.