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G A Puca

Publications and source records attributed to G A Puca.

At least 37 records · Page 2Linked to original sources
PubMed1991

Proteolytic activity of the purified hormone-binding subunit in the estrogen receptor.

The hormone-binding subunit of the calf uterus estradiol receptor was purified as a hormone-free molecule. Immunoaffinity chromatography with a specific monoclonal antibody was used as the final step. The purified subunit was specifically labeled by radioactive diisopropyl fluorophosphate. The diisopropyl fluorophosphate-labeled amino acid was serine. The purified receptor was able to release the fluorogenic or chromogenic group from synthetic peptides containing phenylalanine at the carboxyl terminus. This occurred only in the presence of estradiol and was hampered by aprotinin and diisopropyl fluorophosphate. Estradiol-dependent hydrolytic activity was also found in the eluate from gel slices after SDS/PAGE of purified receptor. This activity comigrated with the renaturable estradiol-binding activity. The estradiol antagonists 4-hydroxytamoxifen and ICI 164,384 as well as other steroid hormones were unable to activate this hydrolytic activity.

Amino Acid Sequence↗
PubMed1991

In vitro binding of the purified hormone-binding subunit of the estrogen receptor to oligonucleotides containing natural or modified sequences of an estrogen-responsive element.

Estrogen receptor (ER) was purified from calf uterus by immunoaffinity chromatography in the absence of the ligand. The purified ER consists of a mixture of monomer and homodimer forms of 67-kDa hormone-binding subunit (no 90-kDa heat shock protein is present). The purified ER was incubated with a 32P-labeled 61-basepair oligonucleotide containing the sequence of the estrogen response element (ERE) of the Xenopus laevis A2 vitellogenin gene. DNA mobility shift assays showed formation of specific complexes of the ERE containing oligonucleotide with ER, formation which did not require and was not affected by estradiol or antiestrogenic molecules. Both the monomer and the dimer were equally able to interact with the ERE-containing oligonucleotide. Sucrose gradient experiments showed that only the ER monomer is able to interact with an oligonucleotide in which a single mutation destroyed the dyad symmetry of ERE. Multiple symmetric mutations which did not alter the dyad symmetry of ERE nevertheless totally destroyed the ability of the oligonucleotide to form complexes with either the monomeric or dimeric form of ER. These results suggest that ER is able to bind to ERE independently of the presence of estradiol or other proteins and, therefore, that estradiol does not act by modulating the ability of ER to bind to ERE on DNA.

Animals↗
PubMed1990

An aprotinin binding site localized in the hormone binding domain of the estrogen receptor from calf uterus.

It has been proposed that the estrogen receptor bears proteolytic activity responsible for its own transformation. This activity was inhibited by aprotinin. Incubation of transformed ER with aprotinin modified the proteolytic digestion of the hormone binding subunit by proteinase K. The smallest hormone-binding fragment of the ER, obtained by tryptic digestion, was still able to bind to aprotinin. These results suggest that aprotinin interacts with ER and the hormone-binding domain of ER is endowed with a specific aprotinin-binding site.

Animals↗
PubMed1989

Aprotinin inhibits the hormone binding of the estrogen receptor from calf uterus.

Micromolar concentrations of the proteinase inhibitor, aprotinin, produced a dose-dependent inhibition in the binding capacity of the estrogen receptor from calf uterus. Aprotinin inhibition was greater at 28 degrees C than at 4 degrees C and only occurred when conditions allowed the receptor transformation. When aprotinin was tested in the presence of transformation inhibitors, its effect was no longer seen. The binding capacity of the highly purified estrogen-binding subunit was similarly inhibited.

Animals↗
PubMed1989

Metal binding sites of the estradiol receptor from calf uterus and their possible role in the regulation of receptor function.

The existence of putative metal binding sites on the estradiol receptor (ER) molecule from calf uterus was evaluated by immobilizing various divalent metals to iminodiacetate-Sepharose. ER from both crude and highly purified preparations binds to metal-containing adsorbents complexed with Zn(II), Ni(II), Co(II), and Cu(II), but not to those complexed with Fe(II) and Cd(II). Elution of ER was obtained by chelating agents or by imidazole, thus indicating that histidine residues on the ER molecule are involved in the interaction with the metal. Analysis of affinity-labeled ER by [3H]tamoxifen aziridine after elution from a column of Zn(II)-charged iminodiacetate-Sepharose showed that ER fragments obtained by extensive trypsinization were also bound. Zn(II) and the same other metals able to bind ER, when immobilized on resins, inhibit the binding of estradiol to the receptor at micromolar concentrations. This inhibition is noncompetitive and can be reversed by EDTA. The inhibition of the hormone binding was still present after trypsin treatment of the cytosol, and it was abolished by preincubation with the hormone. Micromolar concentrations of these metals were able to block those chemical-physical changes occurring during the process of ER transformation in vitro. Furthermore, if added to pretransformed ER-hormone complex, they strongly inhibited the binding of the complex to isolated nuclei. The presence of metal binding sites that modulate the ER activity in the hormone binding domain of ER is therefore speculated. Since progesterone receptor showed the same pattern of binding and elution from metal-containing adsorbents, the presence of metal binding regulatory sites could be a property of all steroid receptors.

Animals↗
PubMed1986

Estradiol receptor has proteolytic activity that is responsible for its own transformation.

We have investigated the effect of various protease inhibitors and substrates on the hormone- and temperature-dependent binding of partially purified estradiol-receptor complex to isolated nuclei. Only serine protease substrates and inhibitors significantly depressed estradiol receptor transformation. At 20 degrees C, we observed 50% inhibition with about 3 microM aprotinin or with 1.4 mM diisopropyl fluorophosphate. Aprotinin also blocked those size and charge modifications of receptor that are characteristic of the transformation process. The estradiol receptor was able to bind to aprotinin-agarose only under transforming conditions; i.e., the interaction was hormone- and temperature-dependent and inhibited by molybdate. Diisopropyl fluorophosphate, a covalent reagent for serine esterases, competitively inhibited the binding and specifically eluted the estradiol-receptor complex that had been bound to aprotinin-agarose. These results indicate that estradiol receptor transformation is due to the effect of a serine protease and that the receptor itself is endowed with this catalytic activity, which is triggered by the steroid.

Alkaline Phosphatase↗
PubMed1986

Interaction between estrogen receptor and subcellular structures of target cells: nuclear localization of unoccupied receptor and its modification induced by estradiol.

Experimental conditions affecting the partitioning of the estrogen receptor were studied. Homogenization of rat uteri at 25 degrees C resulted in a particulate partitioning of the estrogen receptor. The use of frozen tissue (-70 degrees C) or pre-exposure of the tissue to 0 degrees C prior to 25 degrees C homogenization, homogenization at 0 degrees C and tissue dilution all induced soluble partitioning of the receptor. The estrogen receptor found in the particulate fraction was mostly associated with the nuclei, even in the absence of hormone. The interaction between estradiol and the estrogen receptor induced modification in the receptor's charge and size that promoted its cold-insensitive association with the nuclei of target cells. These modifications were studied in a cell-free in vitro system and were reversibly blocked by molybdate. Similar changes occurred in vivo when estradiol interacted with the receptor in the nuclei of target cells.

Animals↗
PubMed1985

Particulate nature of the unoccupied uterine estrogen receptor.

Homogenization of rat uteri at 25 degrees C resulted in a particulate partitioning of the estrogen receptor. Homogenization at 0 degrees C, the use of frozen tissue, or the pre-exposure of the tissue to 0 degrees C prior to 25 degrees C homogenization induced soluble partitioning of the estrogen receptor. Binding of a radiolabeled monoclonal antibody indicated that, in absence of estradiol, the estrogen receptor is particulate and is associated with the nuclei-enriched fraction of the target cell. The presence of receptor in the soluble fraction thus appears to be an artifact of homogenization. The unoccupied receptor, loosely associated with the particulate fraction (cold-sensitive) represents the "native" form of receptor which, upon arrival of the hormone, becomes tightly associated (cold-insensitive). The transition from the cold-sensitive to the cold-insensitive status is accompanied by a modification of the electrical charge of the receptor.

Animals↗
PubMed1984

Interaction of rat liver glucocorticoid receptor with heparin.

When rat liver cytosol containing [3H]dexamethasone-glucocorticoid receptor complex is exposed to immobilized heparin (Sepharose-heparin; Seph-hep) the steroid receptor complex binds to the substituted Sepharose avidly [Kd = 3.5 (+/- 1.7) X 10(-10) M], and 80-90% of the receptor present is adsorbed to the solid phase after 40 min at 0 degree C. The binding is enhanced by Mn2+ (10 mM) and Mg2+, whereas Ca2+ and Sr2+ are ineffective. Sodium molybdate (10 mM) does not influence the reaction but enhances receptor stability. Moreover, binding of the receptor to Seph-hep is dependent on the ionic strength of the medium, because binding is totally reversed by 300 mM KCl. The bound [3H]dexamethasone-receptor complex can be recovered from Seph-hep with solutions (4 mg/mL) of heparin (95% release), dextran sulfate (88%), and chondroitin sulfate (63%); total calf liver RNA is less effective (9%), whereas dextran, D-glucosamine, N-acetyl-D-glucosamine, D-glucuronic acid, and sheared calf thymus DNA are totally ineffective (less than 3%). Both "native" and temperature "transformed" forms of the glucocorticoid receptor interact with immobilized heparin. These results strongly suggest that the receptor site that binds heparin is distinct from that binding DNA. An immediate application of this newly found ability of the glucocorticoid receptor to interact with heparin is the use of Seph-hep for affinity chromatography purification of the glucocorticoid receptor. A purification of 10-fold, with a recovery of 55-65%, can be achieved by using either 4 mg/mL heparin or 300 mM KCl to elute [3H]dexamethasone-receptor bound to the resin.

Animals↗
PubMed1984

Estradiol and progesterone receptors in malignant gastrointestinal tumors.

Estradiol and progesterone receptors were assayed in tumors from 79 patients with primary colorectal and 56 patients with stomach adenocarcinomas. Eighteen of 79 colorectal cancers contained estradiol receptor, while 34 specimens were positive for progesterone receptor. In stomach cancer, the positive samples were 8 for estradiol and 14 for progesterone receptors. In both types of tumors, the Kd was in the range of 10(-10) M for estradiol and 10(-9) M for progesterone receptor, respectively. In colorectal adenocarcinomas, the presence of progesterone receptor seems to be partially correlated to the presence of estradiol receptor while, in stomach tumors, this correlation is lost. The positivity of at least one receptor in colorectal cancers is higher in the female sex. The contrary occurs for stomach cancer. Sucrose gradient centrifugation showed that cytoplasmic estradiol receptor of stomach cancer sedimented at 8S or 4 to 5S at low ionic strength. The isoelectric point of stomach cancer estradiol receptor is 6.5.

Adenocarcinoma↗
PubMed1982

Effect of sodium molybdate on cytosolic estrogen receptor.

Estrogen binding activity of crude calf uterus cytosol is rapidly destroyed in heating. The time course of inactivation at 37 degrees C shows a biphasic pattern; sodium molybdate (5-10 mM) completely blocks one of the components in the estradiol-free cytosol, while it has little effect on cytosolic receptor complexed with estradiol. Partially purified native 8S receptor loses its heat sensitivity, and, as a consequence, the molybdate effect disappears. By sucrose gradient analysis of crude cytosol it is evident that molybdate does not affect the sedimentation properties of the estradiol receptor at low temperature. However, at increasing temperatures, molybdate prevents the disappearance of the receptor peak in the crude cytosol or the formation of large, KCl-resistant, aggregates in the presence of estradiol. The partially purified native 8S receptor does not aggregate on heating; addition to it of receptor-depleted cytosol results in the recovery of heat inactivation and aggregate formation, and this is prevented by molybdate. Molybdate has no protective effect on any other inactivating agent which does not act through aggregation of receptors. A crude cytosolic preparation of the receptor which is unable to form heat-dependent aggregates does not display the fast heat inactivating component.

Animals↗
PubMed1981

Assay of total estradiol receptor in tissue homogenate and tissue fractions by exchange with sodium thiocyanate at low temperature.

After injection of radioactive estradiol to ovariectomized rats, the [3H]estradiol--receptor complex transferred to the nuclei can be solubilized by low concentrations of NaSCN. The extraction by NaSCN is significantly more efficient than that obtained by KCl and is, in fact, complete; i.e., no radioactivity can be found in the nuclei after extraction. Since NaSCN also induces the exchange of receptor-bound estradiol with free hormone [Sica, V., Puca, G. A., Molinari, A. M., Buonaguro, F. M., & Bresciani, F. (1980) Biochemistry 19, 83], a simple assay method has been set up which measures receptor in tissue and tissue fractions, including nuclei and whole homogenate, at 0--4 degrees C, irrespective of whether the receptor is or is not interacting with endogenous hormone. The procedure consists of a simple incubation step at 0--4 degrees C overnight (16 h) of the nuclear fraction, cytosol, and a total homogenate in the presence of excess radioactive estradiol and 0.5 M NaSCN. This method is very easy to carry out, accurate, and precise and avoids the loss of binding sites which results from the heating procedures utilized in other methods. The ability to measure the binding in both the soluble and the particulate fractions of rat uterus permits the determination of the rate of the cytoplasmic to nuclear transfer of estrogen after injection of various hormone concentrations. No nuclear transfer was observed after administration of other nonestrogen hormones such as progesterone, testosterone, or hydrocortisone while a nonsteroid antiestrogen, tamoxifen, was able to translocate the receptor. It was found that 2 h after injection of estradiol into ovariectomized rats total receptor content of uterus shows a decrease which is proportional to the amount of hormone injected. After injection of a hyperphysiological dose of 17 beta-estradiol, a certain amount of the receptor--hormone complex remains in the cytosol for at least 4 h. The nuclear turnover of estradiol receptor related to the progesterone receptor induction has been studied. Actinomycin D and cycloheximide prevent nuclear processing.

Animals↗
PubMed1981

Estrogen and progesterone binding proteins in human colorectal cancer. A preliminary characterization of estradiol receptor.

Estradiol receptor (ER) and progesterone receptor (PgR) were assayed in tumors from 20 patients with primary colorectal cancer. Ten of 20 tumors contained high affinity sites for 17 beta-estradiol and progesterone. The highest concentration of ER was 56 fmol/mg of protein. The ER dissociation constant ranged from 1.6 X 10(-10) M (mean 4.6 +/- 2.6). The highest concentration of PgR was 42 fmol/mg of protein. The PgR dissociation constant ranged from 3 X 10(-9) to 9 X 10(-9) M (mean 5.65 +/- 2.1). Four out of 20 specimens analyzed were from male patients and all resulted negative for both receptors. Sixty per cent of ER positive tumors were also PgR positive, whereas only 20% of ER negative were PgR positive. Sucrose gradient centrifugation showed that cytoplasmic ER of colorectal cancer sedimented at 3 S in the absence of protease inhibitors and at 4.5 S in the presence of 1 mM phenylmethylsulphonyl fluoride (PMSF) both in low and in high ionic strength. When chromatographed on Sephadex G-200 almost all ER was quantitatively recovered in the included fractions. Molecular weights of ER eluted from Sephadex G-200 ranged from 90,000 to 50,000 daltons. Elution profile and molecular weight heterogeneity suggest that, in spite of the presence of PMSF, there is a limited proteolysis of ER. Partially purified colorectal cancer ER did not bind to sepharose-heparin. The isoelectric point of ER was 6.4-6.5.

Adenocarcinoma↗
PubMed1980

Effect of chemical perturbation with NaSCN on receptor-estradiol interaction. A new exchange assay at low temperature.

When 0.5 M sodium thiocyanate is added to uterine cytosol previously labeled with excess [3H]-17 beta-estradiol, no change can be detected in the steady-state cytosol concentration of [3H]estradiol-receptor complex for at least 20 h at 4 degrees C. However, the rate of exchange of bound estradiol in the presence of NaSCN was found to be substantially higher than that in the absence of the chaotropic salt. In the presence of NaSCN, the dissociation rate of the complex increases about 10-fold (K-1 SCN = 1.10 x 10(-2) min-1 vs. K-1 = 1.07 X 10 (-3)min-1) while the rate of association increases about 2-fold (K1 SCN = 1.2 X 10(7) min-1M-1 vs.K1= 7.4 X 10(6) min-1 M-1). The Kd changes 6.4-fold (Kd SCN = 9 X 10(-10) M vs. Kd = 1.4 x 10(-10 M) with no decrease in the number of binding sites as shown by Scatchard plots of saturation experiments. This effect of NaSCN can be exploited to assay preformed estrogen-receptor complex by exchange with [3H]estradiol at low temperature. When the sample containing preformed complex is incubated overnight (16 h) at 4 degrees C with excess [3H]estradiol in the presence of 0.5 M NaSCN, there is a quantitative exchange of nonlabeled for estradiol without loss of binding sites. Hormonal steroids other than estrogens do not interfere, and the exchange estradiol is bound with high affinity. Precision, accuracy, and linearity of the method are highly satisfactory.

Animals↗