Human progesterone receptor binding to mouse mammary tumor virus deoxyribonucleic acid: dependence on hormone and nonreceptor nuclear factor(s)

Using crude progesterone receptor preparations from T47D human breast cancer cells, we show by immunoprecipitation assay that receptor specifically and with high affinity recognizes the hormone response element (HRE) of the mouse mammary tumor virus (MMTV). The use of crude preparations minimizes alterations of receptors or loss of associated factors that may occur during purification. Specific binding was obtained at 1:1 molar ratios of receptor to DNA, and HRE sequences are recognized with an affinity at least 3 orders of magnitude greater than nonspecific DNA. We have compared the DNA-binding activities of different forms of progesterone receptors. The unliganded 8S cytosol receptor had low but detectable binding activity for MMTV DNA. Addition of hormone to cytosol produced a small but consistent 2.5-fold increase. In vitro methods of transforming cytosol receptors from an 8S to a 4S species failed to increase DNA-binding further. By contrast, 4S receptors bound by R5020 in whole cells and extracted from nuclei by salt, displayed a substantially higher (average, 11-fold) binding activity than an equal number of unliganded cytosol receptors. The dissociation constants for cytosol and nuclear receptor binding to MMTV DNA were similar (approximately 2 x 10(-9) M). Thus, nuclear receptors possess a higher capacity for binding to specific recognition sequences. These results suggest that hormone or a hormone-dependent mechanism increases the intrinsic DNA-binding activity of receptors independent of receptor transformation from 8S to 4S. Further experiments indicate that a nonreceptor activity in nuclear extracts can increase the sequence-specific DNA-binding activity of cytosol receptors. This activity is present in both T47D cells and receptor-negative MDA-231 cells. We conclude that the higher DNA-binding activity of the nuclear receptor-hormone complex is due in part to receptor interaction with other nuclear proteins or factors. Such interactions may function to maintain receptors in a disaggregated active complex or to stabilize their binding to specific DNA sites.     

Mechanisms controlling steroid receptor binding to specific DNA sequences.

Mammalian progesterone receptors activated by hormone binding in nuclei of intact cells exhibit substantially higher binding activity for specific DNA sequences than receptors bound with hormone and activated in cell-free cytosol. Differences in DNA-binding activity occur despite the fact that both activated receptor forms sediment at 4S on sucrose gradients and are apparently dissociated from the heat shock protein 90. This suggests that hormone-induced release of heat shock protein 90 from receptors is necessary, but not sufficient for maximal activation of DNA binding. This report is a review of studies from our laboratories that have examined the role of receptor interaction with other nuclear protein factor(s), and receptor dimerization in solution, as additional regulatory steps involved in the process of receptor activation and binding to specific gene sequences.     

Chemical differentiation of type I and type II receptors for adrenal steroids in brain cytosol

Studies outlined here compare the properties of mineralocorticoid (Type I) and glucocorticoid (Type II) receptors in cytosol from adrenalectomized mouse brain. Pretreating cytosol with dextran-coated charcoal (DCC) produced a 4.7-fold increase in the subsequent macromolecular binding of the mineralocorticoid, [3H]aldosterone (20 nM ALDO, in the presence of a 50-fold molar excess of the highly specific synthetic glucocorticoid, RU 26988), whereas it produced a 55% decrease in the binding of the glucocorticoid, [3H]triamcinolone acetonide (20 nM TA). Scatchard analyses revealed that DCC pretreatment had no effect on the affinity or maximal binding of Type I receptors for [3H]ALDO (in the presence of a 0-, 50- or 500-fold excess of RU 26988), whereas it produced a 3- to 6-fold increase in the Kd, and an 8-43% decrease in the maximal binding, of Type II receptors for [3H]TA and [3H]dexamethasone. Optimal stability of unoccupied Type I receptors at 0 degree C was found to be achieved in buffers containing glycerol, but lacking molybdate. Although the addition of molybdate was found to reduce the loss in Type I receptor binding observed after incubating unlabelled cytosol at 12 or 22 degrees C, this stabilization was accompanied by a concentration-dependent reduction in the binding of [3H]ALDO at 0 degree C. Scatchard analyses showed that this reduction was due to a shift in the maximal binding, and not the affinity, of the Type I receptors for [3H]ALDO. The presence or absence of dithiothreitol in cytosol appeared to have little effect on the stability of Type I receptors. In contrast to our finding for Type I receptors, it was possible to stabilize the binding capacity of unoccupied Type II receptors, even after 2-4 h at 12 or 22 degrees C, if the glycerol containing buffers were supplemented with both molybdate and dithiothreitol. In summary, these results indicate distinct chemical differences between Type I and Type II receptors for adrenal steroids.     

The NSILA-s receptor in liver plasma membranes. Characterization and comparison with the insulin receptor.

NSILA-s (nonsuppressible insulin-like activity, soluble in acid ethanol) is a serum peptide that has insulin-like and growth-promoting activities. We have demonstrated previously that liver plasma membranes possess separate receptors for NSILA-s and insulin and have characterized the insulin receptor in detail. In the present study we have characterized the properties and specificity of the NSILA-s receptor and compared them to those of the insulin receptor in the same tissue. Both 125I-NSILA-s and 125I-insulin bind rapidly and reversibly to their receptors in liver membranes; maximal NSILA-s binding occurs at 20 degrees while maximal insulin binding is seen at 1-4 degrees. The pH optimum for NSILA-s binding is broad (6.0 to 8.0), in contrast to the very sharp pH optimum (7.5 to 8.0) for insulin binding. Both receptors exhibit a high degree of specificity. With the insulin receptor, NSILA-s and insulin analogues compete for binding in proportion to their insulin-like potency: insulin greater than proinsulin greater than NSILA-s. With the NSILA-s receptor, NSILA-s is most potent and the order is reversed: NSILA-s greater than proinsulin greater than insulin. Furthermore, six preparations of NSILA-s which varied 70-fold in biological activity competed for 125I-NSILA-s binding in order of their potencies. NSILA-s which had been inactivated biologically by reduction and aminoethylation and growth hormone were less than 1/100,000 as potent as the most purified NSILA-s preparation. Purified preparations of fibroblast growth factor, epidermal growth factor, nerve growth factor, and somatomedins B and C were less than 1% as effective as NSILA-s in competing for the 125I-NSILA-s suggesting that these factors act through other receptors. In contrast, somatomedin A was 10% as active as NSILA-s and multiplication-stimulating activity was fully as active as NSILA-s in competing for the NSILA-s receptor. Analysis of the data suggests that there are approximately 50 times more insulin receptors than NSILA-s receptors per liver cell, while the apparent affinity of NSILA-s receptors is somewhat higher than that of the insulin receptor.     

Evidence that invivo estradiol receptor translocated into nuclei is dephosphorylated and released into cytoplasm

Most of the estradiol binding activity is lost after the receptor-hormone complex migrates into the nuclei. We report that at the same time an equivalent amount of receptor unable to bind hormone appears in cytosol. ATP incubated with uterine cytosol from 17β-estradiol injected mice causes an increase in hormone binding activity. This activation is catalyzed by the ATP-dependent enzyme which reactivates hormone binding of estradiol receptor previously inactivated by a nuclear phosphatase (1). The inactive receptor retains the property to be activated by the ATP-dependent enzyme after a partial purification by heparin-Sepharose. A large dose of cycloheximide does not inhibit the appearance of ATP-activated cytosol receptor. Apparently the 17β-estradiol receptor which migrates into the nucleus is inactivated by the nuclear phosphatase and then released in an inactive, dephosphorylated form into the cyoplasm.     

Characterization of a [3H]methyltrienolone (R1881) binding protein in rat liver cytosol

The binding of radiolabelled methyltrienolone 17 beta-hydroxy-17 alpha-methyl-estra-4,9,11-trien-3-one (R1881) to adult male rat liver cytosol has been characterized in the presence of Na-molybdate to stabilize steroid-hormone receptors, and triamcinolone acetonide to block progestin receptors. Using sucrose density gradient analysis, male liver cytosol contains a [3H] R1881 macromolecular complex which sediments in the 8-9S region. 8S binding of R1881 to male rat serum, female liver cytosol or cytosol from a tfm rat cannot be demonstrated. Further metabolism of [3H] R1881 following 20h incubation with male rat liver cytosol was excluded: In the 8S region 97% of [3H] R1881 was recovered by thin layer chromatography. Characteristics of this [3H] R1881-8S binding protein include high affinity (Kd = 2.3 +/- 41 nM) and low binding capacity (18.8 +/- 3.3 fmol/mg cytosol protein), precipitability in 0-33% ammonium sulfate, and translocation to isolated nuclei following in vivo R1881 treatment. Whereas, the cytosol R1881-receptor is competed for by dihydrotestosterone, testosterone, and estradiol, [3H] estradiol binding in the 8S region is not competitive with androgens but does compete with diethylstilbestrol. The nuclear androgen binding site has a Kd = 2.8 nM for [3H] R1881, and is androgen specific (testosterone greater than 5 alpha-dihydrotestosterone greater than estradiol greater than progesterone greater than cyproterone acetate greater than diethylstilbestrol greater than dexamethasone greater than triamcinolone). Since a number of liver proteins including the drug and steroid metabolizing enzymes are, in part, influenced by the sex-hormone milieu, the presence of a specific androgen receptor in male rat liver may provide valuable insight into the regulation of these proteins.     

应激大鼠肾胞液[^3H]醛固酮结合活性的变化及调节机制的初步研究

为探讨烫伤引起病理性应激时大鼠肾胞液醛固酮结合活性的变化及可能的调节机制,以[～3H]-醛固酮为配体,用放射性配基-受体结合分析法测定了正常对照组、轻度烫伤组和重度烫伤组大鼠肾胞液醛固酮结合活性的结合容量(Rt)和表观解离常数(Kd);采用体内注射TNF-α、IL-1β中和抗体和α-促黑色素细胞刺激激素(α-melanocyte-stimulatinghormone,α-MSH)和合成肽KPV(Ac-D-Lys-L-Pro-D-Val)等措施调节其改变.结果发现,肾胞液存在两种不同结合容量、不同亲和力的醛固酮结合活性受体.与正常对照组的Rt(Rt141.6±7.2fmol/mgpro;Rt2317.6±70.0fmol/mgpro)相比,轻烫组的Rt(Rt141.4±5.0fmol/mgpro;Rt2314.8±45.7fmol/mgpro)无显著差异(P>0.05;P>0.05);重烫组的Rt(Rt122.4±5.4fmol/mgpro;Rt2196.3±32.5fmol/mgpro)则显著下降(P<0.01;P<0.01).体内注射TNF-α与IL-1β中和抗体、α-MSH及KPV均能明显提高重烫组Rt值.结果提示,重度烫伤大鼠肾胞浆醛固酮结合活性降低,TNF-α、IL-1β中和抗体、α-MSH及KPV均可防止重度烫伤引起病理性应激时醛固酮结合活性降低.     

Interaction of ring B unsaturated estrogens with estrogen receptors of human endometrium and rat uterus

The present investigation was undertaken to compare the binding affinities (Ka) of the ring B unsaturated equine estrogens (equilin [Eq], equilenin [Eqn], 17 beta-dihydroequilin [17 beta-Eq], 17 beta-dihydroequilenin [17 beta-Eqn], 17 alpha-dihydroequilin [17 alpha-Eq], and17 alpha-dihydroequilenin [17 alpha-Eqn]) and the classic estrogens (estrone [E1], 17 beta-estradiol [17 beta-E2], and 17 alpha-estradiol [17 alpha-E2]) for estrogen receptors in human endometrium and rat uterus. In both species, the ring B unsaturated estrogens bind with cytosol and nuclear receptors with high affinity (Ka x 10(9) M-1). The relative binding affinities of these estrogens were measured by determining the amount of unlabeled estrogen required to reduce by 50% the specific binding of [3H]17 beta-Eq to endometrial cytosol receptors. The order of activity found was 17 beta-Eq greater than 17 beta-E2 greater than 17 beta-Eqn greater than E1 greater than Eq greater than 17 alpha-Eq greater than 17 alpha-E2 greater than 17 alpha-Eqn greater than Eqn. Essentially the same order of activity was observed when the apparent affinity constants of these estrogens for human and rat cytosol and nuclear receptors were determined by a competitive (inhibition) binding assay. Sucrose density gradient analysis indicated that these estrogens form protein complexes with cytosol and nuclear preparation that sediment at approximately 8S and 4S, respectively. The affinity constants for 17 beta-Eq were approximately two- to six-fold higher than E2 in both species. In a rat uterotropic assay, all nine estrogens were uterotropic. These data indicate that all ring B unsaturated estrogens present in conjugated equine estrogen preparations are biologically active and they express their biologic effects in the human endometrium by mechanisms similar to those described for the classic estrogens.     

Effects of Polyhydric and Monohydric Compounds on the Stability of Type I Receptors for Adrenal Steroids in Brain Cytosol

We have shown previously that unoccupied type I receptors for adrenal steroids in brain cytosol lose their capacity to bind [3H]aldosterone ([3H]ALDO) in a time- and temperature-dependent manner. Based on reports that sugars and polyvalent alcohols are capable of stabilizing a variety of globular proteins, we attempted in the present study to stabilize type I receptors by including polyhydric compounds in our brain cytosol preparations. However, contrary to expectations, adjusting cytosol to a 10% (g/dl) concentration of ethylene glycol, glycerol, erythritol, xylitol, ribitol, or sorbitol failed to stabilize these receptors at 0 degree C and in fact produced a slight reduction in [3H]ALDO binding capacity. The magnitude of this reduction was greater when cytosol was incubated for 2 h at 22 degrees C prior to incubation with [3H]ALDO. In contrast to these results, when brain cytosol was adjusted to a 10% (g/dl) concentration of the monohydric compound, ethanol, a significant increase in [3H]ALDO binding to type I receptors was found. Under identical conditions, methanol and propanol failed to have a significant effect on the binding capacity of these receptors. When cytosol was aged for 2 h at 22 degrees C, all three of these monohydric compounds produced a marked loss in the [3H]ALDO binding capacity of type I receptors. An investigation of various doses of ethanol at 0 degree C on the subsequent binding of [3H]ALDO yielded an inverse U-shaped curve with 10% ethanol producing the highest level of specific binding, as reflected by an increase in maximal binding in Scatchard plots, and 40% ethanol producing a complete loss in type I receptor binding capacity.(ABSTRACT TRUNCATED AT 250 WORDS)     

ATP-dependent enzyme activating hormone binding of estradiol receptor

Mouse uterus estradiol receptor undergoes a inactivation-reactivation process “in vitro”. The specific estrogen binding activity inactivated by nuclei, apparently through a dephosphorylation process (1,2,3), is reactivated by an ATP-dependent process. The enzyme reactivating the receptor has been purified from calf uterus cytosol. It shows high affinity for the inactive receptor (K_m of ～ 0.3 × 10^?9 mol of 17β-estradiol binding sites/l); it is simulated by MgCl₂ and CaCl₂. Present and previous results suggest that in cytoplasm of intact cells a phosphorylation process makes the receptor able to bind hormone and in nuclei dephosphorylation of receptor causes loss of hormone binding activity.     

Characterization of the rat colonic aldosterone receptor and its activation process

Aldosterone increases sodium absorption, short circuit current, and transmural potential difference in rat colon. We studied the rat colonic aldosterone receptor using the synthetic glucocorticoid, 11 beta, 17 beta-dihydroxy-17 alpha-propynylandrosta-1,4,6-triene-3-one, to prevent binding to the glucocorticoid receptor. Specific aldosterone binding was found in proximal and distal colon. Heating to 25 degrees C decreased binding within 15 min, but the protease inhibitor, phenylmethylsulfonyl fluoride, stabilized binding. Binding was highest in terminal distal colon. Competitive binding assay showed aldosterone specificity compared to other competitors was greater at 30 than at 4 degrees C, suggesting temperature-sensitive changes in receptor specificity. Scatchard analysis revealed a straight line with a KD of 2.5 nM at 0 degrees C and 4.1 nM at 30 degrees C. Bmax was higher in distal than in proximal colon (30 degrees C, 156 +/- 33 versus 65 +/- 9 fmol/mg protein) and increased by 36% in proximal and 180% in distal colon at 30 degrees C compared to 0 degrees C. DEAE-cellulose chromatography of unactivated receptor demonstrated a single peak eluting at 200-250 mM KCl. Heat, ATP, and gel filtration did not activate the receptor, whereas increasing cytosolic salt concentration to 300 mM KCl, raising the pH to 8, or adding EGTA and EDTA caused increased DNA-cellulose binding and a new peak eluting at 30-80 mM KCl on DEAE-cellulose chromatography. There is a specific aldosterone receptor in colon with increasing number of binding sites from proximal to most distal segments paralleling aldosterone's physiological effects. Absence of receptor activation with heat, gel filtration, or ATP suggests differences between activation of the aldosterone receptor and other steroid hormone receptors.     

A specific binding protein for 1 alpha,25-dihydroxyvitamin D in the chick embryo chorioallantoic membrane

A 3.7 S binding protein for the steroid hormone and vitamin D metabolite 1 alpha-25-dihydroxyvitamin D (1,25-(OH)2-D) was observed in high salt cytosol extracts of chick embryo chorioallantoic membrane. The binding protein was characterized after partial purification of cytosol extracts by ammonium sulfate fractionation. The binding of 1,25-(OH)2-D was saturable, had a high affinity (Kd = 0.16 nM), and was specific for hormonally active vitamin D metabolites. Analysis of the displacement of [3H]1,25-(OH)2-D by unlabeled analogues showed the affinities of vitamin D metabolites to be in the order of 1,25-(OH)2-D = 1,24R,25-(OH)3-D much greater than 25-OH-D = 1-OH-D greater than 24R,25-(OH)2-D. Hormone binding was sensitive to pretreatment with sulfhydryl-blocking reagents. The chorioallantoic membrane 1,25-(OH)2-D-binding protein associated with the chromatin fraction after homogenization of membranes in low salt buffer, and bound to DNA-cellulose columns, eluting as a single peak at 0.215 M KCl. These findings support identification of this 1,25-(OH)2-D-binding protein as a steroid hormone receptor, with properties indistinguishable from 1,25-(OH)2-D receptors in other chick tissues. The chorioallantoic membrane functions in the last third of embryonic development to reabsorb calcium from the eff shell for deposition in embryonic bone. 1,25-(OH)2-D binding activity in the chorioallantoic membrane increased 4- to 5-fold from day 12 to day 16 of incubation, immediately preceding the onset of shell reabsorption. This finding suggests that 1,25-(OH)2-D may act to regulate shell mobilization and transepithelial calcium transport by the chorioallantoic membrane. Finally, the similarity of shell mobilization to bone resorption, which is also stimulated by 1,25-(OH)2-D, suggests that the chorioallantoic membrane is a useful alternate model for the study of 1,25-(OH)2-D action on bone mineral metabolism.     

Binding sites of iron transferrin on rat reticulocytes. Inhibition by specific antibodies

Rat ventral prostate contains an acidic protein which can bind spermine selectively. The relative binding affinities of various aliphatic amines for the protein are, in decreasing order, spermine greater than thermine greater than greater than putrecine greater than 1,10-diaminodecane, cadaverine and 1,12-diaminododecane. The binding protein has an isoelectric point at pH 4.3 and a sedimentation coefficient of 3 S. Its molecular weight is approx. 30 000. Histones and nuclear chromatin preparations of the prostate can interact with the binding protein. The spermine-binding activity of the purified prostate protein can be inactivated by treatment with intestinal alkaline phosphatases. The phosphatase treated preparation can then be reactivated by beef heart protein kinase in the presence of cyclic AMP and ATP. The spermine-binding activity of the prostate cytosol protein fraction decreases after castration, but increases very rapidly after the castrated rats are injected with 5alpha-dihydrotestosterone. This finding raises the possibility that, in the postate, certain androgen actions may be dependent on the androgen-induced increase in the acidic protein binding of polyamines and their translocation to a functional cellular site such as nuclear chromatin. In the prostate cytosol, spermine also binds to 4-S tRNAs and to a unique RNA which has a sedimentation coefficient of 1.5 S.     

Precocious induction of tryptophan dioxygenase by glucocorticoid in suckling rats and correlation with change in glucocorticoid receptor

When young rats (less than 14 days old) were treated once a day for 2 days with 100 micrograms/100 g body weight of dexamethasone, their liver cytosol showed a sharp new peak of glucocorticoid binding protein (peak C) eluted with 0.14 M NaCl on DEAE-cellulose chromatography. When the young rats were given a single injection of the hormone, the chromatogram showed a dominant peak of binding protein (peak B), eluted with 0.07 M NaCl, which was similar to that in untreated rats. The appearance of peak C on two treatments of young rats with hormone was confirmed by both in vivo and in vitro labeling and also studies on the nuclear fraction. Peaks B and C were specific hormone-binding proteins as shown with excess unlabeled hormone. The appearance of peak C was concomitant with the precocious induction of tryptophan dioxygenase in the liver of the young rats, and pretreatment with two injections of dexamethasone were necessary for maximal enzyme induction. On the other hand, in adult rats a single injection of dexamethasone (of 20 micrograms/100 g body weight or more) was enough to cause the appearance of peak C and induce tryptophan dioxygenase activity maximally; an additional injection of the hormone did not change the chromatographic pattern of the specific binding or the enzyme activity. For this effect in young rats, dexamethasone could not be replaced by other hormones such as glucagon, growth hormone, thyroid hormones, insulin, sex steroids or short-acting glucocorticoid.     

Metal ion dependence of the binding of triiodothyronine by cytosol proteins of bullfrog tadpole tissues.

The binding of triiodothyronine by Rana catesbeiana tadpole tail fin, tail muscle, kidney, and liver cytosol was studied using dextran-coated charcoal to separate bound and free hormone. A metal ion dependency was suggested by the fact that EDTA decreased the binding of triiodothyronine 80 to 90% in tail fin and tail muscle cytosol. Inhibition of binding in kidney or liver was less, 40 to 50%. This inhibition could be restored by adding an excess of divalent cations with an order of potency of Mn2+ greater than Ca2+ congruent to Co2+ greater than Sr2+ greater than Ba2+ greater than Mg2+. Other chelators, e.g. o-phenanthroline, 8-hydroxyquinoline, and ethylene glycol bis(beta-aminoethylether)-N,N'-tetraacetate also decreased the binding of triiodothyronine, whereas citrate, oxalate, imidazole, and glycine had no effect. The triiodothyronine binding capacity of tail fin cytosol was reduced by EDTA treatment and dialysis against buffer. Ca2+ in the 1 to 10 mM range and Mn2+ at 1 mM could restore the binding to normal levels. Higher Mn2+ increased binding 70% above normal or to Ca2+-restored levels. The triiodothyronine cytosol binding activity was nondialyzable, heat-labile. pH-dependent, pronase-digestible, but unaffected by incubation with trypsin, RNase, and DNase, suggesting that the cytosol binding sites are acidic proteins. Scatchard analysis of triiodothyronine binding by the cytosol of different tissues, revealed Kassoc of 7.1 x 10(6) M(-1), 11.6 x 10(6) M(-1), 3.6 X 10(6) M(-1), and 68.0 x 10(6) M(-1) for tail fin, tail muscle, kidney, and liver cytosol, respectively. The corresponding maximal binding capacities in picomoles per mg of crude cytosol protein in these four tissues were 10.4, 0.86, 1.3, and 0.04, respectively.     

Role of sodium and potassium in aldosterone binding of kidney receptors in the rat.

An increase of the sodium concentration produced a diminished binding of labeled aldosterone to kidney receptors in both in vivo and in vitro conditions. The elevation of potassium level did not change the binding capacity of kidney nuclear receptors for aldosterone. Hypernatraemia-induced decrease of aldosterone uptake was significant in the nuclear fraction of both medullary and cortical region of the kidney but did not show remarkable changes in the uptake of cytosol fraction. The transfer of aldosterone from cytosol into the nuclear compartment seems to be changed by the alteration of extracellular sodium concentration.     

Steroidal 21-diazo ketones: photogenerated corticosteroid receptor labels.

The 21-diazo derivatives of 9 alpha-fluoro- and 9 alpha-bromo-21 deoxycorticosterone, 21-deoxycorticosterone, and progesterone were synthesized for use as photoaffinity labels for corticosteroid receptors. In the isolated toad bladder system, 9 alpha-bromo-21-diazo-21-deoxycorticosterone was as active as d-aldosterone and more active than 9 alpha-fluoro-cortisol in augmenting active Na+ transport. The activities of 21-diazoprogesterone and progesterone were equal; both were much less potent than d-aldosterone, however. These results indicate that the 21-diazo derivatives had significant functional activity in the toad bladder system. The rat kidney slice system was used to estimate the relative affinities of the diazo steroids for aldosterone receptor sites by competition experiments. At 100-fold excess of competitor to [3-H]aldosterone, the order of affinities was 9 alpha-fluoro-21-diazo-21-deoxycorticosterone greater than 9 alpha-bromo-21-diazo-21-deoxycorticosterone greater than 21-diazoprogesterone. Moreover, 9 alpha-bromo-21-diazo-21-deoxycorticosterone reduced binding of [3-H]aldosterone to cytoplasmic and nuclear forms of the receptor proportionately. On the basis of competition for [3-H]corticosterone binding, presumably to corticosteroid-binding globulin (CBG), the order of affinities was 21-diazo-21-deoxycorticosterone greater than 21-diazoprogesterone greater than 9 alpha-bromo-21-diazo-21-deoxycorticosterone. These findings indicate that 21-diazo steroids may be suitable as photogenerated affinity labels for mineralocorticoid receptors. The tritiated derivative, [1,2-3-H]-9 alpha-bromo-21-diazo-21-deoxycorticosterone (specific activity 25 Ci/mol) was synthesized and used in model experiments on photogenerated covalent binding to rat plasma proteins. Irradiation with uv light resulted in binding of [1,2-3-H]-9 alpha-bromo-21-diazo-21-deoxycorticosterone to plasma proteins, that was resistant to extraction with methylene dichloride and did not exchange with unlabeled corticosterone. The diazocorticosteroids, therefore, may have the requisite functional and selectivity properties for photoaffinity labeling of corticosteroid-binding proteins. Further studies are needed, however, to assure that photogenerated labeling with these steroids was site specific.     

Glycyrrhizic acid and its hydrolysate as mineralocorticoid agonist

Mineralocorticoid activity of glycyrrhetinic acid (GR) was studied in vivo (electrical potential difference in rat rectum) and in vitro (brush border Mg2+-HCO3- ATPase in rat small intestine, kidney cytosol binding of GR with and without RU-28362, anti-glucocorticoid compound) in order to clarify the mechanism of mineralocorticoid-like activity of GR. Scatchard analysis of [3H]aldosterone showed that Kd of higher affinity site (type I) 6.0 X 10(-9) M, Bmax 1.0 X 10(-14) mol/mg protein, and Kd of lower affinity site (type II) 1.6 X 10(-7) M, Bmax 7.5 X 10(-14) mol/mg protein. GR competed for [3H]aldosterone binding sites in kidney cytosol at the concentration of 10(4) times as that of unlabeled aldosterone. RU-28362 displaced aldosterone binding curve, whereas GR binding kinetic was not affected by this compound. Adrenalectomy caused a significant fall in brush border Mg2+-HCO3- ATPase activity (75% reduction compared with the initial level) which was not restored by GR administration. Electrical potential differences in the adrenalecomized rats were significantly lower than those in the control rats, which did not increase after GR administration.     

Binding and structural properties of oxytocin receptors in isolated rat epididymal adipocytes

Oxytocin initiates its insulin-like action in adipocytes through oxytocin-specific receptors. We have studied binding and structural properties of these receptors with the radioligand [3H]oxytocin. Steady-state binding was reached after 45 min, at 21 degrees C, and 10 min at 37 degrees C. Scatchard analyses of equilibrium binding data indicated a single class of oxytocin binding sites at 21 degrees C (KD = 3.3 nM, RT = 6 X 10(4) sites/cell) and 2 binding sites at 37 degrees C (KD = 1.5 nM, RT = 6 X 10(4) sites/cell; and KD = 20 nM, RT = 30 X 10(4) sites/cell). Insulin, insulin-like growth factor I, and epidermal growth factor increased oxytocin binding (approximately 20-40%), whereas adenosine, a regulator of oxytocin action, did not affect oxytocin binding. Binding activity of oxytocin was impaired by pretreatment of the hormone or adipocytes with dithiothreitol. Dithiothreitol treatment of adipocytes preferentially inactivated high-affinity binding sites. N-ethyl maleimide inhibited oxytocin binding in adipocytes more than dithiothreitol. In contrast to the inhibitory effects of dithiothreitol and N-ethyl maleimide, proteases (trypsin, chymotrypsin and papain) were not able to inhibit fat cell binding activity. These results suggested that in isolated adipocytes: there are high-affinity and low-affinity receptors, but the low-affinity receptors are absent at 21 degrees C; the binding of oxytocin can be regulated by insulin, and growth factors; and the oxytocin receptors contain disulfide bridges and free thiols that are essential for the maintenance of oxytocin binding.     

The CCK receptor on pancreatic plasma membranes: binding characteristics and covalent cross-linking

The cholecystokinin (CCK) receptor in purified plasma membranes prepared from mouse pancreatic acini had a binding affinity of 1.8 nM, an acid pH optimum between 6.0 and 6.5, and an analog specificity of CCK8 greater than CCK33 greater than desulphated CCK8 greater than CCK4. Binding of CCK to its receptor was abolished by pretreatment of plasma membranes with trypsin. When [125I]CCK was cross-linked to its receptors with disuccinimidyl suberate, and the preparation solubilized and subjected to gel electrophoresis and autoradiography, the hormone was associated with Mr 80 000 protein in both the presence and absence of the reducing agent dithiothreitol.