Progesterone Binding Capacity Research Articles

Membrane-Induced Conformational Change of α1-Acid Glycoprotein Characterized by Vacuum-Ultraviolet Circular Dichroism Spectroscopy

The tertiary structure of alpha1-acid glycoprotein (AGP) remains unresolved despite its novel function because AGP is a hard target in X-ray and NMR analyses. To elucidate the membrane-induced conformational change of AGP, the vacuum-ultraviolet circular dichroism (VUVCD) spectra of AGP and its constituent sugars were measured down to 160 nm in the presence or absence of phosphoglyceride liposome using a synchrotron-radiation VUVCD spectrophotometer. The secondary-structure contents and numbers of segments of AGP were estimated from the VUVCD spectra of the protein moiety obtained by subtracting the contributions of the glycan moiety. Further, the positions of secondary structures on the amino acid sequence were predicted by combining the VUVCD data with a neural network algorithm. These comprehensive secondary-structure analyses revealed that AGP consists of 11.4% alpha-helices (3 segments) and 39.9% beta-strands (12 segments) in the absence of liposome (pH 4.5), which are close to the proportions in the secondary structure of native AGP (pH 7.4) predicted by homology modeling, and that it consists of 47.5% alpha-helices (7 segments) and 2.7% beta-strands (2 segments) in the presence of liposome (pH 4.5). Detailed characterization of these alpha-helices of AGP bound to liposome suggested that two alpha-helices (residues 15-27 and 161-175) in the N- and C-terminal regions strongly interact with liposome. Most of the progesterone-binding residues of AGP were involved in the sequences transferring from beta-strands to alpha-helices or unordered structures, which coincided with the large decrease in progesterone-binding capacity of liposome-bound AGP. These results provide the first sequence-level information on the membrane-binding mechanism and structure-function relationship of AGP.

Adrenocortical Nuclear Progesterone-Binding Protein: Identification by Photoaffinity Labeling and Evidence for Deoxyribonucleic Acid Binding and Stimulation by Adrenocorticotropin

Nuclei of the guinea pig adrenal cortex contain a protein that specifically binds progesterone and that, biochemically, is clearly distinct from the classical progesterone receptor. The adrenocortical nuclear progesterone-binding protein has now been purified more than 2000-fold by steroid-affinity chromatography with a 75% yield. The purified protein preparation demonstrated three major bands on sodium dodecyl sulfate-polyacrylamide gel of 79K, 74K, and 50K. To determine which of the three might represent the progesterone-binding protein, steroid photoaffinity labeling was performed which resulted in the specific and exclusive labeling of a 50K band. Thus, the adrenocortical nuclear progesterone-binding protein appears to be distinct from the classical progesterone receptor not only biochemically, but also on the basis of molecular size. To test whether the adrenocortical nuclear progesterone-binding protein can be hormonally stimulated, guinea pigs were treated with ACTH. The chronic administration of ACTH caused a 4- to 6-fold increase in the specific progesterone binding capacity without a change in the binding affinity. There appeared to be no significant difference in nuclear progesterone binding between the zona fasciculata and zona reticularis. This finding suggests a mediating role for the progesterone-binding protein in ACTH action. In addition, the nuclear progesterone-binding protein bound to nonspecific DNA sequences, further suggesting a possible transcriptional regulatory role.

Alteration of lymphocyte reactivity in pregnant women treated with the progesterone receptor inhibitor ZK 98734.

Thirteen women during the 5th and 6th weeks of gestation were treated with a progesterone receptor blocker (ZK 98734) for pregnancy termination. Five patients received 100 mg/day of the compound, and eight patients received 50 mg/day for 4 days. Daily blood samples were obtained during the treatment period as well as on days 8 and 15 after the beginning of drug administration, and cytotoxic activity, progesterone sensitivity, and progesterone binding capacity of the lymphocytes were determined. Determination of SP-1 concentrations monitored the saturation state of trophoblastic progesterone receptors. High and low responders to the progesterone antagonist were identified. Lymphocytes of high responders treated with the 100 mg/day dose demonstrated a rapid increase of cytotoxicity, with a concomitant fall in progesterone sensitivity and progesterone binding capacity. Vaginal bleeding began as early as the 2nd day of treatment, and pregnancy was terminated by the 8th day of the treatment. Low responders receiving 100 mg/day demonstrated similar but attenuated changes. Low responders did not bleed during the first 4 days of treatment, and termination of pregnancy occurred later compared to high responders. Lymphocytes of high responders treated with 50 mg/day behaved similarly to those obtained from the low responders treated with 100 mg/day, and abortion was not completed before the 25th day after the beginning of the treatment. Lymphocytes function of low responders treated with 50 mg/day did not change significantly, the SP-1 concentration did not decline, and pregnancies continued. We conclude that a difference in sensitivity to the progesterone receptor blocker explains the selective block at trophoblastic binding sites only and the inability to block those in the lymphocytes.(ABSTRACT TRUNCATED AT 250 WORDS)

Receptor-binding capacity for estrogens and progesterone in the uterine cervix during the menstrual cycle and in the postmenopausal period

In biochemical studies it was found that the binding capacity of cytoplasmatic estrogen and progesterone receptors in the uterine cervix undergo characteristic changes during the menstrual cycle. The highest estrogen binding values were observed by the eighth day, after which there was a significant drop. The highest progesterone binding values were around the twelfth day, followed by a significant drop after ovulation. In sexually mature subjects the estrogen receptors predominated in the mucosa and the progesterone receptors in the myometrium. Therefore, there is obviously a parallel to the receptor content in the corpus uteri. The maximum estrogen and progesterone binding capacities were in each case ascertained some days before the maximum E2 and Pr values were attained in the serum. Hence, the estrogen receptors have already built up in the uterine cervix before optimal functional activity, measured by the cervix score, has been attained. Anti-estrogens given at the beginning of the cycle can disturb this sequence. The high estrogen receptor content in the endometrium at the beginning of the cycle creates the necessary conditions for the explosive, proliferative gland growth around this time, even though the serum estrogen concentration is still relatively low. In the postmenopause the estrogen binding capacity in the uterine cervix was higher than during sexual maturity, while the reverse was the case with the progesterone binding capacity.

An immunohistochemical study of steroid binding by endometrial glandular epithelial cells throughout the menstrual cycle.

An immunohistochemical technique was used to demonstrate oestrogen and progesterone binding by endometrial glandular epithelial cells at various stages of the menstrual cycle. A cross-section of steroid binding sites, including low affinity, concentration dependent Type II and III binding sites, were demonstrated but only in cells which contain the classical high affinity receptor. The technique demonstrated both free binding sites and receptor-hormone complexes within the cytoplasm but was poorly effective in demonstrating nuclear complexes. Oestrogen binding sites were shown to reach a peak during the late proliferative and early secretory phases of the cycle: oestrogen binding capacity remained high during the mid and late secretory phases. Progesterone binding capacity rose progressively throughout the proliferative and early secretory phase.

Steroidal binding sites in the ampulla of the human fallopian tube — autoradiographic and biochemical study

Most of the morphological changes which occur during the different phases of the reproductive cycle are hormone-dependent. Thus, the aim of this work was to demonstrate the fluctuations in the availability of steroid binding sites in the ampullary segment of the tube during the menstrual cycle. Specimens were obtained during routine gynecological operations and were prepared for histological examination. In addition, determinations of specific binding of estradiol and progesterone (specific activity was about 100 Ci/mmol) were made in soluene-dissolved tissue of ampullary mucosa. Autoradiographic grain counts of tissues which were incubated with [ 3H]estradiol and [ 3H]progesterone were performed on histological sections. The epithelial cell binding capacity was higher than that of the cells in the underlying lamina propria and this was found throughout the cycle. Estrogen and progesterone binding capacity was higher during the proliferative phase and decreased markedly during the secretory phase. Tissue binding of steroids appears to be inversely related to serum levels, which are lower in the proliferative phase and rise during the secretory phase.

Biochemical characterization of the progesterone receptor in the chick bursa of fabricius

In a previous work we demonstrated estrogen-inducible progesterone binding sites in the bursa of Fabricius [1]. In the present study these were characterized and compared to the progesterone receptor (PR) in the chick oviduct. When the size of the binding sites was analyzed with sucrose gradient centrifugation, 2 peaks of bound progesterone were obtained. The sedimentation coefficients of the peaks were 8–9 S and 3–4 S. In size exclusion HPLC only 1 peak was seen with a size corresponding to the 8–9 S in the sucrose gradient. The Stokes radius was 7.7 nm. When the ionic strength was elevated or CaCl 2 was added, smaller steroid binding forms were detected. The sizes of these progesterone binding molecules at low and high ionic strength and in the presence of CaCl 2 were equal in bursa and oviduct when analyzed with HPLC. The Stokes radii of these forms were 5.6 nm in high salt and 2.1 nm with CaCl 2. The steroid binding components in the bursa cytosol eluated as 2 peaks from the DEAE column with KC1 gradient. The peaks corresponded to the so-called A and B components in the chick oviduct. In the presence of molybdate, bound progesterone eluated as one peak from DEAE in both oviduct and bursa. The progesterone binding capacity was shown to be heat labile with equal half-lives in the bursa and the oviduct. Progesterone and ORG 2058 had a high affinity for the binding site and their binding was specific for progestins. It is concluded that the estrogen-inducible progesterone binding site in the bursa of Fabricius resembles the oviductal progesterone receptor in structural and binding properties.

Estrogen receptors in cultured rat uterine cells: induction of progesterone receptors in the absence of estrogen receptor processing.

When cultured rat uterine cells were treated for up to 6 h with 5 nM 17 beta-estradiol, no decrease in the [3H] estradiol-binding capacity of the cells was observed (i.e. no processing). This was true whether the cells were treated directly with 5 nM [3H]estradiol or with 5 nM unlabeled 17 beta-estradiol followed by homogenization and exchange with [3H]estradiol in vitro. In additional experiments, intact cells were treated with medium containing 5 nM [3H]estradiol for 30 min, and then that medium was removed and replaced with medium containing 5 nM unlabeled 17 beta-estradiol. Receptor-bound estradiol in intact cells was totally exchangeable with estradiol in the culture medium (t1/2, approximately 90 min). Six-hour treatment of cells with 5 nM 17 beta-estradiol led to a 50% increase in the [3H]progesterone-binding capacity of the cells, while no loss of estrogen-binding capacity occurred. These results indicate that progesterone receptors can be induced by estrogen in the rat uterus in the absence of estrogen receptor processing.

Effects of molybdate on classification of estrogen and progesterone receptor status in human breast cancer

Incorporation of disodium molybdate in the homogenization buffer for breast cancer specimens was shown to inhibit a time and temperature dependent loss of estrogen (ER) and progesterone (PR) receptor binding capacity in the cytosols. Since positive receptor values are correlated with response to endocrine therapy, the effect of molybdate incorporation on ER/PR classification was further investigated in two separate series of specimens. In one series of 75 consecutive specimens, ER and PR were analyzed before incorporation of molybdate in the asssays, and the results compared with the next 75 consecutive specimens after incorporation of molybdate. The mean ages of the 2 groups of patients were not significantly different (59.1 ± 13.4 versus 59.4 ± 13.3 years). It was found that the PR binding capacity was significantly higher, the proportion of ER+/PR — specimens was significantly reduced, and that a bimodal distribution of ER+ specimens only became apparent when molybdate was incorporated in the ER and PR assays. The possibility exists that ER+ specimens with binding capacities above 175 fmol/mg protein, constituting 24% of all the specimens and representing the second bimodal peak, may represent hormone dependent tumours.

Effects of intravenous progesterone infusions on the epileptic discharge frequency in women with partial epilepsy

Earlier work showed that in a group of women suffering from partial epilepsy, there is a decrease in seizure frequency during the luteal phase of the menstrual cycle. Moreover, iv progesterone infusions decrease the discharge frequency from penicillin epileptic foci in cats, when given in doses that reach plasma concentrations as observed during pregnancy. In the present study, iv progesterone infusions, reaching plasma concentrations as during the luteal phase, were given to 7 women with partial epilepsy. The inclusion criterion was that they should have more than one epileptic discharge per 5 min on an ordinary EEG . A 6 h EEG-registration was made. Blank and progesterone solutions were infused for 2 h respectively and the progesterone infusion was followed by a 2 h EEG recording period. 4 of the 7 patients showed a significant decrease in spike frequency during the infusion. In the patients not showing this effect, the plasma progesterone binding capacity was high. The 2 patients with the most marked progesterone effect had low progesterone binding capacity and no antiepileptic treatment. Somnolence during the test might also have influenced the results in 2 of the patients.

Entrapment of a highly specific antiprogesterone antiserum using polysiloxane copolymers.

Antiprogesterone antiserum was entrapped within a polysiloxane copolymer prepared from a 3:1 mixture of tetraethoxysilane and 3-aminopropyltriethoxysilane monomers. 400 microliters of this monomer mixture entrapped 70 mg of the 140 mg of immunoglobulins which were added, and the protein could not be washed from the highly stable copolymer which formed. Approximately half of the entrapped antiprogesterone antiserum was found to retain progesterone binding capacity with an apparent Ka equal to that of free antiserum in solution and was insensitive to effects of pH between 3 and 7. These preliminary observations and the unique chemistry of polysiloxane polymer formation suggest that such polymers may be useful in the entrapment of proteins for a variety of applications.

Influence of treatment with prostaglandin synthesis inhibitor or progesterone on cytotoxic activity and progesterone binding capacity of lymphocytes during pregnancy

Cytotoxic activity and progesterone binding capacity of lymphocytes obtained from women with threatened preterm labour were tested before and during treatment with acetylsalicylic acid or 17-hydroxyprogesterone caproate. Cytotoxic activity of the lymphocytes significantly decreased (p < 0.001), while progesterone binding capacity significantly increased (p < 0.001) following the introduction of the treatments. No changes in these parameters were observed on lymphocytes of untreated women. Preterm delivery occurred more frequently among untreated women than among women treated with acetylsalicylic acid (p < 0.01) or those treated with 17-hydroxyprogesterone caproate (p < 0.05).

Distribution of progesterone-binding cytochrome P450 and steroid-17α-hydroxylase/C-17,20-lyase within different compartments of the rat testis

Progesterone-binding capacity of cytochrome P450 and rate of progesterone consumption by cytochrome P450-dependent hydroxylases are highest in the Leydig cell fraction but are also detectable in the non-Leydig cell fraction of interstitial cells as well as in seminiferous tubules from rat testis. The Leydig cell compartment, however, contributes only to a minor extent to the total progesterone binding and hydroxylation within the whole testis.

Immunosuppressive effect of serum progesterone during pregnancy depends on the progesterone binding capacity of the lymphocytes

Cytotoxic activity and progesterone binding capacity of the lymphocytes, together with serum progesterone concentrations, were determined in women with normal pregnancy or with a clinical diagnosis of threatened abortion or threatened premature labour. The lymphocytes of women with threatened abortion or threatened premature labour showed significantly higher cytotoxic activity ( P < 0.001) and significantly lower progesterone binding capacity ( P < 0.001) than did lymphocytes obtained from the healthy pregnant women. Significant inverse correlation was found between progesterone binding capacity and cytotoxic activity of the lymphocytes ( P < 0.001), but the progesterone concentration of the pregnancy serum appeared to have no influence on the other two parameters. The findings indicate that intact progesterone binding capacity of the lymphocytes is an essential factor for the manifestation of the blocking effect exerted by pregnancy serum on lymphocyte cytotoxicity in vitro.

Progesterone-binding properties of the microsomal fraction from chick oviduct

Progesterone binds to specific high-affinity and limited-capacity binding sites of chickoviduct microsomes of estiogen-primed chicks. The dissociation constant is 2 × 10 −9 M (range 1.6–3.0) and the number of binding sites 500 femtomoles/mg microsomal protein (range 464–551). Treatment of the estrogen-primed chicks by progesterone had no apparent effect on the progesterone-binding capacity or affinity. Competition studies showed that testosterone, R-5020, Org-2058, D-norgestrel, 5α-dihydrotestosterone and R-2323 were effective competitors of progesterone, in that order, whereas cortisol, estradiol and estrone exhibited only minimal displacement. No displacement of microsome-bound [ 3H]progesterone was found with fenoterol or prostaglandin p 2α. No high-affinity progesterone-binding sites were found in the microsomal fractions of liver, muscle, intestine or brain. On the basis of steroid-binding affinity and steroid-specificity determinations, the microsomal progesterone-binding components seem to be different from the progesterone receptor previously described in chick oviduct cytosol.

The Effect of Copper in vivo on Specific Progesterone Binding by Human Endometrial Cytosol

Copper has been shown to interfere with specific progesterone binding by human endometrial and myometrial cytosol in vitro. These results suggested that one possible mode of action of the copper-bearing intrauterine devices (IUDs) is through interference with the action of progesterone at its target sites. A prospective study was carried out to determine whether the proposed mode of action of copper-bearing IUDs could be demonstrated in vivo. The results of this study revealed a significant difference in specific progesterone-binding capacity between proliferative and secretory endometria (P less than 0.001). However, when secretory endometria of the controlled subjects were compared with those of the copper-bearing IUD wearers, no significant difference was observed in the specific progesterone-binding capacity (P greater than 0.2). These data suggested that copper released from copper-bearing IUDs in vivo may not interfere with the binding of progesterone to its receptors in vitro. It is doubtful that the contraceptive effectiveness of copper-bearing IUDs could be due to the ability of copper to prevent progesterone from exerting its full effects on the endometrium.

Determination of estradiol, estrone, and progesterone in serum and myometrium: correlation with the content of sex steroid receptors and 17 beta-hydroxysteroid dehydrogenase activity throughout the menstrual cycle.

A study has been carried out to compare the influence of estradiol, estrone, and progesterone on the cytoplasmic estradiol and progesterone receptor levels and 17β-hydroxysteroid dehydrogenase (17β-HSD) activity in human myometrium. The steroid hormone concentrations were measured simultaneously in both serum and cytosol of myometrial tissue. The estradiol receptor levels were highest during the midproliferative phase. The highest progesterone-binding capacity was found in myometrial cytosol around ovulation. The midcycle peak of the cytoplasmic progesterone receptor level correlated well with the first peak of tissue concentrations of estradiol and the increase of progesterone tissue concentrations. During the luteal phase, in contrast to the follicular phase, the progesterone receptor level decreased, whereas serum progesterone concentrations were high. The midcycle peak of cytoplasmic progester one-binding capacity and the increase of progesterone tissue levels at the same time were followed by an acti...

Studies on certain aspects of progesterone and cortisol dynamics before, during and after labour.

SUMMARYConcentrations of progesterone, cortisol, free (unbound) progesterone and cortisol binding capacity (CBG) in blood samples were measured serially from 3 weeks preceding delivery to 5 days post partum. There was no change in total plasma progesterone in the last few weeks of pregnancy or during labour but after delivery the concentration of progesterone decreased significantly. Cortisol concentrations showed no change in the last 3 weeks of pregnancy but increased considerably (by 67%) during labour and remained at this high level until at least 2 h post partum, decreasing thereafter. At 5 days post partum the cortisol concentrations were significantly lower than those found a few weeks before labour. The fraction of free progesterone did not change during labour but there was a significant rise in percentage free progesterone 2 h post partum. This rise in percentage free progesterone coincided with the highest concentration of plasma cortisol and a relatively low plasma progesterone. The fraction of free progesterone decreased 1 day post partum to levels that were similar to or slightly lower than those existing before delivery. There was no change in the concentration of CBG before or during labour or at least until 5 days post partum. These results generally support the suggestion that the rise in cortisol concentration during labour can lead to an increase in the fraction of free progesterone.

Interaction of progesterone with amphibian melanosomes

Melanosomes purified by sucrose gradient centrifugation from different Amphibian tissues bind progesterone (K D were respectively 8.2 10 −8M, 4.5 10 −8M and 4.4 10 −8M for Rana skin, retina and ovary), and to a lesser extent other steroid hormones. HCl-hydrolysis removes melanoproteins and increases the progesterone binding capacity of melanin. This result suggests that progesterone binds directly to the melanin polymer. Effects of protein modifiers (SH and SS reagents, proteolytic enzymes, ionic strength) argue for the protein dependence of binding process. Proflavine and Atabrine strongly inhibit progesterone binding to melanosomes and melanin.

PROGESTERONE BINDING IN HUMAN ENDOMETRIAL CARCINOMAS

Abstract By means of dextran-coated charcoal assay, the capacity of various endometrial cytosol preparations for specific binding of 3 H-progesterone was determined. With the use of an arbitrary value of 50 femtomoles of bound 3 H-progesterone per milligram of cytosol protein as the breaking point between high and low binding capacities, 19 out of 20 normal endometria had high progesterone-binding capacities. Two out of 11 Grade 1, 3 out of 8 Grade II, and 2 out of 4 Grade III endometrial carcinomas showed low binding capacities. All 4 endometrial polyps, 7 out of 9 hyperplastic endometria, and 0 out of 7 nonendometrial gynecologic tumors had high binding capacities. These data suggest a progressive loss of specific progesterone-binding activity from normal endometria to hyperplastic endometria, and from the well-differentiated to the anaplastic forms of endometrial adenocarcinoma. There seemed to be an inverse relationship between age and concentration of progesterone receptors in endometrial adenocarcinomas. All irradiated tumors studied had low progesterone-binding capacity.