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J Mester

Publications and source records attributed to J Mester.

At least 109 records · Page 6Linked to original sources
PubMed1984

Purification by affinity chromatography and immunological characterization of a 110kDa component of the chick oviduct progesterone receptor.

A 110kDa component of the chick oviduct progesterone receptor (PR) has been purified to homogeneity according to electrophoretic criteria and specific activity (assuming one progestagen-binding site/110kDa). The procedure involved affinity chromatography of 0.3 M-KCl-prepared cytosol, followed by DEAE-Sephacel chromatography (elution at 0.2 M-KCl). The final yield was about 12% in terms of binding activity. Properties of the 110kDa component indicate that it is identical with the 'B' subunit described previously [Stokes radius approximately 6.1 nm; sedimentation coefficient, (S20, w) approximately 4S; frictional ratio approximately 1.77]. It reacted with the IgG-G3 polyclonal antibody, but not with BF4 monoclonal antibody raised against the 8S molybdate-stabilized chick oviduct PR and reacting with its 90kDa component. Another progesterone-binding component, corresponding to the 'A' subunit, also previously described, was eluted from the DEAE-Sephacel column at approximately 0.08 M-KCl, and contained a peptide of molecular mass approx. 75-80kDa, which had S20, w approximately 4S in a sucrose gradient. This component was also recognized by IgG-G3, but not by BF4; it was very unstable in terms of hormone-binding activity.

Animals↗
PubMed1984

Effects of calcium on the chick oviduct progesterone receptor.

The chick oviduct cytosol progesterone receptor can be transformed to a small form (Rs = 21A, S20,w:2.9) denoted "mero-receptor" by incubation in the presence of Ca2+ [8]. In the molybdate-free cytosol all the progestin binding components could be completely transformed to mero-form by 1 h treatment with 100 mM Ca2+ at 0 degrees C. If EDTA was secondarily added, the ligand was rapidly released. If molybdate (20 mM) containing cytosol was incubated with Ca2+, no radioactivity was found in the meroposition on the Agarose A 0.5 m column, but the bound steroid sedimented at 2.9 S in sucrose gradients containing Ca2+ (and no molybdate). When 20 nM molybdate was added to cytosol containing receptor activated by 0.3 M KCl, complete mero-transformation by Ca2+ was obtained also by the gel filtration criterion, indicating that molybdate does not inhibit the mero-transforming factor. Ligand-free progesterone receptor could also be completely converted to mero-form by endogenous cytosolic transforming factor and calcium. The transforming factor was completely inactivated, when cytosol was run through Agarose A 0.5 m gel. Mero-transformation was found to be irreversible. The purified progesterone receptor subunit 110 K (B) was partially converted to smaller forms by calcium alone (100 mM, 0 degrees C, 1 h) whereas addition of a small amount of cytosol allowed complete conversion to mero-form.

Animals↗
PubMed1984

Chick oviduct progesterone receptor: structure, immunology, function.

Analysis of the purified chick oviduct progesterone receptor using biochemical and immunological approaches indicates that while the 'activated' receptor ('4S') is a mixture of two progestin-binding polypeptides, 'A' (Mr approximately 79 kDa) and 'B' (Mr approximately 110 kDa), the non-activated receptor ('8S') is a population of complexes containing a hormone-binding polypeptide (A or B, but probably not both) bound to a non-hormone-binding protein (Mr approximately 90 kDa). Two molecules of the 90 kDa protein appear to be present in each '8S' receptor molecule. The 90 kDa protein is also associated with the non-activated forms of receptors of other steroid hormones in the chick. Molybdate stabilizes the non-activated receptors, probably by forming weak coordination bonds with radicals provided by the subunits of the '8S' structure. Activation implies separation of the subunits, without a change in their primary structure, and does not require intervention of any protein other than those present in the '8S' receptor form. The presence of ligand at the binding site accelerates the activation process but, in vitro, is not necessary for it to occur. Unlike the non-activated form, activated receptors bind to the cell nuclei. However, histological studies with anti-progesterone receptor antibodies indicate that in the non-hormone-exposed tissue the (non-activated) receptors could be localized in the nuclei.

Animals↗
PubMed1984

The effect of exposure to charcoal and ion-exchange chromatography on the dissociation rate of estrogen from the nuclear estrogen receptor of hen oviduct.

The method of initiating dissociation of 3H-estradiol from the nuclear estrogen receptor of hen oviduct was found to have a profound effect on the dissociation rate. Likewise, prior exposure to charcoal or partial purification by ion-exchange chromatography had an effect on the dissociation rate. When the reaction was initiated by isotopic dilution with the addition of 1 microM unlabeled estradiol, dissociation of the complexes was rapid (t 1/2 approximately 3 min). When the reaction was initiated by the addition of charcoal to adsorb free steroid, the dissociation of the complexes proceeded slowly (t 1/2 approximately 30 min). Partial purification of the receptors by DEAE-Sephacel chromatography or 15 min exposure to charcoal at 0 degree C prior to initiation of the dissociation reaction by isotopic dilution produced a form of the receptor that exhibited an intermediate dissociation rate (t 1/2 approximately 10 min). The partially purified receptor that exhibited an intermediate dissociation rate was reconverted to the rapidly dissociating form in a reconstitution experiment. These data raise the possibility of a nuclear substance that regulates the rates of estrogen dissociation.

Animals↗
PubMed1984

Characterization of the estrogen receptor extracted from hen oviduct nuclei with pyridoxal phosphate.

The estrogen receptor was extracted in high yield from nuclei of laying hen oviduct with 10 mM pyridoxal-5'-phosphate (PLP). The receptor extracted under these conditions, unlike that extracted with 0.4 M KCl, displayed no tendency to aggregate on sucrose gradients in low salt. The receptor was eluted as a single peak from DEAE-Sephacel at an ionic strength of 0.13 M KCl. The receptor after DEAE chromatography had approximately half the molecular weight of that in the nuclear extract. A larger form could be reconstituted by the addition of whole nuclear extract to the DEAE eluate. These data support the notion that the nuclear estrogen receptor is a dimer composed of similar subunits.

Animals↗
PubMed1983

Effects of urea and molybdate on the chick oviduct progesterone receptor.

The chick oviduct cytosol progesterone receptor, when complexed with ligand, can be exposed to urea concentrations as high as 3 M (at 0 degrees C) without loss of steroid binding capacity. The ligand dissociation rate is increased greater than or equal to 10 fold under these conditions. The "native" 8 S form of the receptor is progressively converted to a 4 S species by urea (greater than 2 M) as seen in ultracentrifugation analysis. This conversion is inhibited by Na2MoO4 (5-50 mM) suggesting that molybdate stabilizes the 8 S molecule by direct interaction. At urea concentrations above 2 M, the ligand-free receptor looses progressively its binding capacity. The "transformed", 4 S receptor was less stable than the 8 S species, and could not be protected by molybdate.

Animals↗
PubMed1983

Protein kinase activity of purified components of the chicken oviduct progesterone receptor.

Preparations of the 90K and 110K components of the chick oviduct progesterone receptor (PR) purified to near homogeneity were tested for protein kinase activity. The 90K component was shown to incorporate radioactive phosphate from [gamma-32P]-ATP in the presence of Ca2+ but not of Mg2+ ions, while the 110K component was phosphorylated in the presence of Mg2+, but not of Ca2+. The enzymatic activity of the 90K polypeptide appeared selective, since added proteins (histones) did not become phosphorylated. However, all proteins present in the 110K preparations were phosphorylated in the presence of Mg2+. These data suggest that components of the chick oviduct PR display protein kinase activity.

Animals↗
PubMed1983

Temperature dependence of the dissociation rate constants for the nonactivated (molybdate-stabilized) and activated progesterone receptor-hormone complexes from the chick oviduct.

Both the nonactivated and activated forms of the chick oviduct cytosol progesterone receptor-hormone complexes displayed first-order dissociation kinetics at temperatures between 0 and 25 degrees C. The rate constant was always 2-3-times greater for the nonactivated than for the activated complex. The thermodynamic parameters calculated from the Eyring plot for the nonactivated and activated forms, respectively, were: delta H+ = 28.6 +/- 0.2 and 29.9 +/- 1.5 kcal/mol; -T delta S+ = 7.4 +/- 0.6 and 7.7 +/- 1.6 kcal/mol; and delta G+ = 21.3 +/- 0.5 and 22.1 +/- 0.1 kcal/mol. These values suggest that activation results in an increase in enthalpy of the ligand-receptor interaction, thus stabilizing the complex. The dissociation rate constants for the native complex obtained by two different experimental approaches, namely, isotope dilution ('chase') and dissociation against charcoal, indicated the absence of cooperativity in the receptor-ligand binding.

Animals↗
PubMed1982

Activation of the chick oviduct progesterone receptor by heparin in the presence or absence of hormone.

Activation (transformation) of the chick oviduct progesterone receptor was found to be induced at 0 degrees C by heparin free in solution as well as by chromatography on a column of heparin linked to acrylamide/agarose. The transformed molecule displayed properties of the activated form of [3H]progesterone-receptor complex obtained by heat treatment or by high ionic strength: smaller size (s20,w = 3.9 S, Stokes radius = 5.2 nm), lower rate of dissociation (t 1/2 approx. 50 h at 0 degrees C compared with approx. 20 h for the 'native' form) and increased binding to phosphocellulose. In all cases, molybdate was an effective inhibitor of transformation and stabilized a large 'native' form (s20,w = 7.9 S, Stokes radius = 7.6 nm). Transformation by neither KCl nor heparin depended on the presence of ligand bound to the receptor, and the properties of the receptor molecule produced by treatment of ligand-free receptor with high ionic strength or with heparin were identical with those of the activated progesterone-receptor complex, demonstrating that receptor activation can be obtained experimentally in the absence of hormone. Our data are compatible with a model in which activation implies separation of the 4 S units, which compose the approx. 8 S 'native' form.

Animals↗
PubMed1982

Physiologic and anthropometric aspects of vestibular regulation in rowing.

The role of the vestibular system during physical exercise has only been rarely analyzed by scientific investigations. An investigation was carried out with the purpose of obtaining information on vestibular regulation in rowers. The process of maintaining balance in the boat and the limiting factors were examined. Twenty beginners and 20 well-trained rowers participated in standardized vestibular tests in the laboratory as well as in the boat (single) on the water. The results suggest that several mechanisms in the physiologic field (torque of the body) are affected by training. The well-trained oarsmen showed a significant minor nystagmus frequency, and this criterion for general vestibular regulation also showed a relationship with the specific ability for maintaining balance in the boat.

Electronystagmography↗
PubMed1982

Progesterone receptor from chick oviduct: purification of molybdate-stabilized form and preliminary characterization.

A molydate-stabilized, 'non-activated' form of the progesterone receptor from the cytosol of oestrogen-stimulated chick oviduct has been purified to homogeneity by a three-step procedure. The first step, affinity chromatography using a N-(12-amino-dodecyl)-3-oxo-4-androsten-17 beta-carboxamide-substituted Sepharose gel, purified the receptor 1500-2700-fold with approximately equal to 50% recovery. In the second step, ion-exchange chromatography through a DEAE-cellulose column, progesterone receptor was eluted as a single peak at 0.1 M KCl. Purification after this step was greater than 6700-fold. The third step was filtration through Ultrogel AcA 34, resulting in overall purification approximately equal to 7400-fold with overall recovery approximately equal to 25% of pure receptor on the basis of 1 binding site/molecule of Mr 85000. The purified molybdate-stabilized receptor had a sedimentation coefficient approximately equal to 7.9S +/- 0.1 (n = 4) in 0.15 M or 0.4 M KCl containing sucrose 5-20% gradient and approximately equal to 8.9S +/- 0.2 (n = 6) in 0.15 M KCl containing glycerol 10-35% gradient, and its Stokes radius was 7.05 +/- 0.10 nm (n = 3) (calculated Mr between 240000 and 280000). Binding specificity of the purified receptor was the same as that found in crude cytosol. SDS-PAGE revealed a single band migrating as a polypeptide of Mr approximately equal to 85000 +/- 2300 (n = 9). PAGE under non-denaturing conditions at total acrylamide concentrations 5%, 7% and 9% showed a single [3H]ORG 2058-protein band (ORG 2058 is a high-affinity analogue more suitable than progesterone for electrophoretic studies). The data suggest that the high molecular weight molybdate-stabilized progesterone receptor purified from oestrogen-primed chick oviduct is composed of only approximately equal to 85000-Mr polypeptide chains.

Animals↗
PubMed1982

Combined effects of progesterone and tamoxifen in the chick oviduct.

Tamoxifen, an antiestrogen with no estrogenic effect on growth, differentiation, and egg white protein synthesis in the chick oviduct, has been administered together with progesterone. In estrogen differentiated, hormone-withdrawn animals, biochemical and ultrastructural data indicated that the combined treatment with progesterone plus tamoxifen increased conalbumin (+100%) and ovalbumin (+30%) more than progesterone alone and displayed estrogen-like growth-promoting properties (+50% in DNA content per oviduct). Moreover, when administered with progesterone and estradiol, tamoxifen lost part of its antiestrogenic activity. No obvious change of progesterone clearance from the blood or progesterone metabolism was recorded. The presence of progesterone in amounts similar to those resulting in optimal activity when injected alone (greater than or equal to 1 mg/kg) was necessary to obtain progesterone plus tamoxifen effects. The simultaneous presence of progesterone and tamoxifen was required in order to observe their combined effects, and it was observed that progesterone did not have to commit cells on which tamoxifen could subsequently act. In the immature "undifferentiated" oviduct, contrary to the effects of tamoxifen and progesterone alone, their combination induced cytodifferentiation of tubular gland cells which synthesized conalbumin and ovalbumin, an event never observed before in the absence of estrogen. These results support the suggestion that the hormonal milieu interferes with antiestrogen action.

Animals↗
PubMed1981

Early inhibition by progesterone of oestrogen-induced ornithine decarboxylase activity in the chick oviduct and rat uterus.

The oestradiol-induced increase of ornithine decarboxylase (ODC) activity in the 'withdrawn' chick oviduct was found to be inhibited by progesterone. In vitro (2 h at 37 degrees C), progesterone (1 mumol/l) abolished the effect of oestradiol (20 nmol/l), progesterone alone having no effect. In vivo, progesterone (3 mg/kg) inhibited approximately 70% of the augmentation of ODC activity induced in the oviduct within 2 to 6 h of treatment with oestradiol benzoate (1.5 mg/kg). Administration of progesterone alone in vivo caused an increase in the ODC activity, the maximum level measured after 6 h being similar to that obtained when the chicks were given both oestrogen and progesterone. In the rat uterus in vivo progesterone also inhibited the rise of ODC activity caused by oestradiol, approximately % inhibition being observed between 2 and 6 h after treatment. Progesterone alone had no effect on uterine ODC activity during this period.

Animals↗