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D O Toft

Publications and source records attributed to D O Toft.

At least 91 records · Page 5Linked to original sources
PubMed1984

Characterization of an unusual sex steroid binding component from the chicken oviduct.

In addition to the classical estrogen receptor, chick oviduct cytosol contains a sex steroid binding component (SSB) with specificity for steroidal estrogens, androgens and progestins. We have optimized the measurement of SSB and have further characterized this protein. It was possible to quantitate [3H]estradiol binding to SSB by performing the measurements in the presence of excess diethylstilbestrol, which saturates the estrogen receptor and does not bind to SSB, and by using excess progesterone to determine nonspecific binding. Since SSB appears to be quite unstable with rapid hormone dissociation kinetics, we determined that short incubation times (usually 2 h) at 0 degrees C with 20-30% glycerol in the buffer gave optimal SSB measurements. The affinity of SSB for estradiol (Kd = 20 nM) is about 5% that of the estrogen receptor. In addition to estradiol, several androgens and progestins bind to SSB. However, the nonsteroidal antiestrogen, H1285 does not bind to SSB even though it binds well to the avian estrogen receptor. The tissue content of SSB is about 15-fold greater than for estrogen receptor and is stimulated by estrogen treatment. Whereas labeled SSB cannot be readily resolved by ion-exchange chromatography due to rapid dissociation of hormone from SSB, post-labeling experiments yield binding activity eluting with 0.2 M KCl indicating that SSB is an acidic protein having a chromatography behavior similar to that of estrogen receptor. SSB binding was dramatically reduced by the chaotropic salt, NaSCN, whereas binding to the estrogen receptor was not disrupted. SSB is stabilized by sodium molybdate, a property which is characteristic of steroid receptors. Although the role of SSB in the chick oviduct is yet to be determined, an understanding of its properties is essential for accurate determinations of the estrogen receptor.

Animals↗
PubMed1984

The avian progesterone receptor: isolation and characterization of phosphorylated forms.

Methods have been developed for isolation of the avian progesterone receptor in the nontransformed, molybdate-stabilized state. The final step in this procedure. DEAE-Sephadex chromatography, resolves the receptor into two forms, components I and II. Analysis of these components by polyacrylamide gel electrophoresis under denaturing conditions shows that both contain a peptide with Mr = 90,000. These peptides contain phosphorylated serine residues, as shown by [32P]orthophosphate incorporation studies. When the cytosol receptor is treated with alkaline phosphatase, its steroid binding capacity is abolished. These studies show that the nontransformed progesterone receptor is a phosphoprotein and indicate that receptor phosphorylation may be important to the maintenance of steroid binding capacity.

Alkaline Phosphatase↗
PubMed1984

Transformation of highly purified avian progesterone receptor.

Transformation of the avian progesterone receptor to the nuclear form was studied using highly purified receptor preparations. The progesterone receptor was purified to near homogeneity in the presence of 10 mM sodium molybdate by affinity chromatography on deoxycorticosterone-Sepharose followed by DEAE-Sephadex chromatography. This latter step resolved the receptor into two 8S forms, I and II. Receptor transformation was measured by the binding of receptor to the polyanion resins (DNA-cellulose, phosphocellulose, or ATP-Sepharose) and to isolated nuclei and by the change in sedimentation coefficient from 8S to 4S. Molybdate was removed from the purified receptor preparations by agarose gel filtration. This step resulted in transformation of a major portion of receptor, as indicated by the above criteria. The extent of transformation was enhanced slightly by further incubation of the receptor in 0.2 M KCl. Control samples, which contained 10 mM molybdate, remained nontransformed, as tested by sedimentation or binding analysis. However, receptor transformation could not be reversed by adding molybdate back to transformed receptor. Although transformation of both receptor components I and II was observed, the extent of component I transformation was generally 2- to 5-fold greater than that of component II. About 50-90% of component I could be converted to a form that bound DNA-cellulose, ATP-Sepharose, and phosphocellulose. Since the progesterone receptor is a phosphoprotein, 32P-labeled receptor was tested for any loss of phosphate during transformation or receptor inactivation by incubation at 37 C. No observable loss of 32P occurred with either treatment. Our results show that transformation of the 8S receptor components I and II can be achieved in the absence of other cytosolic factors.

Animals↗
PubMed1983

Interaction of chick oviduct progesterone receptor with the 2',3'-dialdehyde derivative of adenosine 5'-triphosphate.

Avian oviduct progesterone receptor was treated with the 2',3'-dialdehyde derivative of ATP (oATP) in an attempt to demonstrate the presence of nucleotide binding sites on the receptor. oATP, when added to cytosol, inhibited binding by transformed receptor to ATP-Sepharose, DNA-cellulose, phosphocellulose, or isolated nuclei in an irreversible manner. oATP did not disrupt the steroid-receptor complex, but it did alter the ionic properties of the receptor. This was demonstrated by an increased affinity of receptor for DEAE-cellulose and for hydroxylapatite. oATP mimicked the effect of ATP on progesterone receptor with regard to two properties: it altered the rate of receptor inactivation that occurs in the absence of progesterone, and it promoted receptor conversion from an 8S complex to lower sedimenting forms (4-6 S). The action of oATP on the receptor could be blocked by the addition of pyridoxal 5'-phosphate, which has been shown previously to interact with the progesterone receptor. A partial interference of oATP action was also observed when ATP was added. These results indicate that oATP interacts with the progesterone receptor and may be used as an affinity-labeling agent for receptor characterization.

Adenosine Triphosphate↗
PubMed1983

Characterization and physiological variation of estrogen receptors in rabbit corpora lutea throughout pregnancy and pseudopregnancy: the effect of hysterectomy and sustained estradiol treatment.

Previous studies suggest that regression of the rabbit corpus luteum is associated with a uterine-induced loss of responsiveness to estradiol. To determine if this is due to loss of estrogen receptor, cytoplasmic and nuclear estrogen receptors were measured in pseudopregnant, hysterectomized-pseudopregnant and pregnant rabbits throughout luteal life. Estrogen receptor levels were higher in corpora lutea than in nonluteal tissue and were generally higher in nuclei compared to cytosol. Estrogen receptor levels were low on Day 3, increased 2- to 3-fold by Day 6-8, reached peak levels by Days 8-10, and then gradually decreased in a pattern similar to the pattern of serum progesterone typical of each group. Hysterectomy was not associated with elevated cytoplasmic or nuclear estradiol receptor levels. When hysterectomized rabbits were treated with estradiol-filled Silastic implant on Day 1, nuclear estradiol receptor levels fell by Day 20 to levels seen in untreated hysterectomized rabbits. Despite substantial losses in nuclear estrogen receptor, serum progesterone remained elevated on Days 16 and 20. Thus, the ability of estradiol to maintain serum progesterone in hysterectomized rabbits did not correlate directly with the level of estrogen receptor.

Animals↗
PubMed1983

Comparison of estrogen receptors in human premenopausal and postmenopausal uteri using isoelectric focusing.

Previous studies have suggested that the estrogen receptor in the postmenopausal uterus, although capable of binding estrogen, may be inactive, that is, incapable of translocating and binding to nuclear acceptor sites. The present study uses isoelectric focusing to characterize differences in the estrogen receptor between premenopausal and postmenopausal uterine tissue. Human myometrium (15 premenopausal and ten postmenopausal samples) was examined by isoelectric focusing in the presence and absence of diisopropylfluorophosphate (DFP) and molybdate, which inhibit proteolysis and stabilize the receptor. As demonstrated previously, receptor forms could be identified in two regions of the pH gradient, one in the pH 4 to 5 region and another at pH 6 to 7. When prepared without DFP and molybdate, the percentage of receptors focusing in the pH 4 to 5 range was significantly greater for premenopausal than postmenopausal tissue (P less than .01). The addition of DFP and molybdate significantly altered the focusing pattern of postmenopausal tissue (P less than .01), increasing the percentage of binding in the pH 4 to 5 range. In the presence of DFP and molybdate, the focusing patterns of receptor from premenopausal and postmenopausal tissue were not significantly different. These data suggest that endogenous proteases are more prevalent in postmenopausal tissue and can degrade the receptor to forms that focus at a higher pH. With the inhibition of these proteases, the focusing patterns of receptors in premenopausal and postmenopausal tissue become very similar, representing the undegraded and perhaps native form of the receptor.

Adult↗
PubMed1982

Phosphorylation in vivo of chicken oviduct progesterone receptor.

Progesterone receptor from an oviduct tissue mince incubated with (32P)orthophosphate was purified using affinity chromatography, gel filtration, and DEAE-Sephadex chromatography. Two receptor peaks were eluted from the DEAE column. Peak I contained one major protein band with a molecular weight of 90,000 and a 32P band at 90,000. Peak II also had a major 90,000-dalton protein band and a 32P band at the same position. In addition, peak II contained a major protein band at 104,000 daltons and a 32P band which appeared to be slightly larger. Peaks I and II from chickens injected with (32P)orthophosphate showed the same pattern of labeling. The 90,000-molecular weight proteins from peaks I and II and the 32P-labeled component of II with Mr greater than 104,000 all contained phosphoserine. On two-dimensional gels, the 90,000-dalton proteins from peaks I and II were indistinguishable. Both contained labeled phosphate. Two-dimensional gels separated the 104,000-dalton peptide from the slightly larger 32P-labeled protein which traveled close to it on a one-dimensional gel. The relationships of the 104,000-dalton protein and this phosphoprotein to the progesterone receptor are not known. The 90,000-dalton receptor form common to both peaks is a phosphoprotein.

Animals↗
PubMed1982

Purification of "nontransformed" avian progesterone receptor and preliminary characterization.

The nontransformed (molybdate-stabilized) avian oviduct progesterone receptor has been purified to near homogeneity by a simple four-step procedure: ammonium sulfate fractionation, affinity chromatography, gel filtration, and DEAE-Sephadex A-25 chromatography. The affinity resin (deoxycorticosterone-agarose) is very resistant to chemical breakdown and enzymatic destruction and can be used repeatedly. The material obtained after gel filtration was separated into two receptor components by DEAE-chromatography. The two components, termed I and II, are obtained in about 18% yield and are purified by about 6000-fold (I) and 4000-fold (II). They are highly purified as only a single major Coomassie blue-stained polypeptide of about Mr = 90,000 in both components I and II is seen on sodium dodecyl sulfate-gel electrophoresis plus a second band of Mr = 104,000 which is seen only in component II. Both purified receptor components have sedimentation coefficients of 8 S in glycerol gradients containing molybdate (10 mM). In the absence of molybdate, however, both receptor forms have sedimentation coefficients of about 4 S in gradients containing 300 mM KCl. Therefore, stabilization of the 8 S form by molybdate is still evident in highly purified receptor preparations. The Stokes radii, binding specificity, and steroid dissociation rate of purified receptor are similar to those found in crude cytosol. These results confirm our previous observation on the existence of two 8 S forms of the progesterone receptor and show that these molybdate-stabilized forms remain intact throughout purification.

Animals↗
PubMed1982

Characterization of two 8 S forms of chick oviduct progesterone receptor.

Progesterone receptor from the chick oviduct was characterized in the presence of sodium molybdate, an agent which blocks receptor transformation and maintains the receptor in an aggregated state. In sucrose gradients containing 10 mM molybdate, receptor from total cytosol sedimented with a peak at 8.9 +/- 0.1 S in the presence of 10 mM KCl and at 7.9 +/- 0.1 S in the presence of 300 mM KCl. Two receptor forms could be separated on DEAE-cellulose. One eluted at 100 mM KCl (type 1) and the other at 160 mM KCl (type 2). Both forms sedimented at 7.8 to 7.9 S and gel filtration in the presence of 300 mM KCl showed that type 1 had a Stokes radius of 73 A and type 2, of 76 A. Calculations based on the Stokes radii and sedimentation coefficients yielded estimated molecular weights of 234,000 (type 1) and 244,000 (type 2). These two 8 S receptor forms were analyzed for their content of the smaller A and B receptor components which had been identified previously in cytosol. After removal of molybdate, the type 1 and type 2 receptors were dissociated into 3.5 S forms by treatment with pyridoxal 5'-phosphate followed by borohydride reduction. The smaller forms were then identified by DEAE-cellulose chromatography. The type 2 receptor contained only the B component. Type 1 receptor also yielded only one smaller hormone-binding component. This form resembled the A receptor but appeared to be slightly larger and more acidic. Therefore, under these conditions, the A and B forms of progesterone receptor are not found together in a dimer. Instead, B is found in one 8 S complex and A, or a precursor of A, is found in another 8 S complex.

Animals↗
PubMed1982

A new affinity resin for purification of non-transformed avian progesterone receptor.

A new steroid affinity resin has been developed which can be successfully used to partially purify the non-transformed species of the progesterone receptor from chick oviduct. Deoxycorticosterone has been modified through its 21-carbon and linked to a stable spacer arm which is attached to Sepharose 2B by an epoxide technique. The affinity resin is very resistant to chemical breakdown or to enzymatic destruction in crude tissue extracts. We have standardized the capacity and optimum conditions for elution of receptor which is bound to the affinity resin. Receptor purification of several hundred-fold can be obtained routinely with good yield. The cytosol receptor prepared in the presence of sodium molybdate remains in the non-transformed 8S state following affinity chromatography.

Animals↗
PubMed1981

Transformation of mammary cytoplasmic glucocorticoid receptor under cell-free conditions.

The transformation of glucocorticoid--receptor complex in the cytosol from lactating mouse mammary tissue was studied by using elevated temperature and KCl as promoters of the transformation reaction. The transformed receptor was identified from the nontransformed receptor by the following criteria: (a) increased binding to DNA--cellulose, (b) increased binding to ATP--Sepharose, (c) higher affinity for the steroid as determined by steroid dissociation kinetics, and (d) different sedimentation profiles on sucrose gradients containing KCl and sodium molybdate. A greater percentage of the nontransformed receptor was converted to the transformed state by an increased KCl concentration as opposed to increased temperature. Pretreatment of cytosol with 10 mM sodium molybdate prevented both the temperature- and salt-mediated transformation of the receptor.

Animals↗
PubMed1980

Concentration of cytosolic estrogen receptors in patients with postmenopausal osteoporosis.

Although all postmenopausal women are relatively estrogen-deficient, osteoporosis develops in only some of them. This could be related to a greater-than-normal degree of postmenopausal failure in sex hormone production or to a decrease in biologic effect at target tissue sites. We compared the concentration of sex steroids in serum and the concentration of cytosolic estrogen receptors in one target tissue (obtained by biopsy of the uterine cervix) in 18 women with postmenopausal osteoporosis and in 18 age-matched postmenopausal control subjects. Serum androstenedione and estrone were not significantly lower and serum estradiol was only marginally lower in the osteoporotic patients. Estrogen-receptor concentration in cervical tissue from both postmenopausal groups was not significantly different. We concluded that abnormalities of residual postmenopausal production of sex steroids and binding of sex steroids to cervical target tissue are not the major factors accounting for the decreased bone mass in women with postmenopausal osteoporosis.

Aged↗
PubMed1978

An improved method for the measurement of 1,25-(OH)2D3 in human plasma.

Here we report a highly sensitive and convenient ligand binding assay for the determination of 1,25(OH)2D3 in small volumes of human plasma. This method involves: (1) extraction of vitamin D3 and its metabolites using methanol-methylene chloride with separation of phases by centrifugation; (2) gel chromatography and high pressure liquid chromatography for the quantitative isolation of 1,25-(OH)2D3; and (3) a sensitive ligand binding assay for 1,25-(OH)2D3 employing cytosol receptor from the intestinal mucosa of rachitic chicks. Using modified rachitogenic chick diets allows early (less than 4 wks) harvesting of active receptor for 1,25-(OH)2D3 in high yield. The method includes a rapid and effective procedure for stable and long-term storage of the active cytosol receptor. A convenient dextran-charcoal means is used for the separation of receptor bound from free 1,25-(OH)2D3 resulting in the achievement of a lower (less than 5%) background (i.e., nonspecific binding) than reported for other 1,25-(OH)2D3 assays. Analysis of this receptor shows it to be a saturable, single class of binding sites with a dissociation constant (Kd) of approximately 3.7 x 10-11. The final recovery of 1,25-(OH)2D3 following extraction and chromatography is 80 +/- 3% and triplicate determinations can be made on a 3 ml plasma sample. The ligand binding assay routinely detects less than or equal to 5pg of 1,25-(OH)2D3 per assay tube and the inter- and intraassay variation, based on repeated determinations of 1,25-(OH)2D3 in pooled normal human plasma, is less than 5%. Preliminary studies indicate that our methodology will permit measurement of plasma 1,25-(OH)2D3 levels in all normal subjects and in pathophysiologic states where 1,25-(OH)2D3 levels may be below or above normal values. 1,25-(OH)2D3 values (pg/ml +/- SEM) in human plasma obtained from both normals and patients with various untreated calcium homeostatic disorders were: normals = 33.5 +/- 1.8; end-stage chronic renal failure = 5.1 +/- 1.2; primary hypoparathyroidism = 18.3 +/- 2.8; primary hyperparathyroidism = 61.4 +/- 7.1; and hyperthyroidism with associated hypercalcemia = 42.1 +/- 8.4.

Animals↗
PubMed1977

Interaction of progesterone receptor with immobilized adenosine triphosphate.

Affinity chromatography has been used to study the binding of ATP to cyto-plasmic progesterone receptors of hen oviduct. A resin which selectively binds the receptor protein was prepared by linking ATP covalently to Sepharose 4B through a 6-carbon bridge of adipic acid dihydrazide. Receptor bound to the affinity resin was recovered in a single peak upon gradient elution with KCl (0.2-1 M) or ATP (0-0.1 M). While affinity chromatography was normally accomplished using the [3H]progesterone receptor complex, the hormone was not necessary for ATP binding under the conditions employed. The chromatography of crude receptor preparations allowed up to 100-fold purification with greater than 80% recovery of the receptor. The semipurified receptor appeared intact when analysed by sucrose gradient centrifugation, polyacrylamide gel electrophoresis, and DEAE-cellulose chromatography. The latter procedure separated the receptor into two components, A and B, both of which were capable of binding ATP. Although a specific biochemical role of ATP in hormone receptor action has not been demonstrated, the present studies support this possibility and, in addition, offer a convenient and reliable step for the purification of progesterone receptors.

Adenosine Triphosphate↗
PubMed1976

ATP-PPi exchange activity of progesterone receptor.

Progesterone receptor preparations from avian oviduct catalyze a pyrophosphate (PPi)-exchange reaction between ATP and 32P-labeled PPi. The reaction requires ATP exclusively and is Mn++-dependent. This enzyme activity is detectable in receptor preparations that have been purified extensively by chromatography on ATP-Sepharose and DEAE-Sephadex columns. Polyacrylamide gel electrophoresis of purified preparations reveals a comigration of [3H]progesterone-receptor complex and the enzyme activity. The PPi-exchange reaction is inhibited by both o-phenanthroline and rifamycin AF/013, which also block the nuclear binding of progesterone receptor. These findings indicate that progesterone receptor may be an enzyme or a subunit of an enzyme that is active in nucleotide metabolism.

Adenosine Triphosphate↗