Evidence that 5 S intermediate state in glucocorticoid receptor transformation contains hsp90 in addition to the steroid-binding protein

Previous studies have shown that the exposure of molybdate-stabilized nontransformed glucocorticoid receptor (GR) of the chick embryonic neural retina to 0.4 M KCl dissociated the 9.5 S complex to a 5 S GR complex, which is an intermediate state in GR transformation. The present study was designed to characterize the 5 S GR complex. It shows that molybdate-stabilized nontransformed 9.5 S GR complex and 5 S GR interact with monoclonal antibodies (MAb) directed against 90 kDa heat shock protein (hsp90), as evidenced by the increase in the sedimentation velocity of these GR-complexes. Electrofocusing of the partially purified molybdate-stabilized nontransformed GR, prepared from [32P]-labeled neural retinas, and of the 5 S GR (derived from molybdate-stabilized preparation) showed that nontransformed GR complex, which has an apparent pI (pI') value of 5.0 +/- 0.2, and 5 S GR, which was resolved in a major peak with a pI' value of 5.8, are phosphorylated. Partially purified 5 S GR, cleared of molybdate and exposed to 25 degrees C, was resolved by electrofocusing into two phosphorylated fractions, one with a pI' value of 6.5, representing the monomeric GR form and the other with a pI' value of 5.1, apparently representing the acidic hsp90. The dissociation of hsp90 from the molybdate-cleared 5 S heterodimer seems to account for the decrease in the negative net charge of 5 S GR from pI' 6.5. Monomeric GR, derived from a molybdate-cleared, partially purified GR preparation, by the exposure to 25 degrees C, did not retain glucocorticoid-binding activity. Molybdate-stabilized 5 S GR was apparently re-assembled into the oligomeric nontransformed state when the salt concentration was reduced. This phenomenon was evident under the low-salt conditions of electrofocusing, by the shift in pI' value of GR from 5.8 to 5.0; and in glycerol density gradients containing 0.15 M KCl, by the shift in the sedimentation of the GR complex from 5 S to 9.5 S.     

Subunit composition of the molybdate-stabilized non-activated glucocorticoid receptor from rat liver

A monoclonal IgG 2a antibody directed against the activated rat liver glucocorticoid receptor (GR) was used to prepare an immunoaffinity matrix of high capacity. The molybdate-stabilized GR from rat liver cytosol was immunoadsorbed on this gel. A non-hormone-binding protein of Mr approximately 90,000, as determined after denaturing gel electrophoresis, was eluted from this matrix following removal of molybdate and exposure to heat (25 degrees C) and salt (0.15 M NaCl). Subsequently, the Mr approximately 90,000 protein was purified to homogeneity using high-performance ion-exchange chromatography, covalently radiolabelled, and analyzed by high-performance size-exclusion chromatography and sucrose gradient ultracentrifugation. Hydrodynamic characterization indicates that, under our experimental conditions, the molybdate-stabilized rat liver GR (Rs approximately 7.4 nm, s20,w approximately 9.1 S, calculated mol. wt Mr approximately 285,000) includes one steroid-binding unit (Rs approximately 5.5 nm, S20,w approximately 4.3 S, calculated Mr approximately 100,000) and a dimer of Mr approximately 90,000 non-hormone-binding protein (Rs approximately 6.9 nm, S20,w approximately 6.1 S, calculated native Mr approximately 180,000).     

Characterization of a hormone receptor defect in the androgen-insensitivity mutant

Mouse kidney cytosol contains specific receptors that reversibly bind dihydrotestosterone at a concentration of 43 f moles/mg protein. [Nonstandard abbreviation: DHT, dihydrotestosterone, 17 β-hydroxy-5 α-androstan-3-one.] The equilibrium dissociation constant of the receptor-dihydrotestosterone complex is 1.3 × 10^?9M for females and 1.7 × 10^?9M for castrated males. The complex sediments at 8–9S in glycerol gradients. In males bearing the androgen-insensitivity mutation (analogous to human testicular feminization), the specific dihydrotestosterone receptor activity is decreased about 8 fold. The residual binding activity has wild type affinity (K_D = 1.5 × 10^?9M) for dihydrotestosterone and also sediments at 8–9S. Kidney cytosol from castrated mutant mice displays a new binding component with low affinity and high capacity for dihydrotestosterone.     

Further theoretical refinement on the internal thermodynamics of perfect enzymes.

By solving simultaneously the equation for ''uniform binding'' [Albery & Knowles (1976) Biochemistry 15, 5631-5640] and the equation for ''differential binding'' [Chin (1983) J. Am. Chem. Soc. 105, 6502-6503], I derived the following simple equation for perfect enzymes (with single substrate and single product) under irreversible conditions: K2 = beta(1 + Rs)/1-beta(1 + Rs) where K2 is the internal equilibrium constant and beta is the Brönsted coefficient of the elementary catalytic step, and Rs is defined as [S]0/Ks, with [S]0 being the physiological substrate concentration and Ks being the substrate dissociation constant. The equation suggests that the perfect enzyme can have different internal thermodynamic properties depending on physiological conditions.     

Low temperature solution behaviour of Methylophilus methylotrophus electron transferring flavoprotein: a study by analytical ultracentrifugation

The solution behaviour of electron transferring flavoprotein (ETF) from Methylophilus methylotrophus was investigated at low temperature (4 °C) by analytical ultracentrifugation. The concentration dependence of the apparent weight average molecular weight, M_w,app, established the existence of the protein in heterodimeric state (M = 63,700 Da), but also signified the possible dissociation of the heterodimer at lower concentrations into its constituent subunits (M = 28,900 Da and 33,700 Da, together with FAD and AMP cofactors of collective M = 1120 Da). This similarity in subunit size allows approximate quantification of the dissociation in terms of expressions for a monomer-dimer equilibrium. The dissociative behaviour was confirmed by determination of the point average molecular weight, M_w,app(r), as a function of the ETF concentration, c(r), throughout the sedimentation equilibrium distributions obtained with loading concentrations of 0.4 and 0.7 mg/ml. By means of the recently formulated ``psi' procedure for direct analysis of solute self-association a value of (1.5 ± 0.1) μM has been obtained for the dissociation constant K_d. Sedimentation velocity experiments yielded an estimate of the heterodimer sedimentation coefficient, s⁰ _20,w, of (4.5 ± 0.2) S which for M = 63,700 Da suggests a globular structure. Received: 29 November 1996 / Accepted: 2 December 1996     

The purified activated glucocorticoid receptor is a homodimer

The structure of purified preparations of activated (DNA-binding) glucocorticoid receptor (GR) was analyzed in the presence or absence of DNA. A 35-base pair DNA fragment harboring a strong GR-binding site from the mouse mammary tumor virus promoter (-189/-166) was used for stoichiometric analysis of the GR.DNA complex. Glycerol gradient centrifugation was utilized in order to separate the 6 S GR.DNA complex from the 4 S GR and the 3 S DNA fragment. Synthetic glucocorticoid [3H]triamcinolone acetonide bound to GR and 32P-5'-end-labeled DNA fragment were used as probes for quantitation of each component. Such experiments demonstrated that two hormone molecules (two 87.5-kDa GR peptides) are associated with each cognate DNA site. Quantitative DNase I footprinting confirmed this result. The formation of the GR.DNA complex was ligand-dependent, but once formed the complex remained stable after ligand dissociation. Incubation of GR with 0.01-0.1% (w/v) glutaraldehyde resulted in a shift in its sedimentation rate from 4 to 6 S. Gel filtration chromatography of glutaraldehyde-treated GR resulted in a complex of slightly larger size than the gamma-globulin standard (158 kDa). Gel filtration of GR without glutaraldehyde treatment gave the identical result. This suggests that a GR multimer, probably a homodimer, is stable during gel filtration chromatography but needs to be stabilized by glutaraldehyde cross-linking or DNA during glycerol gradient centrifugation. We conclude that the activated GR exists as a homodimer when unbound as well as when bound to DNA.     

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 1 alpha,25-dihydroxyvitamin D3 receptor from rat intestinal cytosol.

The 1 alpha,25-dihydroxyvitamin D3 receptor from rat intestinal cytosol has been partially characterized. Sucrose density gradient sedimentation and analytical gel filtration analyses of this receptor yielded values of 3.1 S, 80,000, and 36 A for the sedimentation coefficient, molecular weight (Mr), and Stokes molecular radius (Rs), respectively. The receptor was found to be a protein by its susceptibility to protease but not nuclease digestion, and studies with N-ethylmaleimide and iodoacetamide revealed the presence of a reduced cysteine residue near the ligand binding site of the receptor. Kinetic and equilibrium binding studies showed an equilibrium dissociation constant of 7.4 x 10(-10) M (4 degrees C), an association rate constant of 1.7 x 10(7) M-1 min-1 (0 degrees C) and a dissociation rate constant of 7.2 x 10(-4) min-1 (4 degrees C, t1/2 = 16 h).     

Estrogen receptor in chicken oviduct: receptor dissociation kinetics and transformation

Cytosolic and nuclear estrogen receptor forms of chicken oviduct have been studied by (1) measuring hormone dissociation kinetics and by (2) sucrose density gradient analysis on high salt gradients. Estradiol dissociates from the receptor in chicken oviduct cytosol at 22 degrees C following a two-phase exponential process. The fraction of receptor with a fast dissociation rate (k = 120 X 10(-3) min-1) decreases as a function of the pre-incubation at 22 degrees C; after prolonged pre-incubation only the slowly dissociating (k = 12.3 X 10(-3) min-1) form remains. Dissociation of moxestrol, a synthetic estrogen with a higher affinity, from the cytosol receptor at 30 degrees C is similar, showing a transition of a fast dissociating form (k = 120 X 10(-3) min-1) to a slowly dissociating form (k = 7.6 X 10(-3) min-1) as a result of pre-incubation at 30 degrees C. A concomitant temperature-dependent shift of the estrogen receptor from a 4.8 S to a 6.1 S form was observed with moxestrol but not with estradiol as a ligand. Sodium molybdate (20 mM) and NaSCN (400 mM) inhibit the temperature-dependent increase in sedimentation coefficient, but molybdate allows the formation of a receptor form which shows intermediary dissociation kinetics. Estrogen receptor, precipitated with ammonium sulfate (0-35%) shows monophasic dissociation kinetics of estradiol (k = 39.5 X 10(-3) min-1) and for moxestrol (k = 10.8 X 10(-3) min-1), suggesting full receptor activation only with moxestrol as a ligand. Moxestrol-receptor complexes obtained by ammonium sulfate precipitation sediment at 0 degree C at 4.8 S. Only after subsequent incubation at 30 degrees C a shift from 4.8 S to 5.9 S is observed, suggesting that the formation of the slowly dissociating form of the receptor may precede the formation of a stable transformed receptor complex. The nuclear estrogen receptor with estradiol as a ligand shows biphasic dissociation kinetics at 22 degrees C (k = 70 X 10(-3) min-1; k = 14.0 X 10(-3) min-1). The ratio of both components (1:1) does not change after preincubation of the nuclear receptor extract at 22 degrees C. Moxestrol dissociates from the nuclear receptor at 30 degrees C monophasically with a slow rate (k = 6.1 X 10(-3) min-1), suggesting that it is extracted as an activated hormone-receptor complex.(ABSTRACT TRUNCATED AT 400 WORDS)     

Phosphorylation of immunopurified rat liver glucocorticoid receptor by the catalytic subunit of cAMP-dependent protein kinase

We have examined phosphorylation of the rat liver glucocorticoid receptor (GR) and GR-associated protein kinase (PK) activity in the immunopurified receptor preparations. Affinity labeling of hepatic cytosol with [³H]dexamethasone 21-mesylate showed a covalent association of the steroid with a 94 kDa protein. GR was immunopurified with antireceptor monoclonal antibody BuGR2 (Gametchu & Harrison, Endocrinology 114: 274–279, 1984) to near homogeneity. A 23° C incubation of the immunoprecipitated protein A-Sepharose adsorbed GR with [-³²P]ATP, Mg²⁺ and the catalytic subunit of cAMP-dependent PK (cAMP-PK) from bovine heart, led to an incorporation of radioactivity in the 94 kDa protein. Phosphorylation of GR was not evident in the absence of the added kinase. Of the radioinert nucleotides (ATP, GTP, UTP or CTP) tested, only ATP successfully competed with [-³²P]ATP demonstrating a nucleotide specific requirement for the phosphorylation of GR. Other divalent cations, such as Mn²⁺ or Ca²⁺, could not be substituted for Mg²⁺ during the phosphorylation reaction. Phosphorylation of GR was sensitive to the presence of the protein kinase inhibitor, H-8, an isoquinoline sulfonamide derivative. In addition, the incorporation of radioactivity into GR was both time- and temperature-dependent. The phosphorylation of GR by cAMP-PK was independent of the presence of hsp-90 and transformation state of the receptor. The results of this study demonstrate that GR is an effective substrate for action of cAMP-PK and that the immunopurified protein A-Sepharose adsorbed GR lacks intrinsic kinase activity but can be conveniently used for the characterization of the phosphorylation reaction in the presence of an exogenous kinase.Abbreviations BUGR2 anti-GR monoclonal antibody - cAMP-PK cAMP-dependent protein kinase - DMSO dimethyl sulfoxide - EDTA ethylenediamine tetra acetic acid - GR glucocorticoid receptor - H-8 Isoquinoline sulfonamide derivative - hsp-90 90 kDa heat-shock protein - PMSF phenylmethylsulfonyl fluoride - PR progesterone receptor - NaF sodium fluoride - SDS sodium dodecyl sulfate - SDS-PAGE SDS-polyacrylamide gel electrophoresis - SR steroid receptor - TA triamcinolone acetonide     

Characterization of the purified activated glucocorticoid receptor from rat liver cytosol

The activated glucocorticoid receptor (GR) from rat liver cytosol was purified by sequential chromatography on DNA-cellulose and DEAE-Sepharose. Analysis by sodium dodecyl sulfate-gel electrophoresis demonstrated a main band with Mr = 94,000 (94K band). Two minor bands with Mr = 79,000 (79K band) and 72,000 (72K band) were also seen in this preparation. Photoaffinity labeling showed that the hormone is bound to the 94K and 79K components but not to the 72K component. Immunoblotting using antibodies raised against the 94K protein demonstrated cross-reactivity between the 94K and 79K components but not with the 72K species. The 72K species could be partially separated from the 94K and 79K components by density gradient centrifugation. Limited proteolysis of the purified GR with trypsin or alpha-chymotrypsin led to degradation of the 94K and 79K components and appearance of a 39K fragment which still retained the hormone and could be bound to DNA-cellulose. The 72K component was not affected by digestion with trypsin or alpha-chymotrypsin. However, chromatography on DNA-cellulose of the alpha-chymotrypsin-treated GR resulted in elution of the 72K component in the flow-through of the column while the 39K fragment was retained on the column and eluted with 0.18 M NaCl. In the control experiment where no alpha-chymotrypsin treatment was performed, the 72K component could not be detected in the flow-through fraction but was eluted together with the 94K and 79K components at 0.18 M NaCl. These results suggest that the 72K protein might be bound to the 94K and/or 79K component. The 39K fragment did not bind antibodies raised against the 94K protein. The 39K fragment was further degraded by trypsin but not by alpha-chymotrypsin to a 27K and a 25K fragment while both still retained the ligand. These data obtained with limited proteolysis of the purified GR are in agreement with previous findings on proteolysis of the GR in crude cytosol (Wrange, O., and Gustafsson, J.-A. (1978) J. Biol. Chem. 253, 856-865; Carlstedt-Duke, J., Okret, S., Wrange, O., and Gustafsson, J.-A. (1982) Proc. Natl. Acad. Sci. U.S.A. 79, 4260-4264).     

Catalytic properties of glutathione reductase from liver at norm and toxic hepatitis

Catalytic properties of glutathione reductase (GR; EC 1.6.4.2) have been investigated using homogenous preparations of this enzyme purified from livers of control rats and rats with toxic hepatitis. Some properties of this enzyme remained unchanged under conditions of toxic hepatitis; these included eletrophoretic mobility (R_f = 0.23 ± 0.01), molecular mass (104.5 ± 5.2 kDa), pH optimum (7.4 ± 0.37), as well as close pK values of functional groups. However, enzyme isolated from toxic liver was characterized by lower affinity for substrate and coenzyme as well as by appearance of substrate inhibition. In addition there are some differences in regulation of GR activity by metabolites of tricarboxylic acid cycle.     

Partial characterization and ontogeny of renal cytosolic glucocorticoid receptors in mouse kidney

The glucocorticoid receptor (GR) was partially characterized in mouse renal cytosol. A sensitive and reproducible [3H]dexamethasone binding assay suitable for use with small quantities of cytosolic protein, was developed. Studies defined the optimal equilibrium binding conditions, metabolism of [3H]dexamethasone in adult renal cytosol, specificity of binding of the GR, and molecular weight of the GR-[3H]dexamethasone complex by gel filtration chromatography. The assay was subsequently used to measure the renal GR during different stages of foetal and postnatal development, as well as in glomerular and renal tubular preparations from adult mice. An almost linear increase in GR occurred from day 13 to day 18 of gestation with levels rising from 100 to 201 fmol/mg cytosol protein; this was followed by a sharp rise in receptor concentration just after birth to 343 fmol/mg cytosol protein. Adult levels, 410-433 fmol/mg cytosol protein, were reached by 2 weeks after birth. The equilibrium dissociation constants (Kd) of the [3H]dexamethasone-receptor complex were similar in adult and in embryonic cytosols (range, 2.8-11.8 nM; mean +/- SD = 6.5 +/- 2.9 nM). Specific binding was assessed to be 3- to 5-fold greater in tubular than in glomerular preparations. These data on the localization and ontogeny of GR during murine metanephric development provide a basis for study of glucocorticoid-mediated effects on various models of congenital and acquired renal disease.     

Solubilization of γ-aminobutyric acid receptor from rat brain

Gamma-aminobutyric acid (GABA) binding sites were solubilized from rat brain synaptosomal fractions by extraction with a combination of sodium deoxycholate and potassium chloride. Specific ³H-GABA binding to the solubilized fraction was saturable with the apparent dissociation constant, Kd = 23.4 ± 0.2 nM. GABA agonists and an antagonist inhibited the binding. The relative potencies of these drugs in competing for ³H-GABA binding to the solubilized fraction are in good agreement with findings with the membrane fraction, suggesting that the binding sites in the solubilized fraction retain the characteristics of membrane-bound GABA receptor. The sedimentation coefficient value of ³H-GABA binding site was estimated to be 11.3S by sucrose density gradient centrifugation, and this value was identical with that of ³H-flunitrazepam binding site in the same solubilized fraction.     

Covalent stabilization of the nontransformed chick oviduct cytosol progesterone receptor by chemical cross-linking

The nontransformed forms of the chick oviduct cytosol progesterone receptor of sedimentation coefficient approximately 8 S (8S-PR) are heterooligomers including one hormone binding molecule, either B, approximately 110,000, or A, approximately 79,000, and two non-hormone binding subunits recently identified as heat-shock protein Mr approximately 90,000 (hsp 90) [Renoir, J. M., Buchou, T., Mester, J., Radanyi, C., & Baulieu, E. E. (1984) Biochemistry 23, 6016-6023]. In the crude cytosol, bisimidates reacted under mild conditions and gave rise to complexes, binding progesterone and reacting with BF4, an anti-hsp 90 monoclonal antibody. These complexes have a sedimentation coefficient of 8.4 S and Rs of 8.1 nm in the presence of 0.4 M KCl and in the absence of molybdate ions, i.e., in conditions that would transform non-cross-linked 8S-PR to Rs approximately 5 nm forms of approximately 4-S sedimentation coefficient. All bisimidates tested, of an effective reagent length between 0.73 and 1.09 nm, gave comparable results in the cytosol prepared with or without molybdate ions, confirming that the latter were not responsible for the formation of the cross-linked 8S complexes. It was found that the dimethyl pimelimidate cross-linked 8S-PR was more resistant to inactivating conditions, urea, or heat treatment than the non-cross-linked 8S-PR. The 8S-PR cross-linked in the cytosol was purified by affinity chromatography in the absence of molybdate ions. After purification, it also reacted with the monoclonal antibody BF4 and had the same Rs (8.0 nm), sedimentation coefficient (approximately 8.5 S), and thus Mr (approximately 290,000) as the original cytosol.(ABSTRACT TRUNCATED AT 250 WORDS)     

Temperature dependence of the dissociation rate constants for the nonactivated (molybdate-stabilized) and activated progesterone receptor-hormone complexes from the chick oviduct

Both the nonactivated and activated forms of the chick oviduct cytosol progesterone receptor-hormone complexes displayed first-order dissociation kinetics at temperatures between 0 and 25 degrees C. The rate constant was always 2-3-times greater for the nonactivated than for the activated complex. The thermodynamic parameters calculated from the Eyring plot for the nonactivated and activated forms, respectively, were: delta H+ = 28.6 +/- 0.2 and 29.9 +/- 1.5 kcal/mol; -T delta S+ = 7.4 +/- 0.6 and 7.7 +/- 1.6 kcal/mol; and delta G+ = 21.3 +/- 0.5 and 22.1 +/- 0.1 kcal/mol. These values suggest that activation results in an increase in enthalpy of the ligand-receptor interaction, thus stabilizing the complex. The dissociation rate constants for the native complex obtained by two different experimental approaches, namely, isotope dilution ('chase') and dissociation against charcoal, indicated the absence of cooperativity in the receptor-ligand binding.     

A cellular activator of catecholamine-sensitive adenylate cyclase in rat reticulocytes and erythrocytes: changes during reticulocyte development and effects on the beta receptor

Rat reticulocytes contain an isoproterenol-sensitive adenylate cyclase activity which is lost with maturation to erythrocytes despite no change in the density of β-adrenergic receptors. To explore this observation, a cytosol factor, previously shown to be important in the expression of catecholamine-sensitive adenylate cyclase in the reticulocyte, was compared to a cytosol factor obtained in a similar manner from mature erythrocytes. The cytosol factor from reticulocytes augmented isoproterenol-responsive adenylate cyclase activity in reticulocyte and erythrocyte membranes half-maximally at 0.7 ± 0.1 (SEM) and 1.1 ± 0.3 μg/ml, respectively. These concentrations of reticulocyte-derived cytosol factor were significantly lower (P < 0.01) than those concentrations of the factor from erythrocytes necessary to augment isoproterenol-responsive adenylate cyclase activity in reticulocyte (9.7 ± 2.3) and erythrocyte (7.5 ± 1.0) membranes. Cytosol factor from reticulocytes also caused greater total isoproterenol responsiveness than that from erythrocytes both in reticulocyte (784 ± 107 vs 525 ± 65 pmol/mg protein) and in erythrocyte membranes (54 ± 6 vs 36 ± 3); P < 0.05. Neither reticulocyte nor erythrocyte cytosol factor affected the concentration at which isoproterenol half-maximally stimulated adenylate cyclase in either set of membranes. However, the cytosol factor from reticulocytes markedly decreased the binding affinity of isoproterenol for β receptors in reticulocytes from 0.8 ± 0.2 to 6.9 ± 1.4 μm; P < 0.001. This reticulocyte factor had no significant effect on the binding affinity of isoproterenol for erythrocyte membranes. Erythrocyte factor did not change the binding affinity for isoproterenol in either reticulocyte or erythrocyte membranes.     

Hormone dependency of transformation of rat liver glucocorticoid receptor in vitro: effects of heat, salt, and nucleotides

A majority of the untransformed glucocorticoid-receptor complexes (GRc) from rat liver cytosol sedimented in the 9S region in 5-20% sucrose gradients containing 0.15 M KCl and 20 mM Na2MoO4. Incubation of the cytosol at 23 degrees C, or at 0 degree C with 10 mM ATP or 0.3 M KCl caused appearance of a slower migrating (4S) form which exhibited an increased affinity toward DNA-cellulose and ATP-Sepharose. Presence of 20 mM Na2MoO4 blocked this 9S to 4S transformation of GRc. A complete conversion of the 9S to the 4S form occurred upon a 2 h incubation of GRc with 10 mM ATP at 0 degree C. Other nucleoside triphosphates (GTP, CTP, and UTP), ADP and PPi (but not AMP or cAMP) were also effective in transforming the 9S form. The heat transformation occurred in a time-dependent manner and was complete within 1 h at 23 degrees C; presence of 10 mM ATP during this 23 degrees C incubation period allowed a complete 9S to 4S alteration in 10-20 min. Addition of ATP also accelerated the rate of salt activation of the GRc; a 50% conversion to the 4S form occurred in 20 min or 3 min in the absence or the presence of 10 mM ATP during the 0 degree C incubation of GRc with 0.15 M KCl. An absolute requirement of the hormone for 9S to 4S transformation of glucocorticoid receptor (GR) was evident, as no conversion of the 9S form to the 4S form could be achieved with the ligand-free GR under any of the above conditions. Incubation of cytosol preparations at 23 degrees C or at 0 degree C with KCl or ATP caused dissociation of the GRc and reduced the steroid binding capacity of GR. Although aurintricarboxylic acid, pyridoxal 5'-phosphate, Na2MoO4, Na2WO4, o-phenanthroline, Rifamycin AF/013 and heparin inhibited the ATP-Sepharose and DNA binding of the GRc, only Na2MoO4 and Na2WO4 selectively blocked the 9S to 4S conversion. We suggest that the 9S to 4S transformation in vitro of rat liver GRc represents an acquisition of DNA and ATP-Sepharose binding ability and may involve a separation of subunits from an oligomeric receptor structure.