Rapid analysis of estrogen and progesterone receptors using gel-exclusion high-performance liquid chromatography

Pavlik, Edward J.; Nagell, J.R. van; Muncey, Mark; Donaldson, Elvis S.; Hanson, M.; Kenady, Daniel E.; Reese, Erica; Talwalkar, V. R.

doi:10.1021/bi00530a024

Cited by 45 publications

(11 citation statements)

References 30 publications

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“…Cells were harvested by centrifugation and washed three times with saline. Yeasts were disrupted at 40C by vigorous agitation with 250-to 300-gm glass beads on a Vortex mixer for about 15 min in a homogenization medium containing 250 mM sucrose, 10 mM Tris, 1.5 mM EDTA, 12 mM monothioglycerol, 10 mM Na2MoO4, and 10% glycerol (pH 7.8). Tubes were -kept cold by immersion in an ice slurry.…”

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confidence: 99%

Estradiol binds to a receptor-like cytosol binding protein and initiates a biological response in Paracoccidioides brasiliensis.

Loose¹,

Stover²,

Restrepo³

et al. 1983

Proc. Natl. Acad. Sci. U.S.A.

126

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Paracoccidioidomycosis, a disease caused by Paracoccdioides brasilensis, which is endemic to Latin America, is much more common in men than women, suggesting a role for hormonal factors. We recently showed that two other yeasts possess steroid binding proteins and postulated that these receptorlike molecules represented a mechanism by which the hormonal milieu of the host might influence an infecting pathogen. Therefore, we examined P. bralensis for a sex steroid binding protein. Mr of =60,000. Competition experiments revealed that estrone, estriol, and progesterone had 25% of the affinity of estradiol, whereas diethylstilbestrol, androgens, and corticosteroids had low affinity. Investigation of steroid hormone actions in P. brasiienis indicated that estradiol inhibited the fungal transformation from mycelial form to yeast form, the initial step of infection. This suppressive effect was dose-dependent and not found with testosterone. We hypothesize that endogenous estrogens in the host, acting through the cytosol binding protein in the fungus, inhibit mycelialto-yeast transformation, thus explaining the resistance of women *to paracoccidioidomycosis.Paracoccidioides brasiliensis is a pathogenic fungus, the etiologic agent of paracoccidioidomycosis (South American blastomycosis). Although paracoccidioidomycosis is 13-87 times more common in men than women (1), contact with P. brasiliensis is essentially the same for the two sexes (2). The possibility has been considered that the hormonal milieu of the host might directly influence P. brasiliensis, affecting its pathogenicity (1, 3). Although this hypothesis is speculative, our recent findings (4-6) suggest a basis for a mechanism by which host hormones could affect an invading pathogen and alter its infectious properties. We have demonstrated the existence of intracellular proteins in two fungal genera that bind vertebrate steroid hormones with high affinity and specificity. Candida albicans possesses a protein that binds corticosterone and progesterone (4, 5), and Saccharomyces cerevisiae contains a different protein that selectively binds estrogens (6). We have postulated that these macromolecules may represent primitive hormone receptors in fungi. These proteins appear not only to bind endogenous fungal ligands but also to bind vertebrate steroid hormones, a fact we have exploited in selecting the radioprobes for these studies. Therefore, we considered the possibility that P. brasiliensis could possess either an androgen or an estrogen binding protein which, when occupied by the appropriate host sex steroid, might result in altered pathogenicity, thus explaining the predominance of infections in males. The data to be presented in this report demonstrate that P. brasiliensis does contain an estrogen binding protein. We also show that estradiol inhibits the fungal, transformation from mycelial form to yeast form (mycelial-to-yeast transformation), the initial step in the establishment of infection (7). Although the concordance of binding in vitro and bi...

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confidence: 99%

Estradiol binds to a receptor-like cytosol binding protein and initiates a biological response in Paracoccidioides brasiliensis.

Loose¹,

Stover²,

Restrepo³

et al. 1983

Proc. Natl. Acad. Sci. U.S.A.

126

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“…Progesterone receptors were determined on preparations homogenized in TEMG buffer (1 uterine equiv/mL) after charging with [3H]R5020 (10 nM) and using radioinert progesterone as competitor (2 µ ) (Pavlik et al, 1982a).…”

Section: Methodsmentioning

confidence: 99%

Estrogenicity of coumestrol in the mouse: fluorescence detection of interaction with estrogen receptors

Nelson¹,

Pavlik²,

Nagell³

et al. 1984

Biochemistry

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The estrogenicity of coumestrol, a fluorescent phytoestrogen, has been examined in murine uteri. Coumestrol competed with 17 beta-[3H]estradiol for binding to cytoplasmic estrogen receptors, caused cytoplasmic estrogen receptors to associate with chromatin in the nucleus, and induced progesterone receptors. By use of size-exclusion high-performance liquid chromatography (SEHPLC), the interaction of coumestrol with estrogen receptors was examined directly by monitoring the fluorescence associated with macromolecules having properties characteristic of estrogen receptors. These analyses were made possible by the addition of dimethylformamide to the elution buffer, at a concentration (7.5%) which improved recoveries but did not interfere with estrogen receptor binding. It was possible to detect fluorescent coumestrol at approximately 0.5 nM. All determinations were performed with preparations in which estrogen receptor activity was 3-10 nM. Exposure of these preparations to coumestrol (50 nM) resulted in the elution of increased fluorescent activity in the regions where estrogen receptors eluted during SEHPLC. This fluorescent activity was reduced when diethylstilbestrol, 17 beta-estradiol, hexestrol, or tamoxifen was present as a competitor (2 microM) but was unaffected by testosterone or progesterone. Diethylstilbestrol reduced fluorescence below endogenous base lines and thereby displayed a fluorescence quench property which was not observed with other ligands. When hepatic and renal estrogen receptor preparations were used, the injected receptor activity was observed to be the major limiting factor in detecting the interaction of coumestrol with estrogen receptors. These observations are relevant to attempts to visualize estrogen receptors in tumor cells and demonstrate that accepted biochemical criteria for ligand-receptor interaction can be satisfied when fluorescent ligands are examined.

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“…A breakthrough in this area of research is the finding that sodium molybdate, an inhibitor of phosphatases and proteases, can stabilize estrogen receptors [2][3][4], Several high-pressure liquid chromatogra phy (HPLC) methods have been shown to be useful tools, for both qualitative and quanti tative analyses of ERC owing to rapid elution. sensitivity and reproducibility [5][6][7][8][9]. By HPLC techniques, ERC of different molecular weights and surface charges were shown to be present in human breast cancers [5], Iactating rat mammary glands [5] uterine tumors [5] and rabbit uterine tissues [6][7][8].…”

Section: Introductionmentioning

confidence: 99%

“…sensitivity and reproducibility [5][6][7][8][9]. By HPLC techniques, ERC of different molecular weights and surface charges were shown to be present in human breast cancers [5], Iactating rat mammary glands [5] uterine tumors [5] and rabbit uterine tissues [6][7][8].…”

Section: Introductionmentioning

confidence: 99%

Analysis of Rat Uterine Estrogen Receptors by High-Pressure Liquid Chromatography Methods

Madhok¹,

Leung²

1986

Horm Res

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Cytosolic (ER_c) and nuclear (ER_n) estrogen receptors prepared from rat uteri were characterized by size-exclusion and ion-exchange HPLC. The oligomeric ER_C eluted as a single, sharp peak near the exclusion volume of the gel column; ER_n eluted as a broad peak. When salt-extracted ER_n was partially purified sequentially by Sephadex G-200, DEAE-cellulose chromatography and polyacrylamide gel electrophoresis, the partially purified receptor moieties were not distinguishable by the sucrose gradient method, but showed characteristic retention times in the size-exclusion HPLC column. Further distinction in net surface charges was observed between ER_C and ER_n moieties by ion-exchange high-pressure liquid chromatography (HPLC). Molybdate-stabilized ER_C was eluted as sharp peak at 0.27 M salt gradient. In contrast, fresh extracts of ER_n emerged as a broad peak in the region of 0.1–0.2M salt gradient. In the absence of molybdate, ER_c dissociated into several 4–5 S molecules, which were well resolved in the DEAE column. This report, therefore, demonstrates the usefulness of size-exclusion and ion-exchange HPLC for steroid receptor analysis.

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Rapid analysis of estrogen and progesterone receptors using gel-exclusion high-performance liquid chromatography

Cited by 45 publications

References 30 publications

Estradiol binds to a receptor-like cytosol binding protein and initiates a biological response in Paracoccidioides brasiliensis.

Estradiol binds to a receptor-like cytosol binding protein and initiates a biological response in Paracoccidioides brasiliensis.

Estrogenicity of coumestrol in the mouse: fluorescence detection of interaction with estrogen receptors

Analysis of Rat Uterine Estrogen Receptors by High-Pressure Liquid Chromatography Methods

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