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Biomedical subjects

M Beato

Publications and source records attributed to M Beato.

At least 163 records · Page 9Linked to original sources
PubMed1980

Interaction of S-carboxymethylated uteroglobin with progesterone.

S-Carboxymethylated uteroglobin, a small progesterone-binding globular protein, was studied by means of high-field proton magnetic resonance spectroscopy. Conformational changes induced by the steroid have been observed and indicate a well-defined rearrangement of the structure of the protein. The unusual stoichiometry of one progesterone to two uteroglobin dimers is confirmed by 1H NMR. His-8 plays a central role in the mechanism of interaction of uteroglobin with progesterone. The pH dependence of affinity constant for the complexation of the steroid parallels the titration of this histidine. Although it is not a part of the active site, it influences a crucial conformational transition of the protein through the charge carried by its imidazole ring. Formation of a tetramer of uteroglobin subunits able to bind progesterone is critically compared to a kinetic scheme involving only dimers.

Animals↗
PubMed1980

Synthesis and secretion of uteroglobin in rabbit endometrial explants cultured in vitro.

Explants from rabbit endometrium incorporate [35S] methionine into uteroglobin and other proteins in an essentially linear fashion for at least 8 h. Newly synthesized uteroglobin was characterized by gel chromatography, sodium dodecyl sulphate gel electrophoresis and immunological techniques. Labelled uteroglobin accumulates in the tissue explants during the first 3 h and then reaches a plateau level. Secretion of uteroglobin into the medium is detected after a 1-h lag period and proceeds linearly thereafter. Explants from animals treated with estradiol alone or estradiol plus progesterone (pseudopregnancy) synthesized respectively 2--3-fold and 10--20-fold more uteroglobin than explants from control animals. The rate of degradation of uteroglobin in the in vitro system was not affected by the hormonal treatment of the animals. There is a good correlation between the uteroglobin content in the uterine fluid and the rate of synthesis of uteroglobin in endometrial explants, suggesting that the in vitro system faithfully reflects the in vivo hormonal effects on uteroglobin synthesis.

Animals↗
PubMed1980

The activated glucocorticoid receptor of rat liver. Purification and physical characterization.

The activated form of the hepatic glucocorticoid receptor has been purified 60 000-fold taking advantage of the differential affinity for phosphocellulose of the activated and the nonactivated forms of the receptor. Rat liver cytosol was incubated with [3H]triamcinolone acetonide at low temperature and low ionic strength and adsorbed to a large excess of phosphocellulose. The unbound fraction was heat-activated, passed through a column of DEAE-cellulose and precipitated with ammonium sulfate. The activated receptor was then bound to a small phosphocellulose column and eluted with a gradient of NaCl. Additional steps included chromatography on DNA-cellulose, precipitation with ammonium sulfate and centrifugation through a sucrose density gradient. The final preparation was identified as the steroid-receptor complex by electrophoresis on polyacrylamide gels in the presence of heparin; it exhibits a single band of molecular weight 40 000 +/- 4000 in gels containing sodium dodecyl sulfate. An independent calculation of the molecular weight under nondenaturing conditions based on the Stokes, radius (2.7 nm) and the sedimentation coefficient (S20, w = 3.0 S) yields similar results, indicating that the activated form of the receptor we have isolated is composed of a single polypeptide chain and contains a single steroid-binding site per molecule. Since this homogenous preparation of receptor has retained its affinity for DNA it may be useful for cell-free studies.

Animals↗
PubMed1980

RNA synthesis in rabbit endometrial nuclei. Hormonal regulation of transcription of the uteroglobin gene.

Endometrical nuclei, prepared from rabbits subjected to different hormonal treatments, were used for the cell-free synthesis of RNA. Optimal conditions for the incorporation of [3H]UMP into RNA are described, leading to the synthesis of relatively undegraded RNA molecules. Under these conditions there is virtually no initiation of new RNA chains in vitro, and RNA chain elongation is inhibited up to 60% by low concentrations of alpha-amanitin and up to 90% by actinomycin D. The synthesis of RNA is slightly inhibited in the presence of Hg-CTP and monothioglycerol, but newly synthesized mercurated RNA can be efficiently separated from endogenous RNA upon chromatography on sulfhydryl-Sepharose under stringent conditions. The RNA synthesized in vitro by endometrial nuclei from pseudopregnant rabbits contains RNA sequences transcribed from the uteroglobin gene, as demonstrated by hybridization to an excess of purified preuteroglobin cDNA. In endometrial cells from pseudopregnant animals the number of RNA polymerase II molecules transcribing the uteroglobin gene is 12-fold higher than in control animals, demonstrating that at least part of the hormonally induced accumulation of preuteroglobin mRNA is due to an increased rate of transcription of the uteroglobin gene.

Animals↗
PubMed1979

Activation of the progesterone receptor of rabbit uterus.

The influence of several parameters on the kinetics of activation of the progesterone receptor in the cytosol of rabbit uterus is described. The estimation of the proportion of activated receptor is based on the differential affinity of the activated and non-activated forms of the receptor for phosphocellulose. Under appropriate conditions binding to phosphocellulose can be used as a test of activation and gives results similar to those obtained with DNA--cellulose, or isolated cell nuclei. The kinetics of receptor activation is temperature-dependent and compatible with a first-order reaction at all temperatures tested. The thermodynamic activation energy of this reaction is 67.8 kcal mol-1. The progesterone receptor can be activated to various extents by increased ionic strength or by dilution of the cytosol with buffers of low ionic strength, and in all cases the activation follows apparent first order kinetics. At a concentration of 0.4 M NaCl, 70--80% of the receptor can be converted into the activated form. The activated and non-activated forms of the receptor appear to be in equilibrium. Salt-activated and heat-activated receptor can be transformed to a non-activated form by decreasing either the salt concentration, or the temperature of incubation. The rate of dissociation of the steroid from the activated form of the receptor is indistinguishable from that observed with the non-activated form, but the activated receptor is more thermolabile. Upon centrifugation on sucrose gradients there are no major differences in the sedimentation behaviour of the two forms of the receptor.

Animals↗
PubMed1979

Synthesis and characterization of a DNA complementary to pre-uteroglobin mRNA.

Uteroglobin, a progesterone-induced uterine protein of the rabbit, is synthesized in cell-free systems as a precursor containing 21 additional amino-acids at its N-terminal end. The mRNA for pre-uteroglobin has been purified from the membrane-bound polysomes of induced endometrium and used as template for the synthesis of a full copy complementary DNA. Final purification of the cDNA was based on hybridization to the template mRNA up to a low value of r0t (0.01 M . s) and digestion of the non-hybridized cDNA by S1 nuclease. A comparison of the hybridization kinetics of the pre-uteroglobin cDNA and rabbit globin cDNA to their respective templates indicates a nucleotide sequence complexity of 650 for pre-uteroglobin mRNA, in agreement with the values obtained by sucrose gradient centrifugation and polyacrylamide gel electrophoresis in formamaide. The melting temperature of the hybrids of pre-uteroglobin cDNA to its template reflects the absence of mismatched sequences. This cDNA has been used to quantify pre-uteroglobin mRNA sequences in the endometrial RNA from control animals and from animals treated sequentially with estradiol and progesterone. In agreement with the induction of uteroglobin-synthesizing activity, there is a dramatic increase in the uterine content of pre-uteroglobin mRNA after hormonal treatment. Part of this effect can be accounted for by hormonally induced cell proliferation. When expressed on a DNA basis there is a 50--100-fold increase in the cellular content of pre-uteroglobin mRNA following hormonal treatment.

Animals↗
PubMed1978

Amino acid sequence of progesterone-induced rabbit uteroglobin.

Uteroglobin, a steroid-binding protein of the uterine secretion of the rabbit which is induced by progesterone, comprises two identical polypeptide chains of 70 amino acid residues linked by two disulfide bonds. The primary structure has been determined by using both automated and manual methods of Edman degradation. Overlapping peptides were isolated from tryptic, and CNBr digests. The sequence is not homologous to any known protein except for a small acidic region (residues 22-29) resembling a sequence found in somatotropin. The C-terminal half is relatively basic. Implications for the secondary structure are discussed.

Amino Acid Sequence↗
PubMed1978

Effect of phospholipases and lysophosphatides on partially purified steroid hormone receptors.

Treatment with phospholipase A2 of crude or partially purified preparations of the glucocorticoid receptor of rat liver results in an inactivation of the receptor, which cannot be attributed to contaminating proteases. Similar enzymatic treatment of the progesterone receptor of rabbit uterus does not affect its steroid-binding activity. At various stages during purification the preparations of glucocorticoid receptor contain 10 to 50-fold higher concentrations of lipid phosphate than the corresponding preparations of progesterone receptor, suggesting that the effect of phospholipase A2 on the hepatic receptor could be mediated by lysophosphatides produced during hydrolysis of endogeneous phospholipids. In fact, mixing experiments show that in the presence of the glucocorticoid receptor, phospholipase A2 also inactivated the progesterone receptor. Both partially purified receptors are inactivated by similar concentrations of added lysophosphatides but are not affected by incubation with phospholipase C, which does not produce ionic detergents. In addition, the effects of phospholipase A2 and of added lysophosphatides can be overcome by an excess of bovine serum albumin, indicating that free lysophosphatides are involved in receptor inactivation, possibly due to their strong detergent properties.

Animals↗
PubMed1978

Characteristics of the purified uteroglobin-like protein from rabbit lung.

A uteroglobin-like protein was prepared from lung extracts of female rabbits by absorption to immobilized anti-uteroglobin immunoglobulin and purified to homogeneity by gel filtration on Sephacryl S-200. The final preparation is indistinguishable from uteroglobin according to its behaviour in Ouchterlony double-diffusion, polyacrylamide gel electrophoresis under denaturing and non-denaturing conditions, ultraviolet spectrum, tryptic peptide analysis, and progesterone-binding properties. Progesterone binding to the lung protein exhibits an affinity similar to that observed with authentic uteroglobin and is equally enhanced by reduction of the protein with dithiothreitol. Competition experiments with non-radioactive steroids demonstrate a similar steroid-specificity for both proteins. Progesterone binding causes a perturbation in the ultraviolet absorbance of tyrosine residues of the lung protein similar to that observed with uteroglobin. These data suggest that the proteins prepared from both sources are biochemically identical.

Animals↗
PubMed1977

Properties of the partially purified activated glucocorticoid receptor of rat liver. Binding to chromatin subunits.

The activated glucocorticoid receptor from rat liver has been purified over 3000-fold by repeated adsorption to phosphocellulose. The partially purified receptor-triamcinolone complex is stable for sever weeks at -75 degrees C in the presence of 0.1% bovine serum albumin, and sediments at 2.9 (+/- 0.1) S in sucrose gradients containing 0.15 M Na Cl. The Stokes' radius of the partially purified receptor is 26.5 A, and the frictional ratio is 1.14, indicating that at near physiological salt concentration the activated receptor is slightly elongated and has a molecular weight of 33 500. These preparations of receptor are free of exo- and endonucleases and bind to DNA and chromatin, as well as to the native chromatin subunits, the nucleosomes. Removal of histone H1 and further digestion of the nucleosomal DNA to the core size of about 140 base pairs indicate that the integrity of the nucleosome structure determines the extent of interaction with the glucocorticoid receptor. Although the binding of receptor to unfractionated rat liver chromatin is more efficient than to chicken erythrocyte chromatin, the nucleosomes of both tissues bind equal amounts of receptor. Therefore, the factors responsible for this tissue difference do not reside in the nucleosomes, but rather in a higher order structure of the chromatin or in that part of the genome which is not organized as nucleosomes.

Animals↗
PubMed1977

Binding of the partially purified glucocorticoid receptor of rat liver to chromatin and DNA.

The binding of the glucocorticoid receptor of rat liver to chromatin and DNA has been studied with crude and partially purified preparations of cytosol receptor labelled with [3H]-triamcinolone acetonide in vitro. The use of crude preparations of receptor and increasing protein concentrations leads to an apparent saturation of chromatin and DNA, suggesting a limited number of high affinity nuclear acceptor sites for the receptor. Appropriate controls indicate that the observed saturability of chromatin acceptor sites is due to the presence in crude receptor preparations of heat-stable protein factors which interfere with the binding of the receptor to the genome; whereas the apparent saturation of DNA is due to contamination with deoxyribonucleases. If the activated complex of receptor and triamcinolone acetonide (R-TA) is partially purified to a step where it is free from nucleases and inhibitors, its binding to both chromatin and DNA is linearly dependent on the concentration of free (R-TA) in the incubation medium. There is no absolute specificity with respect to the source of DNA or chromatin, although liver chromatin has considerably higher receptor binding capacity than chromatin from avian erythrocytes. The rate kinetics of association and dissociation for the binding of (R-TA) to DNA and chromatin are very similar, but DNA exhibits a 10-fold higher receptor binding capacity than chromatin. These data, in conjunction with the effect of poly-(D)-lysine and and NaCl on the binding of (R-TA) to chromatin and DNA, suggest that most of the receptor molecules bound to chromatin in vitro interact with the "accessible" DNA stretches. Although a small population of receptor molecules may bind specifically to target tissue genome, the detection of these specific sites against the background of unspecific binding is not possible with unfractionated chromatin or DNA preparations.

Animals↗